key: cord-005892-3yuznrdv authors: hübener, p.; braun, g.; fuhrmann, v. title: das akut-auf-chronische leberversagen als diagnostische und therapeutische herausforderung der intensivmedizin date: 2017-02-16 journal: med klin intensivmed notfmed doi: 10.1007/s00063-017-0263-3 sha: doc_id: 5892 cord_uid: 3yuznrdv acute-on-chronic liver failure (aclf) is an emerging clinical syndrome in patients with underlying liver disease that is usually triggered by one or multiple insults and characterized by progressive hepatic and nonhepatic organ failure, a significant risk of infections, and high short-term mortality rates. despite our incomplete understanding of the underlying pathophysiology, aclf requires timely diagnostic and therapeutic measures aiming at the identification and elimination of causative factors as well as the prevention of complications to improve the prognosis of affected patients. das akut-auf-chronische leberversagen (aclf) wird im zusammenschluss der european association for the study of the liver (easl) und der american association for the study of liver diseases (aasld) als "akute verschlechterung einer vorbestehenden chronischen lebererkrankung, die üblicherweise im zusammenhang mit einem auslösenden ereignis steht und aufgrund eines multiorganversagens mit erhöhter 3-monats-letalität verbunden ist" definiert [1] . der fokus der definition liegt dabei eindeutig auf dem organversagen, das zumeist extrahepatische organe betrifft (. abb. 1) und die prognose betroffener patienten am stärksten beeinflusst [2, 3] . zur identifizierung, risikostratifizierung sowie definition in abhängigkeit von klinisch relevanten outcomeparametern wurden in 2 großen prospektiven multizentrischen untersuchungen -davon eine in europa (chronic liver failure consortium acute on chronic liver failure in cirrhosis, canonic) und eine in kanada/den usa (north american consortium for the study of end stage liver disease, nacseld) -risikogruppen p. hübener, g. braun und v. fuhrmann repräsentieren die sektion gastroenterologie der deutsche gesellschaft für internistische intensivmedizin und notfallmedizin (dgiin). mit hoher kurzfristiger mortalität identifiziert. gleichzeitig wurde die prognostische wertigkeit klinischer scores für das aclf evaluiert [4, 5] . hierbei zeigte sich ein in der tendenz erwarteter, in der tatsächlichen ausprägung jedoch alarmierender anstieg der kurzzeitletalität bis hin zu über 70 % [5] . klar abzugrenzen ist das aclf daher vom akuten leberversagen (alv), das durch den funktionsausfall einer zuvor gesunden leber definiert ist und bei dem durch intensivmedizinische und supportive maßnahmen in über 70 % der fälle ein überleben ohne organtransplantation erreicht werden kann [6, 7] . die identifikation spezifischer auslöser des aclf ist für die diagnosestellung nicht zwingend erforderlich (. tab. 1). die häufigsten auslöser des aclf sind geographisch unterschiedlich verteilt und oft für den krankheitsverlauf relevant [4) . die häufigsten auslöser des aclf sind geographisch unterschiedlich verteilt und oft für den krankheitsverlauf relevant [4, 5] . sie beinhalten lokale und generalisierte infektionen, neue oder fortgesetzte leberschädigung durch alkohol und/oder andere hepatotoxische substanzen, operationen, zirkulationsstörungen sowie seltenere ursachen wie beispielsweise einen schub einer autoimmunen hepatitis (. tab. 2). während die varizenblutung traditionell eher als dekompensation einer vorbestehenden leberzirrhose gewertet und ihre mortalität in den letzten jahren durch moderne evidenzbasierte behandlungsstrategien (einschließlich endoskopischer blutstillung, medikamentöser und interventioneller senkung des portosystemischen druckgradienten und vermeidung infektiöser komplikationen) gesenkt werden konnte, können sowohl hämorrhagischer als auch septischer schock nach einer blutung ein aclf auslösen. in bis zu 40 % der fälle kann kein auslösendes ereignis identifiziert werden. in diesen fällen liegen möglicherweise subklinische infektionen, passagere störungenderintestinalen barriere oder unerkannte hepatotoxische effekte als mögliche auslöser zugrunde [9] . unabhängig davon, ob das aclf durch direkte oder indirekte schädigung der leber ausgelöst wird, scheinen 2 faktoren im zentrum der pathophysiologie der erkrankung zu stehen: 1) hämodynamische veränderungen und 2) systemische inflammationsreaktionen ("systemic inflammatory response syndrome", sirs; [9, 11] [12] . zudem wurden auch andere pro-und antiinflammatorische zytokine (stnf-α r1, il-2, il-8, il-10, ifn-γ) in stark erhöhten plasmatischen konzentrationen bei patienten mit aclf nachgewiesen [13] . auch routinelaborparameter, wie leukozyten und crp, sind bei patienten mit aclf erhöht und korrelieren in ihrer höhe mit der anzahl von organausfällen und dem aclf-grad [5] . bei patienten, die eine erste immunaktivierung im rahmen des auslösenden stimulus überleben, wird, ähnlich wie bei der sepsis ohne lebererkrankung, ein übergang in einen immunsupprimierten bzw. immundysregulierten zustand ("immune paralysis"; [14] ) beobachtet, der das risiko für weitere infektionen signifikant erhöht. angesichts der hohen letalität hospitalisierter zirrhotiker bei infektionen (etwa 15 %) bzw. bei schweren bakteriellen infektionen mit septischem schock (60-100 %; [15] [16] [17] ) erscheint eine aggressive antiinfektive präventions-und therapiestrategie bei aclf-patienten von zentraler bedeutung. die canonic-studie ging der frage nach, welcher anteil von -zunächst meist auf einer normalstation -hospitalisierten zirrhosepatienten ein aclf aufweisen oder im verlauf entwickeln und welche prognostischen parameter sich in dieser patientengruppe ausmachen lassen, um besonders gefährdete subgruppen frühzeitig zu erkennen. das aclf wurde dabei als häufigste indikation für eine aufnahme von zirrhosepatienten auf eine intensivstation identifiziert. die anzahl und das jeweilige ausmaß von organversagen wurden mittels eines modifizierten sequentialorgan-failure-assessment(sofa)-scores erfasst, da sich dieses system in der vergangenheit zuverlässiger in der mortalitätsabschätzung von kritisch kranken zirrhosepatienten erwiesen hatte als die meld-oder child-pugh-klassifikation. der sofa-score wurde für die studie modifiziert (. tab. 3) und schließlich in eine vereinfachte form ("clif consortium organ failure score", clif-c-ofs) mit identischen organparametern und ähnlichem prognostischem wert modifiziert. der schweregrad des aclf wurde anhand der anzahl der organausfälle (of) stratifiziert: aclf 1: 1 of, aclf 2: 2 of, aclf 3: 3-6 of. in die außerordentlich hohe dynamik des krankheitsbilds aclf spiegelt sich auch in der tatsache wider, dass während der hospitalisierung in etwa 50 % eine besserung und in 20 % eine verschlechterung auftrat [18] . die heilungsrate sank mit dem aclf-grad von 55 % (aclf 1) über 35 % (aclf 2) bis auf 15 % (aclf 3). da sich der aclf-grad zumeist in den ersten beiden bis maximal 7 tagen änderte, wird hier ein "goldenes zeitfenster" für therapeutische interventionen vermutet (. abb. 1). für eine risikoabschätzung von patienten mit akuter dekompensation einer leberzirrhose mit oder ohne aclf wurden klinische und laborchemische parameter identifiziert, die mit erhöhter mortalität assoziiert waren. hier wurden 1) die anzahl von organversagen (nach clif-kriterien), 2) das patientenalter sowie 3) die konzentration zirkulierender leukozyten als unabhängige variablen identifiziert. diese resultate gingen wiederum in eine erweiterung des clif-c-ofs ein ("clif consortium aclf score", clif-c aclfs), der einen punktwert von 0-100 annehmen kann und eine deutlich bessere vorhersagekraft hinsichtlich der 28-, 90-, 180-und 360-tages-mortalität besitzt als meld-, meld-na-und child-pugh-score [19] . nachteilig ist hierbei, dass der score (verglichen zum ursprünglichen sofa-oder auch clif-sofa-score) nicht ohne eine komplexe formel berechnet werden kann; die kalkulation kann jedoch beispielsweise durch einen onlinerechner des "clif consortium" erfolgen [20] . in der weiteren aufarbeitung der canonic-kohorte zeigte sich eine 100 %ige letalität von patienten mit ≥4 organversagen und/oder einem "clif-aclfs score" ≥64 nach 3-7 tagen therapie, sofern keine lebertransplantation erfolgte [18] . die zahlen unterstreichen den stellenwert eines frühen und raschen -insbesondere intensivmedizinischen -therapiebeginns, um das fortschreiten des multiorganversagens zu durchbrechen. ob eine frühzeitige lebertransplantation bei patienten mit aclf zielführend ist, ist derzeit thema kontroverser debatten und wird erst durch zukünftige studien geklärt werden können. zusätzlich kann die wiederholte risikostratifizierung mittels scores bei patienten mit aclf in dem schwierigen entscheidungsprozess hinsichtlich der art des weiteren therapeutischen vorgehens (kurativer oder palliativer therapieansatz) helfen. wir möchten jedoch betonen, dass derartige entscheidungen definitiv ärztliche entscheidungen sind, die niemals von einem score übernommen werden dürfen. im rahmen dieses ärztlichen entscheidungsprozesses müssen neben der unmittelbaren schwere der erkrankung beispielsweise auch der mutmaßliche patientenwunsch, jeweilige lokale und nationale überlebensraten, eine potenzielle reversibilität der or-das akut-auf-chronische leberversagen als diagnostische und therapeutische herausforderung der intensivmedizin zusammenfassung das akut-auf-chronische leberversagen ("acute-on-chronic liver failure", aclf) ist ein emergentes krankheitssyndrom, das durch einen oder mehrere akute trigger bei vorgeschädigter leber ausgelöst wird und vom progressiven hepatalen und nichthepatalen organversagen, einem gravierenden risiko infektiöser komplikationen sowie hoher kurzfristiger letalität gekennzeichnet ist. wenngleich pathophysiologisch noch weitgehend unverstanden erfordert das aclf frühzeitige diagnostische und therapeutische maßnahmen, die sich auf zugrunde liegende ursachen sowie das verhindern von komplikationen richten, um die prognose betroffener patienten zu verbessern. leberversagen · zirrhose · infektion · organversagen · transplantation acute-on-chronic liver failure: a diagnostic and therapeutic challenge for intensive care abstract acute-on-chronic liver failure (aclf) is an emerging clinical syndrome in patients with underlying liver disease that is usually triggered by one or multiple insults and characterized by progressive hepatic and nonhepatic organ failure, a significant risk of infections, and high short-term mortality rates. despite our incomplete understanding of the underlying pathophysiology, aclf requires timely diagnostic and therapeutic measures aiming at the identification and elimination of causative factors as well as the prevention of complications to improve the prognosis of affected patients. liver failure · cirrhosis · infection · organ failure · transplantation acute-on chronic liver failure acute-on-chronic liver failure toward an improved definition of acute-on-chronic liver failure survival in infectionrelated acute-on-chronic liver failure is defined by extrahepatic organ failures acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis lessons from look-back in acute liver failure? a single centre experience of 3300 patients acute liver failure the pathogenesis of aclf: the inflammatory response and immune function acute-on-chronic liver failure: a new syndrome that will re-classify cirrhosis mechanisms of decompensation and organ failure in cirrhosis: from peripheral arterial vasodilation to systemic inflammation hypothesis tumor necrosis factor alpha and interleukin 6 plasma levels in infected cirrhotic patients pathophysiological effects of albumin dialysis in acute-on-chronic liver failure: a randomized controlled study patients with acute on chronic liver failure display "sepsis-like" immune paralysis short-term prognosis of community-acquired bacteremia in patients with liver cirrhosis or alcoholism: a population-based cohort study bacterial infection in patients with advanced cirrhosis: a multicentre prospective study infections in patients with cirrhosis increase mortality fourfold and should be used in determining prognosis clinicalcourseofacuteon-chronic liver failure syndrome and effects on prognosis development and validation of a prognostic score to predict mortality in patients with acute-on-chronic liver failure acute-on-chronic liver failure: consensus recommendations of the asian pacific association for the study of the liver (apasl) glucocorticoids plus n-acetylcysteine in severe alcoholic hepatitis prednisolone or pentoxifylline for alcoholic hepatitis management of critically-ill cirrhotic patients intensive care of the patient with cirrhosis acute-on-chronic liver failure before liver transplantation: impact on posttransplant outcomes acute-onchronic liver failure: excellent outcomes after liver transplantation but high mortality on the wait list acute-on-chronic liver failure: terminology, mechanisms and management bacterialinfectionsincirrhosis: a position statement based on the easl special conference management of acute-on-chronic liver failure transfusion strategies for acute upper gastrointestinal bleeding albumin for bacterial infections other than spontaneous bacterial peritonitis in cirrhosis. a randomized, controlled study deleterious effects of betablockers on survival in patients with cirrhosis and refractory ascites nonselective beta blockers increase risk for hepatorenal syndrome and death in patients with cirrhosis and spontaneous bacterial peritonitis lack of consensus for usage of beta-blockers in end-stage liver disease treatment with non-selective beta blockers is associated with reduced severity of systemic inflammation and improved survival of patients with acute-onchronic liver failure intensive care unit admissions with cirrhosis: risk-stratifying patient groups and predicting individual survival prognostic importance of the cause of renal failure in patients with cirrhosis acutekidneyinjuryandacute-onchronic liver failure classifications in prognosis assessment of patients with acute decompensation of cirrhosis diagnosis and management of acute kidney injury in patients with cirrhosis: revised consensus recommendations of the international club of ascites albumin dialysis in cirrhosis with superimposed acute liver injury: a prospective, controlled study randomized controlledstudyofextracorporealalbumindialysis for hepatic encephalopathy in advanced cirrhosis extracorporeal albumin dialysis with the molecular adsorbent recirculating system in acute-on-chronic liver failure: the relief trial prometheus versus molecular adsorbents recirculating system: comparison of efficiency in two different liver detoxification devices effect of the molecular adsorbent recirculating system and prometheus devices on systemic haemodynamics and vasoactive agents in patients with acute-onchronic alcoholic liver failure effects of fractionated plasma separation and adsorption on survival in patients with acute-on-chronic liver failure high-volume plasma exchange in patients with acute liver failure: an open randomised controlled trial extracorporeal liver support and liver transplant for patients with acute-on-chronic liver failure international liver transplant society practice guidelines: diagnosis and management of hepatopulmonary syndrome and portopulmonary hypertension pulmonary complications in liver diseases therapeutic options in pulmonary hepatic vascular diseases hepatocardiac disorders : interactions between two organ systems hepatic encephalopathy in patients with acute decompensationofcirrhosisandacute-on-chronic liver failure coagulation parameters and major bleeding in critically ill patients with cirrhosis management of acute-on-chronic liver failure: rotational thromboelastometry may reduce substitution of coagulation factors in liver cirrhosis nutrition for the liver transplant patient nutritional management of acute and chronic liver disease the nutritional management of hepatic encephalopathy in patients with cirrhosis: international society for hepatic encephalopathy and nitrogen metabolism consensus the safety of intubation in patients with esophageal varices granulocyte colony-stimulating factor mobilizes cd34(+) cells and improves survival of patients with acute-on-chronic liver failure key: cord-261608-4sjlg0p0 authors: trejo-paredes, camila; mohammed, turab jawaid; salmon, adrian title: covid-19-induced liver injury: a clinical distraction? date: 2020-10-31 journal: chest doi: 10.1016/j.chest.2020.08.901 sha: doc_id: 261608 cord_uid: 4sjlg0p0 nan a 51-year-old male with a history of hypertension and diabetes was admitted with fever and breathlessness secondary to covid-19 and started on hydroxychloroquine (hcq) and azithromycin. his routine blood work, including a complete metabolic panel, was unremarkable. he was intubated for progressive hypoxia, and given the elevated ferritin-5088 ng/dl and d-dimer >2500mg/l, he was started on high-dose heparin infusion for anticoagulation. his ferritin and d-dimer continued to uptrend, and on day 10, he was noted to have a total bilirubin of 8.4 mg/dl. highest peak values noted, aspartate aminotransferase-229, alanine aminotransferase-167, alkaline phosphatase-162, total bilirubin-22, and direct bilirubin-15.5. tests for hepatitis a, b, c, hsv, ebv, hemolysis workup, and imaging studies were unremarkable. after a week, the liver enzymes and bilirubin down trended and returned to baseline spontaneously. no clear etiology was ascertained and was presumed multifactorial likely from viral hepatitis, sepsis or drug-induced cholestasis. his hospital course was also complicated by acute renal failure requiring renal replacement therapy, coagulopathy, and encephalopathy which coincided with liver injury trailing behind the peak of inflammatory markers. he underwent tracheostomy and percutaneous endoscopic gastrostomy tube placement prior to discharge after 7 weeks. emerging data support the hypothesis that liver injury in covid-19 is often the result of sars-cov-2 directly binding to ace2+ cholangiocytes, leading to cholangiohepatitis. in addition, cytokine storm may further exacerbate the hepatic injury in covid-19 (2). although our patient lacked any known risk factors for liver injury and preexisting liver disease, he developed a significant hyperbilirubinemia and transaminitis without sequalae. hepatic congestion in ventilated patients, shock liver and particularly, drug-induced liver injury (dili) remains an important consideration in covid-19 patients. initiating antiviral therapy and curtailing cytokine dysregulation at an early stage could be beneficial to curb the disease progression (3). conclusions: covid-19 induced viral hepatitis is now being increasingly identified as a self-resolving complication and the physician should be mindful of it and in the right setting, it may only be a clinical distraction. we should be cognizant of other potential causes of liver injury in covid-19 patients like concurrent infection, sepsis-induced and dili. liver injury in covid-19: the current evidence clinical characteristics and mechanism of liver damage in patients with severe acute respiratory syndrome covid-19 and the liver: little cause for concern. the lancet gastroenterology & hepatology disclosures: no relevant relationships by turab jawaid mohammed, source¼web response no relevant relationships by adrian salmon, source¼web response no relevant relationships by camila trejo-paredes american college of chest physicians key: cord-280234-anlytu3q authors: memar, elmira haji esmaeil; mamishi, setareh; ekbatani, meisam sharifzadeh; alimadadi, hosein; yaghmaei, bahareh; chegini, victoria; janani, somayeh; mahmoudi, shima title: fulminant hepatic failure: a rare and devastating manifestation of coronavirus disease 2019 in an 11-year-old boy date: 2020-09-29 journal: arch pediatr doi: 10.1016/j.arcped.2020.09.009 sha: doc_id: 280234 cord_uid: anlytu3q although several typical manifestation of novel coronavirus disease 2019 (covid-19) including respiratory symptoms, weakness, fever, and fatigue have been reported, some rare and novel manifestations have also been observed, particularly in children. we report a pediatric case of fulminant hepatic failure associated with covid-19. although the patient was treated for acute fulminant hepatic failure in the context of covid-19, he died following the progression of the disease to stage 4 hepatic failure with encephalopathy and brain death. in december 2019, novel coronavirus disease 2019 (covid-19) caused by a novel coronavirus, sars-cov-2, emerged in wuhan, china, and since then has spread around the world. although at the start of the outbreak it was thought that children were less likely to be infected with covid-19, over times the number of reports on pediatric patients increased [1] [2] [3] . there are three main categories of sars-cov-2 infection including asymptomatic, non-severe symptomatic, as well as severe respiratory and systemic presentations [4] , and the incidence of symptomatic infection and critical disease in children is known to be lower than that of adults [3, 5] . the typical manifestations of covid-19 that have been reported include respiratory symptoms, weakness, fever, and fatigue; however, rare and novel manifestations are also seen, particularly in children [6] . in this study, we report a pediatric case with fulminant hepatic failure associated with covid-19. the patient, an 11-year-old boy, presented to the emergency department for fever, icterus, and abdominal pain that had started 7 days earlier. he had no past medical history of chronic liver j o u r n a l p r e -p r o o f 3 disease or of using potentially hepatotoxic drugs or of food poisoning. none of his family members had symptoms of covid-19 or of hepatic diseases. on admission, he was icteric, dyspneic, and with a decreased level of consciousness. he had been fully alert and responsive 1 week before admission, but severe fatigue, confusional state, and decreased level of consciousness had gradually developed. he had spent 1 month in quarantine owing to the covid-19 outbreak; however, approximately 10 days before admission, he visited a ranch and a few hours later had fever, chills, and malaise. after 1-2 days, icterus appeared primarily in the eyes and subsequently spread to the rest of the body. there were no complaints of coughing, sneezing, symptoms of upper respiratory tract infection, vomiting, and diarrhea. unfortunately, due to a lack of resources, he had not been visited by any physician. after 7 days of illness, he was admitted to a general hospital with the primary diagnosis of hepatitis that led to the diagnosis of fulminant hepatic failure after initial evaluation. he was then transferred to our hospital, an iranian pediatric referral hospital, for liver transplantation. the laboratory findings are shown in table 1 . at the original hospital the stage of disease progression was 2 and at our center it progressed to stage 3 or 4. upon admission to our emergency room, the score on the glasgow coma scale (gcs) was 7-8 and he had active gastrointestinal and nasal bleeding. thus, immediately after arrival, our decision was to intubate the patient and immediately transfer him to the intensive care unit (icu) where he underwent mechanical ventilation. in addition, to rule out other probable causes of fulminant hepatitis, tests for other viruses including hepatitis a, hepatitis b, herpes virus, epstein-barr virus, cytomegalovirus were performed, with all results being negative. moreover, to rule out metabolic disease, additional tests were run, including amino acid chromatography, organic acid chromatography, and measurement of succinylacetone in urine, all of which yielded negative results. treatment for covid-19 was initiated with hydroxychloroquine and kaletra, and we also prescribed vancomycin and ceftazidime for other probable bacterial infections. in the icu, the patient's gcs was 4-5 and decreased further on the following day.. owing to unstable hemodynamics, we added norepinephrine and epinephrine but the patient's condition progressed to hepatorenal syndrome and he underwent dialysis. unfortunately, after 2-3 days he died in spite of all the medical and supportive measures. in this study, we report a novel pediatric case of fulminant hepatic failure associated with covidalthough there have been a significantly smaller number of reported cases of covid-19 in the pediatric population compared with the adults, the number of infected children has seen a moderate increase [2, 7] . children with covid-19 infection may be asymptomatic or have fever, dry cough, and fatigue with a few upper respiratory symptoms including nasal congestion and runny nose; other patients might show gastrointestinal symptoms including abdominal discomfort, nausea, vomiting, abdominal pain, and diarrhea [2, 8, 9] . in addition to the typical manifestation, rare and novel symptoms have also been seen, particularly in children such as our patient. fulminant hepatic failure is considered the fourth main cause of liver transplantation in children, preceded by extrahepatic cholestatic liver diseases, intrahepatic cholestasis, and metabolic diseases followed by primary liver tumor and cystic fibrosis [10] . acute liver failure is a life-threatening condition with sudden onset of liver injury, decreased liver function, hepatic encephalopathy, and coagulopathy in patients without preexisting liver disease. however, etiologic factors might change depending on geographic variation, age, and socioeconomic status. viral agents and drugs mostly cause acute liver failure, but sometimes no etiologic factor can be found. however, viral infections are the most common reasons [7] . because of the unusual manifestation of covid-19 in children, it should be ruled out as the cause of acute liver failure. the incidence of hepatic abnormalities significantly increases after infection with sars-cov-2 and during the course of the disease [11] . elevated liver biochemistry profiles may reflect a direct virus-induced cytopathic effect and/or immune damage from the provoked inflammatory response [12] . in addition, sars-cov-2 infection might induce rhabdomyolysis with an initial increase in creatinine kinase level [13] . increasing evidence has demonstrated the frequent incidence of liver injury in covid-19, especially in patients with multiple organ injury. liver injury itself often manifests as transient elevation in serum aminotransferases. however, acute liver failure and intrahepatic cholestasis have been reported [14] . liver damage in patients with coronavirus infections might be directly caused by the viral infection of liver cells. patients with severe covid-19 seem to have higher rates of liver dysfunction [15] . hepatic dysfunction in severe covid-19 is accompanied by a greater activation of the coagulative and fibrinolytic pathways, relatively lower platelet counts, rising neutrophil counts and neutrophil-to-lymphocyte ratios, and high ferritin levels [16] . j o u r n a l p r e -p r o o f 6 in fulminant hepatic failure, hepatic transplantation is the only therapy that has been proven to improve survival in the majority of patients, despite specific therapeutic options for distinct orthotopic etiologies. the result is determined by complications such as severe coagulopathy, infection, renal dysfunction, or increased intracranial pressure. the decision regarding transplantation depends on the probability of spontaneous hepatic recovery, which can be determined by multiple factors. the most important variables for predicting the need for transplantation during fulminant hepatic failure include the degree of encephalopathy, the age of the patient, and the underlying cause of hepatic failure [17] . owing to the acute fulminant hepatic failure in our patient, the only treatment option was liver transplantation; however, because of the progressive course of the disease and its rapid progression to stage 4 with encephalopathy and brain death, he died. in conclusion, in patients with fulminant hepatic failure, especially in cases with symptoms including fever, respiratory distress, and diarrhea, we should rule out covid-19 infection as the underlying cause. the need for emergency liver transplantation in fulminant hepatic failure due to covid-19 and the indications for transplantation should be further evaluated. the authors have no conflict of interest relevant to this paper to disclose. clinical and ct features in pediatric patients with covid-19 infection: different points from adults the coronavirus disease 2019 (covid-19) in children: a study in an iranian children's referral hospital covid-19 epidemic: disease characteristics in children immunologic features in coronavirus disease 2019: functional exhaustion of t cells and cytokine storm severe and fatal forms of covid-19 in children atypical and novel presentations of coronavirus disease 2019: a case series of three children liver transplant for fulminant hepatic failure: a singlecenter experience systematic review of covid-19 in children shows milder cases and a better prognosis than adults novel coronavirus disease 2019 (covid-19) outbreak in children in iran: atypical ct manifestations and mortality risk of severe covid-19 infection pediatric liver transplantation liver and kidney injuries in covid-19 and their effects on drug therapy; a letter to editor clinical insights for hepatology and liver transplant providers during the covid-19 pandemic weakness and elevated creatinine kinase as the initial presentation of coronavirus disease 2019 (covid-19). m laboratory abnormalities in children with novel coronavirus disease 2019 liver injury in covid-19: management and challenges covid-19 and the liver: little cause for concern fulminant hepatic failure: etiology and indications for liver transplantation key: cord-005949-8po9xe5g authors: streetz, k.l.; tacke, f.; koch, a.; trautwein, c. title: akutes leberversagen: übersicht zur aktuellen diagnostik und therapie date: 2013-11-06 journal: med klin intensivmed notfmed doi: 10.1007/s00063-013-0285-4 sha: doc_id: 5949 cord_uid: 8po9xe5g although acute liver failure is a rare disease with a prevalence of 5 per 1 million people, it has a considerablely high mortality rate of 34 %. the main causes in western civilizations are drug overdose (acetaminophen) and viral hepatitis. patients are affected by the loss of liver synthesis function and are at risk of developing hepatic encephalopathy and possible multiorgan failure. specific therapies consisting of the administration of n-acetylcysteine (acetaminophen) or of nucleotide/nucleoside analogs (hepatitis b) are possible, but are often not adequate. orthotopic liver transplantation is, therefore, frequently the only remaining effective therapy for severe acute liver failure. due to organ shortage, new prognostic tools, e.g., the acute liver failure study group (alfsg) score, have been developed to improve patient selection using sufficiently stringent selection criteria. das alv ist als eine potenziell reversible leberfunktionsstörung (ikterus, koagulopathie "international normalized ratio", inr, >1,5) mit rascher entwicklung einer hepatischen enzephalopathie ohne vorliegen eines chronischen leberschadens definiert. die amerikanische "acute liver failure study group" unterscheidet in bezug auf die zeit zwischen dem auftreten von koagulopathie und beginnender hepatischer enzephalopathie weiterhin zwischen dem hyperakuten (<7 tage), dem akuten (7-28 tage) und dem subakuten (28 tage -6 monate) leberversagen [9] . in deutschland geht man aktuell von etwa 4-500 fällen eines alv aus [4] . insgesamt liegt die prävalenz bei etwa 5 pro 1 mio. einwohner. epidemiologische daten zur genese des alv zeigen bezüglich der zugrunde liegenden ursachen ein diversifiziertes bild. eine kürzliche erhebung an 155 patienten einer deutschen kohorte zeigte, dass die ursache in 32% nicht durch acetaminophen (paracetamol) induziert medikamentös toxisch, in 21% viral und in 9% durch acetaminophen bedingt war. in 24% der fälle konnte sie sogar nicht genau bestimmt werden [6] . eine lebertransplantation erfolgte hier in 47% der schweren fälle mit begleitender enzephalopathie. in nordamerika und mitteleuropa dominieren intoxikationen (hauptsächlich paracetamol, antibiotika u. a.) vor viralen hepatitiden und selteneren ursachen (. tab. 1). in etwa 20% der fälle bleibt die ursache noch unklar, wie durch eine große aktuelle auswertung des eurotransplant-registers (. abb. 1) verdeutlicht wird [5] . die dem alv zugrunde liegende pathogenese ist abhängig von der auslösenden ursache vielfältig. getriggert durch zytotoxische botenstoffe (tumornekrosefaktor-α, tnf-α) und mediatoren (reaktive sauerstoffradikale) kommt es zum hauptsächlich periportalen zelluntergang, der durch nekrotischen und apoptotischen zelltod von hepatozyten bedingt ist. begleitet wird dies von einer variablen entzündungsreaktion. bei unkontrolliertem untergang von hepatozyten kommt es zu einer progredienten organdysfunktion mit entwicklung einer koagulopathie. durch die verringerte synthese von gerinnungsfaktoren und deren inhibitoren verlängert sich die prothrombinzeit. eine begleitende plättchendysfunktion und thrombopenie führt dann zu klinisch vermehrter blutungsneigung. der ausfall der hepatischen entgiftungsfunktion geht mit einem messbaren anstieg des bilirubins und des ammoniaks einher. es kommt weiterhin zu einer erhöhung des splanchnischen druckgraausreichend effektiv. die durchführung einer lebertransplantation ist hier oft die einzige verbleibende therapieoption. neue prognosescores, wie der acute-liver-failure-studygroup(alfsg)-score, ermöglichen eine verbesserte patientenselektion, um dem organmangel durch eine ausreichend stringente indikationsstellung gerecht zu werden. paracetamol · transplantation · hepatische enzephalopathie · multiples organversagen · organdysfunktionsscore acute liver failure. diagnosis and therapy abstract although acute liver failure is a rare disease with a prevalence of 5 per 1 million people, it has a considerablely high mortality rate of 34%. the main causes in western civilizations are drug overdose (acetaminophen) and viral hepatitis. patients are affected by the loss of liver synthesis function and are at risk of developing hepatic encephalopathy and possible multiorgan failure. specific therapies consisting of the administration of n-acetylcysteine (acetaminophen) or of nucleotide/nucleoside analogs (hepatitis b) are possible, but are often not adequate. orthotopic liv-er transplantation is, therefore, frequently the only remaining effective therapy for severe acute liver failure. due to organ shortage, new prognostic tools, e.g., the acute liver failure study group (alfsg) score, have been developed to improve patient selection using sufficiently stringent selection criteria. acetaminophen · transplantation · hepatic encephalopathy · multiple organ failure · organ dysfunction scores problemen) und ggf. rekombinanter faktor viia sowie kryopräzipitat (fibrinogen, faktor viii, xiii, von willebrandfaktor; bei hypofibrinogenämie <100 mg/dl) verwendet. zur prävention der statistisch häufigen oberen gastrointestinalen blutung wird die durchführung einer säuresuppression mittels protonenpumpenhemmern oder h 2 -blockern empfohlen. die behandlung des alv sollte bei entsprechender schwere der erkrankung (anhaltende leberfunktionseinschränkung, beginnende enzephalopathie) nach möglichkeit in einem zentrum mit transplantationsmöglichkeit erfolgen. sofern möglich sollte natürlich unverzüglich eine kausale therapie eingeleitet werden (. tab. 5). die etablierte therapie des häufigen paracetamolinduzierten alv besteht in der intravenösen gabe von n-acetylcystein (nac) in form eines 72-stündigen reduktionsschemas (nac: 150 mg/kg/h für 1 h, dann 12,5 mg/kg/h für 4 h und 6,25 mg/kg/h für 67 h). interessanterweise wurde in einer prospektiven multizentrischen studie gezeigt, dass es beim nicht durch paracetamol bedingtem alv unter gabe von nac zumindest bei patienten mit niedriggradiger hepatischer enzephalopathie (°i-ii) ebenfalls zu einer verbesserung des transplantatfreien überlebens kommt [10] . daher empfiehlt sich das nebenwirkungsarme nac-schema generell auch bei allen anderen entitäten des alv. eine spezifische behandlung sollte bei der akuten hepatitis-b-infektion eingeleitet werden. sinnvoll erscheint aktuell die einleitung einer behandlung mittels hochpotenter antiviraler medikamente, wie entecavir und tenofovir [15] . bei sicherung der diagnose einer autoimmunen hepatitis ist eine steroidtherapie indiziert [7] . im rahmen eines herpes-simplex-virus(hsv)-induzierten alv wurde die wirksamkeit von aciclovir gezeigt. die wenigsten therapien sind jedoch durch gute randomisierte studien abgesichert, da aufgrund der niedrigen fallzah-len oft nicht ausreichend patienten rekrutiert werden können. da der verlauf des alv in seiner geschwindigkeit kaum vorhersagbar ist, empfiehlt sich die frühzeitige verlegung in ein transplantationszentrum. kontraindikationen für eine lebertransplantation, wie aktiver alkoholabusus, drogenkonsum, schwere systemische und maligne erkrankungen sowie eine fehlende soziale einbindung, die für die komplexe anschließende nachbetreuung notwendig ist, müssen ausgeschlossen werden. lebertransplantation sollte rasch und unverzüglich erfolgen die listung für eine lebertransplantation sollte rasch und unverzüglich erfolgen, um auf den möglicherweise rapiden verlauf des alv entsprechend reagieren zu können. durch alv bedingte lebertransplantationen machen etwa 9% aller lebertransplantationen aus, wie aktuelle daten des europäischen lebertransplantationsregisters zeigen [1] . diese rate blieb über die letzten 20 jahre ungefähr konstant. daten einer prospektiven amerikanischen studie belegen die effektivität und notwendigkeit dieser therapieoption [12] . hier wurde gezeigt, dass 84% der patienten mit alv nach erhalt einer frühen transplantation überlebten, während die überlebensrate ohne lebertransplantation bei nur 34% lag. in der regel beeinflussen 3 faktoren den erfolg einer notfalllebertransplantation im rahmen einer high-urgency(hu)-listung eines patienten mit alv: das alter des empfängers, die schwere der lebererkrankung und die qualität des spenderorgans. je schlechter der klinische zustand des patienten ist, umso schwieriger sind die allgemeinen transplantationsbedingungen. patienten mit paracetamolinduziertem alv sind hierbei am meisten beeinträchtigt. ebenso zeigen patienten über 50 jahre eine 2-fach erhöhte morta koch geben an, dass kein interessenkonflikt besteht. f. tacke erhielt vortragshonorare von den firmen gilead trautwein erhielt vortragshonorare von den firmen gilead dieser beitrag beinhaltet keine studien an menschen oder tieren evolution of indications and results of liver transplantation in europe. a report from the european liver transplant registry (eltr) extracorporeal albumin dialysis with the molecular adsorbent recirculating system in acute-onchronic liver failure: the relief trial population-based surveillance for acute liver failure acute liver failure: a life-threatening disease liver transplantation for acute liver failure in europe: outcomes over 20 years from the eltr database etiologies and outcomes of acute liver failure in germany usefulness of corticosteroids for the treatment of severe and fulminant forms of autoimmune hepatitis effects of fractionated plasma separation and adsorption on survival in patients with acute-on-chronic liver failure acute liver failure: summary of a workshop intravenous n-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure extracorporeal detoxification using the molecular adsorbent recirculating system for critically ill patients with liver failure results of a prospective study of acute liver failure at 17 tertiary care centers in the united states development of an accurate index for predicting outcomes of patients with acute liver failure keratin variants predispose to acute liver failure and adverse outcome: race and ethnic associations management of severe acute to fulminant hepatitis b: to treat or not to treat or when to treat? key: cord-324509-5c6fzdjm authors: huang, haijun; chen, shanshan; li, hong; zhou, xian‐long; dai, yining; jia, wu; zhang, jun; shao, lina; yan, rong; wang, mingshan; wang, jiafeng; tu, yuexing; ge, minghua title: the association between markers of liver injury and clinical outcomes in patients with covid‐19 in wuhan date: 2020-07-22 journal: aliment pharmacol ther doi: 10.1111/apt.15962 sha: doc_id: 324509 cord_uid: 5c6fzdjm background: the outbreak of coronavirus disease 2019 (covid‐19) is a critical challenge for public health. the effect of covid‐19 on liver injury has not been fully presented. aims: to evaluate the dynamic changes in liver function and the relationship between liver function damage and prognosis in patients with covid‐19. methods: retrospective analysis of clinical data of 675 patients with covid‐19 in zhongnan hospital of wuhan university from january 3 to march 8, 2020. patients were classified as normal, abnormal liver function and liver injury. results: of 675 patients, 253 (37.5%) had abnormal liver function during hospitalisation, and 52 (7.7%) had liver injury. the dynamic changes of alt and ast levels were more significant in patients with liver injury and in those who died. ast >3‐fold uln had the highest risk of death and mechanical ventilation. compared to patients with normal ast levels, mortality and risk of mechanical ventilation significantly increased 19.27‐fold (95% confidence interval [ci], 4.89‐75.97; p < 0.0001) and 116.72‐fold (95% ci, 31.58‐431.46; p < 0.0001), respectively, in patients with ast above 3‐fold uln. increased leucocytes, decreased lymphocytes and female sex were independently associated with liver injury. conclusions: the dynamic changes in liver function may have a significant correlation with the severity and prognosis of covid‐19. increased index of liver injury was closely related to mortality and need for mechanical ventilation. therefore, these indicators should be closely monitored during hospitalisation. in december 2019, unexplained pneumonia cases emerged in wuhan, hubei province, china, 1,2 which spread rapidly throughout the country and became a public health emergency of international concern. on january 7, a novel coronavirus was detected in a swab sample of a patient by the china center for disease control and prevention (cdc). the disease was subsequently named the novel coronavirus disease 2019 (2019-ncov). 3 the pathogen of covid-19 pneumonia is severe acute respiratory syndrome coronavirus 2 (sars-cov-2), 1,4 which mainly causes respiratory, intestinal, liver and nervous system diseases. 5, 6 a study showed that more than 50% of patients with covid-19 have different degrees of liver injury. 7 some studies have reported the clinical characteristics of patients with coronavirus disease 2019 (covid19) , including some factors that may lead to covid-19-related liver damage and the relationship between liver function damage and disease prognosis. 1, 3, [8] [9] [10] [11] in these studies, different degrees of elevated levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were reported. 1, 3, 8, 12 however, the effect of covid-19 on liver injury has not been fully presented. liver injury is related to the severity and mortality of covid-19. 1316 cai et al systematically described the clinical characteristics of covid-19 patients with liver injury and revealed that liver injury was related to disease severity. 14 in addition, a study reported that liver injury was related to death in patients with covid-19, and mortality was related to an increase in liver enzyme levels. 13 however, mechanical ventilation, which is the main auxiliary treatment for critical patients and an important clinical outcome of covid-19, was not involved. on the other hand, dynamic changes in liver functions may indicate a certain relationship between liver injury and mortality. there were few studies on the dynamic changes of liver functions in covid-19-related liver injury. 13, 17 nevertheless, the dynamic changes in liver function based on fatal and nonfatal individuals have never been reported. moreover, there is little research on what abnormalities occur at what time and how those may relate to clinical outcomes. therefore, we retrospectively analysed the clinical characteristics and dynamic changes in liver function based on different liver function levels at admission and different prognosis, in the purpose of finding out risk factors related to liver injury, and associations between markers of liver injury and clinical outcomes in covid-19, including mortality and mechanical ventilation. from january 3 to march 8, 2020, the medical records of in-patients diagnosed with covid-19 were analysed retrospectively at zhongnan hospital of wuhan university. information on epidemiological, demographic, clinical symptoms or physical signs and comorbidities was extracted from the electronic medical records. according to the diagnosis and treatment standard of covid-19 18 issued by the national health committee, the disease severity was divided into three groups: mild, severe and critical. patients with mild type might have fever and respiratory symptoms, and pneumonia was revealed by imaging. severe covid-19 was defined when the patients met any of the following criteria: (a) respiratory distress (≥30 breaths/min); (b) resting oxygen saturation ≤93%; and (c) arterial blood oxygen partial pressure (pao2)/fio2 ≤300 mm hg. in the critical group, at least one of the following three diagnostic criteria fisher's exact test. dynamic changes in liver function based on different liver function levels at admission and different prognosis were presented using locally weighted scatterplot smoothing (loess). the mixed-effect cox proportional risk regression model was used to study the relationship between liver enzyme level and mortality and mechanical ventilation. the mixed-effect cox model was adjusted for gender, age, smoking, chronic liver disease and comorbidities (including hypertension, diabetes mellitus, coronary heart disease and chronic obstructive pulmonary disease). to explore the factors associated with covid-19-related liver injury, logistic regression analysis was performed. the variables with p < 0.1 in a univariate analysis were then included in a forward stepwise regression model. a twosided p of less than 0.05 was considered statistically significant. table 1 . among the 675 patients, 370 (54.8%) patients had normal liver function, 253 (37.5%) patients had abnormal liver function and 52 (7.7%) patients had liver injury. in patients with liver injury, the median age was 51.50 (35.75-60.25), and the ratio of males to females was 4:1. the body mass index (bmi) of patients with liver injury was 24.66 (23.14-26.37), which was higher than that of patients with normal and abnormal liver function. the incidences of hypertension, diabetes mellitus (dm), coronary heart disease (chd) and chronic obstructive pulmonary disease (copd) were 12 (23.08%), 6 (11.54%), 6 (11.54%) and 1 (1.92%), respectively, in these patients with liver injury. among 52 patients with liver injury, 42 (80.77%) patients had fever, and 20 (38.46%) had dyspnoea, which were significantly higher than those of patients without liver injury (p < 0.001). twenty-eight (53.85%) patients with liver injury were in the mild group, 8 (15.38%) patients were in the severe group and 16 (30.77%) patients were in the critical group. the median values of alt, ast and tbil were 105.00 (49.25-159.50), 58.50 (45.00-90.50) and 12.55 (9.50-17.67), respectively, which were much higher than those of patients with normal and abnormal liver function (p < 0.001 for all). the number of lymphocytes in patients with liver injury was 1.08 (0.58-1.63), which was significantly lower than that in patients without liver injury. figure 3c depicted that the fluctuation in tbil levels was mild and normal in the nonfatal group, and the tbil levels increased much more slowly than the alt and ast in the fatal group. nevertheless, tbil continued to rise slowly until it surpassed the uln at the third week. kaplan-meier survival curves were used to evaluate the survival probability and mechanical ventilation-free survival probability during hospitalisation in patients of covid-19 with different levels of alt, ast and tbil. among these indexes of liver function, ast over 3-fold uln had the highest risks of death and mechanical ventilation. in addition, abnormal levels of alt and tbil were also significantly associated with the risk of death and mechanical ventilation (figures 4 and 5) . the relationship between impaired liver function, mortality and mechanical ventilation was evaluated by a mixed-effect cox model adjusted for age, gender, smoking, chronic liver disease and comorbidities, with the hazard ratios of alt, ast and tbil to mortality and the risk of mechanical ventilation showed in logistic regression analysis of the influencing factors of liver injury, such as epidemiological and clinical characteristics, and laboratory variables was performed to select the predictor parameters of covid-19 patients. factors significantly associated with liver injury were increased leucocytes, decreased lymphocytes and female (table 3 ). in this study, we retrospectively and systematically analysed the there were few studies on the dynamic changes of liver functions in covid-19-related liver injury. 13, 17 one study had suggested that the dynamic changes in liver enzyme levels in severe patients were more significant, and ast was the parameter most correlated with mortality. 13 another study indicated that the pattern of liver biochemical was consistent with the damage of hepatocytes, especially ast. the correlation between ast and alt was very strong on admission and throughout the hospitalisation. this suggested that liver injury was the predominant source of aminotransferase elevation. 17 it is in agreement with our findings. in our study, the dynamic changes of alt and ast levels were more significant in patients with liver injury and in the fatal group. moreover, ast over 3-fold uln had the highest risks of death and mechanical ventilation. in the group of patients with liver injury, alt and ast levels disease. the sight increase of ast levels in the early stage of disease may be related to immune-mediated inflammation in the liver. 13 on the other hand, a multicentre study has reported that the incidence of increased tbil in covid-19 was 10%. 12 in our study, level of tbil increased only in the later stage of increased leucocytes and decreased lymphocytes were proved to be risk factors for liver injury. [21] [22] [23] this occurred because of inflammatory response having some effect on the occurrence of covid-19-related liver injury. 23 a study has showed that lymphopenia may be a key factor related to disease severity and mortality, 24 and this is consistent with the conclusion of our research. the study has several limitations. firstly, this is a retrospective study. the data are not able to assess the causality of covid-19related liver injury and poor clinical outcomes. secondly, some cases did not have enough clinical data on past liver injury. thirdly, the sample size of this study is small. a large cohort study is needed to clarify the association of dynamic changes in liver function and clinical outcomes. in conclusion, the dynamic changes in the markers of liver injury have a significant correlation with severity and prognosis of covid-19. elevated liver function was closely related to mortality and risk of the study was supported by the national natural science foundation a new coronavirus associated with human respiratory disease in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study coronavirus pathogenesis a novel coronavirus from patients with pneumonia in china sars-associated viral hepatitis caused by a novel coronavirus: report of three cases clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical characteristics of 140 patients infected with sars-cov-2 in wuhan clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19): a multi-center study in wenzhou city characteristics of covid-19 infection in beijing clinical characteristics of coronavirus disease 2019 in china longitudinal association between markers of liver injury and mortality in covid-19 in china covid-19: abnormal liver function tests multicenter analysis of clinical characteristics and outcome of covid-19 patients with liver injury cardiodynamic state is associated with systemic inflammation and fatal acute-on-chronic liver failure liver biochemistries in hospitalized patients with covid-19 a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) sars-cov-2 infection of the liver directly contributes to hepatic impairment in patients with covid-19 exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia clinical and immunological features of severe and moderate coronavirus disease 2019 dysregulation of immune response in patients with covid-19 in wuhan, china. clin infec dis clinical characteristics of covid-19 in patients with liver injury a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the association between markers of liver injury and clinical outcomes in patients with covid-19 in wuhan shanshan chen: department of infectious disease, zhejiang provincial people's hospital & people's hospital affiliated of hangzhou medical college zhang: department of orthopaedic surgery, zhejiang provincial people's hospital & people's hospital affiliated of department of nephrology, zhejiang provincial people's hospital & people's hospital affiliated of hangzhou medical college yuexing tu: department of intensive care unit, zhejiang provincial people's hospital & people's hospital affiliated of hangzhou medical college key: cord-275637-ea6w2kqv authors: roca-fernandez, a.; dennis, a.; nicolls, r.; mcgonigle, j.; kelly, m.; banerjee, r. title: high liver fat associates with higher risk of developing symptomatic covid-19 infection initial uk biobank observations date: 2020-06-05 journal: nan doi: 10.1101/2020.06.04.20122457 sha: doc_id: 275637 cord_uid: ea6w2kqv background a high proportion of covid-19 patients develop acute liver dysfunction. early research has suggested that pre-existing fatty liver disease may be a significant risk factor for hospitalisation. liver fat, in particular, is a modifiable parameter and can be a target for public health policy and individual patient plans. in this study we aimed to assess pre-existing liver disease as a risk factor for developing symptomatic covid-19. methods from 502,506 participants from the uk biobank, 42,146 underwent mri (aged 45-82), and had measures of liver fat, liver fibroinflammatory disease and liver iron. patients were censored on may 28th to determine how many had tested for covid-19 with symptomatic disease. uk testing was restricted to those with symptoms in hospital. covid-19 symptoms included fever, dry cough, sore throat, diarrhoea and fatigue. univariate analysis was performed on liver phenotypic biomarkers to determine if these variables increased risk of symptomatic covid-19, and compared to previously described risk factors associated with severe covid-19, including to age, ethnicity, gender and obesity, findings increased liver fat was associated with a higher risk for symptomatic confirmed covid-19 in this population in univariate analysis(or:1.85, p=0.03). in obese participants, only those with concomitant fatty liver([≥]10%) were at increased risk(or:2.96, p=0.02), with those having normal liver fat (<5%) showing no increased risk(or:0.36, p=0.09). conclusions uk biobank data demonstrated an association between pre-existing liver disease and obesity with severe covid-19, with higher proportions of liver fat in obese individuals a likely risk factor for symptomatic disease and severity. public policy measures to protect patients with liver disease who may have almost double the risk of the general population should be considered, especially as dietary and pharmacological strategies to reduce body weight and liver fat already exist. funding university of oxford, innovate uk, uk biobank. authors are employees of perspectum ltd. coronavirus disease is an infectious disease caused by the recently discovered sars-cov-2 coronavirus. risk factors leading to severe infection have been identified in previous studies around the world (1-3) including increased age, male sex, non-caucasian ethnicity and the presence of pre-existing co-morbidities, in particular cardiovascular and metabolic conditions, particularly those conditions involving liver function. the majority of patients experiencing covid-19 developed abnormal liver function(4), with studies reporting abnormal alanine transaminase (alt), aspartate aminotransferase (ast), albumin levels, and increased gamma-glutamyl transferase (ggt) serum in severe patients (6) . the development of liver injury was more common in severe covid-19, with liver injury in mild disease often transient and resolving without treatment. the mechanistic link between covid-19 and the liver may relate to the expression of the ace2 protein, through which sars-cov-2 is believed to enter cells (7) in cholangiocytes (8) . however, recent studies have also shown a hepatocellular profile of damage rather than cholestasis (9) . alternative hypotheses suggest that liver injury in severe cases is being caused by an immune response triggered by the infection (6) or due to a drug-induced liver injury from the antivirals, antibiotics and steroids widely being used for treating covid-19 (10) . traditionally, characterisation of chronic liver disease has relied on invasive liver biopsy to evaluate the pathological hallmarks of disease: fibrosis, inflammation, and steatosis (11) . more recently, scalable, non-invasive alternatives with precise and accurate quantitative measurements have been developed using mri biomarkers of liver fat (also known as proton density fat fraction, pdff, and fibroinflammation, ct1). multiparametric mri measuring liver fat and fibroinflammation has demonstrated value in characterising liver disease (12, 13) and predicting clinical outcomes (14) . this has allowed for determination of the current prevalence . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . of fatty liver disease in the uk adult population, along with genetic associations with liver fat and fibroinflammation (15) . the uk biobank has recently released covid-19 data on testing status for a subset of participants, thus offering a unique resource for investigating the associations between demographic, phenotypic and genotypic factors with covid-19 disease severity and related healthcare outcomes. the aim of this study was to test the hypothesis that liver disease, and specifically liver fat accumulation, is a risk factor for developing symptomatic covid-19. 42,146 participants were extracted from the uk biobank (ukb) imaging study with complete liver imaging to measure fat and fibroinflammatory disease. of these, 287 had a covid-19 test result available with 79 testing positive and 208 testing negatives (figure 1 ). patient meta-data including information on covid-19 testing status, demographics, diabetes status, cardiometabolic risk factors and genetic variants associated with liver disease were available. no biochemistry data was available for alt, ast, albumin or ggt levels. mri measurement of liver fat (proton density fat fraction, pdff, measured in %) and fibroinflammation (iron corrected t1 mapping, ct1, measured in ms) biomarkers were obtained. both liver fat and ct1 have been included in the large cohort uk biobank study (ukb) (16, 17) due to the repeatability and reproducibility of livermultiscan (18) , and suitability for large-scale population imaging. liver fat and ct1 are both continuous variables, with population reference ranges and cut-offs for disease previously described. liver fat >5% is diagnostic of fatty liver disease (19) ; liver fat >10% is considered to qualify as severe fatty liver disease and is a commonly used inclusion criteria in clinical trials of patients with non-alcoholic steatohepatitis (nash) (20) . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint a liver ct1 greater than 825ms has been shown to predict liver-related clinical outcomes (14) , comparably to histological fibrosis staging. ukb has released covid-19 test results from 16 march 2020 onwards, with the majority of sars-cov-2 positive tests coming from patients with symptoms in hospital due to the nature of uk testing. we accessed ukb to determine datasets related to liver health using application 9914 (determining the outcomes of people with liver disease). the ukb has approval from the north west multi-centre research ethics committee (mrec) and obtained written informed consent from all participants prior to the study. statistical analysis was performed using r software (version 3.6.1) with a p-value less than 0.05 considered statistically significant. descriptive statistics were used to summarise baseline participant characteristics. mean and standard deviation (sd) were used to describe normally distributed-continuous variables, median with interquartile range for non-normally distributed, and frequency and percentage for categorical variables. mean difference in biomarker values between those who tested positive for covid-19 and those who did not were compared using the mann-whitney-u test, and difference in the counts of the binary outcomes of the self-reported conditions were compared using chi-squared tests. to discriminate patients who tested positive from those who did not, we measured for count data computing the summary measures of risk and a chi-squared test for difference in the observed proportions from count data, presented in a 2 by 2 table with the function epi.2by2 from the library epir in r. subsequently, univariable logistic regression analysis was performed for all the potential predictors which included liver fat, ct1, age, sex, ethnicity, body mass index (bmi), self-reported diabetes and self-reported hypertension status. this was performed using . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint the glm function in r and the brglm2 tool that aims to reduce the bias observed when results are sparse. risk scores were extracted from the odds ratio estimates of having a positive rest as calculated in the logistic regression model with confidence intervals calculated with the wald's test. pearson's chi-square test of independence was used to investigate the relationship of the genetic variation and the result on the covid-19 test. participant characteristics for each of the groups evaluated in this study are reported in table1. in the symptomatic and tested population, male participants were more likely to test positive for covid-19. comparing those that tested positive to the untested population, being male, non-white-british, and diabetic all increased likelihood of being symptomatic and receiving a positive covid-19 test. the median age of those being symptomatic and testing positive was lower than in the untested population. table 2 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint furthermore, the 32.7% of obese patients with liver fat ≥10% had a higher likelihood of being symptomatic and testing positive for covid-19 (or: 2.96, p=0.02). in contrast, the 37.2% of obese patients with normal liver fat (or: 0.36, p=0.09) did not (table3). the aim of this study was to explore the hypothesis that pre-existing liver disease increases the risk of having symptomatic covid-19. we report on a cohort of 42,146 participants from the ukb with complete liver imaging, of which 287 have covid-19 test results available with 79 participants testing positive. our study demonstrates that in addition to the previouslyreported risk factors of male gender, non-white-british ethnicity, and obesity (1-3), liver fat is also a significant risk factor for having symptomatic covid-19, with a person testing positive for covid-19 being 1.85 times more likely to have pre-existing severe fatty liver disease. to put that into context, 4743 people in this cohort of 42,146 had severe fatty liver disease, suggesting that 11% of the uk population carries this higher risk for covid-19. several risk factors have been identified from early studies in china (1), europe (2) and the us (3) that lead to severe infection, including increased age, male sex, non-caucasian ethnicity and the presence of pre-existing co-morbidities, such as cardiovascular and metabolic conditions, including diseases of the liver. unlike previous studies, our research does not find increasing age as a risk factor in this cohort, but this is likely an artefact of the limited age range in the ukb population and increasing likelihood of exposure to the virus in people of working age versus those that had recently retired. in line with our findings, previous studies have reported bmi as a risk factor for covid-19, but the mechanism(s) of excess weight in conferring additional risk remain unclear. in this study, more than a third of obese participants (37.2%) had normal liver fat and were not at increased . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint risk of being symptomatic and testing positive for covid-19. in contrast, obese patients with liver fat ≥10%, representing approximately one third of the obese population, did have significantly higher odds of being symptomatic and testing positive for covid-19. this highlights the important independent role of liver fat, in addition to obesity, in the severity of, and susceptibility to, covid-19. due to the nature of uk testing for covid-19 over the period these data were collected, only those symptomatic for the disease to the extent that hospitalisation was warranted will have had test results available. given the high prevalence of fatty liver disease in the general population, combined with the utility of lifestyle-interventions (22) , and new therapeutics that have demonstrated clear reversal of fatty liver disease in weeks, patients with high liver fat may benefit from going on calorie restriction diets and pharmacotherapeutic interventions. investigational therapies such as ngm282 and resmetirom have been shown to reduce liver fat in a matter of weeks (23, 24) , but need to be trialled specifically for utility in this context of use. more generally, public health measures should be considered to protect the 11% of the uk population that has liver fat greater than 10%, as their risk of experiencing covid-19 with sufficient severity to require hospitalisation has more than doubled. the main limitation of our study is the relatively small proportion of ukb participants for which testing results are available. this analysis was performed on the second release of covid-19 testing data and subsequent analyses as more data are released will provide additional power to investigate a larger number of exposures. this limitation is largely a result of uk policy at the time to only test those patients requiring hospitalisation; however, one benefit of this is policy is the ability to infer that those participants who tested positive had severe covid-19 disease requiring hospitalisation. this inference will not be possible in future releases of . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint covid-19 testing data following changes in the uk testing policy to support widespread testing. this study highlights the potentially important role of liver disease in covid-19 severity. as more data becomes available through the ukb, further investigations into the associations of liver health with other demographic characteristics and co-morbidities may reveal more detailed relationships, for example, between multiple risk factors and the development of severe covid-19. given the high prevalence of fatty liver disease, and the availability of new treatments specifically for fatty liver disease, these results have the potential to inform public health policy around the management of this at-risk population. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint abbreviations: covid-19, coronavirus disease. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet liver injury in covid-19: management and challenges cell entry mechanisms of sars-cov-2 specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection. biorxiv non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study covid-19 and drug-induced liver injury: a problem of plenty or a petty point? the nas and the histopathologic diagnosis in nafld: distinct clinicopathologic meanings. hepatol baltim md multiparametric magnetic resonance for the non-invasive diagnosis of liver disease multiparametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease severity multiparametric magnetic resonance imaging predicts clinical outcomes in patients with chronic liver disease genomewide and mendelian randomisation studies of liver mri yield insights into the pathogenesis of steatohepatitis uk biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age characterisation of liver fat in the uk biobank cohort repeatability and reproducibility of multiparametric magnetic resonance imaging of the liver dallas steatosis index identifies patients with nonalcoholic fatty liver disease hepatic fat-content assessment using magnetic resonance-based methods opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients. medrxiv nutrition and nonalcoholic fatty liver disease: current perspectives. gastroenterol clin ngm282 improves liver fibrosis and histology in 12 weeks in patients with nonalcoholic steatohepatitis. hepatol baltim md mgl-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial key: cord-033914-a9e3rncp authors: kauffman-ortega, e.; valdovinos-díaz, m.a. title: in memoriam ludwig van beethoven. clinical history and possible diagnoses of the genius of musical composition in silence() date: 2020-10-17 journal: nan doi: 10.1016/j.rgmxen.2020.10.006 sha: doc_id: 33914 cord_uid: a9e3rncp nan carried out one day after his death by dr. johann wagner and dr. karl von rokitansky, the father of modern pathologic anatomy. 1 ludwig van beethoven was born on december 16, 1770, in the small german city of bonn, situated on the banks of the rhine. he was the second of 7 children, of whom only 3 lived beyond infancy (ludwig, kaspar anton karl, and nikolaus johann). his paternal grandmother, josepha, and his father, johann van beethoven, suffered from alcohol use disorder, which led to his father's death when ludwig was 21 years old. complications from tuberculosis claimed the lives of his mother, maria magdalena keverich, and his younger brother, kaspar anton karl. during his childhood, ludwig contracted smallpox, which resulted in facial scarring, and he experienced recurrent respiratory infections, facilitated by underlying bronchial asthma. for the rest of his life, up to his death at the age of 56, he was tormented by numerous illnesses, the majority of unknown etiology. the childhood suffering he endured due to paternal violence and abuse, his mandatory role of providing for and supporting his family as an adolescent, the inner silence and confinement brought on by deafness, and his multiple health problems as an adult, interwoven with his immense commitment to music, resulted in beethoven's leaving us a musical legacy whose melodies, in addition to their beauty, have the power to embrace millions of souls (fig. 1 ). beethoven's deafness, one of the most interesting medical enigmas, with a still uncertain etiology, was first described in ludwig's personal correspondence with his close friend, franz gerhardt wegeler, in 1801. beethoven described a 3-year history of progressive bilateral hypoacusis to high frequency sounds, such as those produced by the violin, piccolo, and piano, that initially began in the left ear. the hypoacusis was associated with unbearable tinnitus, poor discrimination, and recruitment, characteristics that are consistent with sensorineural hearing loss. 2, 3 the hearing defect caused beethoven to have low self-esteem, emotional lability, and progressive isolation, leading to suicidal ideations (the heiligenstadt testament). he was irritable, combative, and arrogant. like his father and paternal grandmother, ludwig was a drinker, with important periods of alcohol consumption that began intermittently when he was 17 years old, particularly during his periods of depression. his favorite drink was hungarian wine, which at that time was of poor quality and tainted with lead to improve its aroma and flavor. the progression of his hearing loss culminated in complete deafness at 47 years of age. during that period of his life, beethoven composed the missa solemnis, the ninth symphony, which debuted in 1824, and his last sonatas for piano and string quartets, all of them important works that were written when he was completely deaf. the autopsy report described cranial thickness of twice the normal size, with a prominence of the frontal bone and irregularity of the zygomatic bones, thinning of the auditory nerves, predominantly of the left nerve, devoid of medullary substance, and a narrowed eustachian tube with retracted mucosa at the level of the bone portion. 1 numerous pathologies in the differential diagnosis have been proposed for beethoven's sensorineural hearing loss, and the most sustainable are: 1) lead poisoning, based on the presence of residuals of lead 100 times higher than normal in his hair and bones, according to an analysis performed in the united states in the mid 1990s, 4 2) cogan's syndrome, characterized by bilateral sensorineural hearing loss and interstitial keratitis secondary to vasculitis, albeit there is no evidence of vestibular dysfunction in beethoven's texts; that syndrome can be associated with idiopathic inflammatory bowel disease and reactive arthritis, 5 and 3) paget's disease is supported by the frontal bone prominence, tinnitus, and headache. other less probable diagnoses are otosclerosis, sarcoidosis, and syphilis. 1 after his mother's death, estimated to have occurred when he was between 17 and 22 years of age, beethoven began to have numerous gastrointestinal symptoms that would accompany him for the rest of his life. his clinical symptoms were characterized by generalized colicky abdominal pain with periods of exacerbation and remission. at times the pain was incapacitating, and he had changes in the frequency of bowel movements and stool consistency, with a predominance of watery diarrhea but no malabsorption or inflammatory features. he sometimes complained of hyporexia, headache, arthralgia, and meteorism. the pain episodes were exacerbated during periods of stress or depression and improved with analgesics, such as quinine and salicin, baths with cold or lukewarm river water (''danube baths''), or with alcohol consumption. the episodes of pain increased in frequency and intensity over time. between 34 and 37 years of age, beethoven presented with a purulent soft tissue infection in a toe, consistent with an abscess, that almost required amputation, followed by a submandibular abscess that was resolved in three months. at 53 years of age, he also presented with painful eye redness, most likely due to scleritis, uveitis, or interstitial keratitis. 6, 7 despite the gastrointestinal symptomatology described, beethoven had a robust physique. he was short, with broad shoulders, and had a short neck, round nose, dark skin, and a wide and prominent forehead. he leaned a bit forward when walking but had no signs of the chronic malnutrition that became apparent during the last years of his life, when he presented with complications associated with liver disease. at autopsy, the chest cavity and contents were normal, the stomach and intestines were distended with air, with no relevant macroscopic alteration, and the pancreas was large and hard. the main pancreatic duct was dilated and appeared ''as wide as a goosequill''. 1 the semiology of those signs and symptoms suggests that beethoven suffered from diarrhea-predominant irritable bowel syndrome, given that he had no alarm symptoms, such as gastrointestinal bleeding, or anatomopathologic findings of organic disease in the digestive tract. the description of the pancreas at autopsy is consistent with chronic pancreatitis, probably secondary to alcohol consumption. other less likely diseases in the differential diagnosis explaining his gastrointestinal symptomatology have been proposed, such as idiopathic inflammatory bowel disease, sarcoidosis, intestinal tuberculosis, whipple's disease, lead poisoning, and selective iga deficiency. the first manifestations of liver failure became evident in 1821, when beethoven was 51 years old. he presented with progressive jaundice, nausea, vomiting, abdominal pain, asthenia, and adynamia, related to a probable viral hepatitis or alcoholic hepatitis, that remitted after 3 months. in 1822, he had an episode of unilateral pleuritic chest pain, described as ''thoracic gout''. in 1826, he presented with complications that could be attributed to cirrhosis of the liver with portal hypertension, such as epistaxis that was probably thrombocytopenia-related, altered mental status, hepatic encephalopathy, and tense ascites. he underwent 4 paracentesis procedures, performed by his physician, dr. andreas ignaz wawruch, through laparotomy. twenty-two liters of ascites were drained, but no asepsis measures were utilized, nor was there adequate closure of the abdominal wall, and the patient developed an infected ascitic fistula and possible bacterial peritonitis. 8 despite numerous medical recommendations, beethoven, now emaciated, continued to eat and drink as he pleased, with intentions of composing a tenth symphony, a requiem, and music for goethe's faust. finally, in march of 1827, he presented with progressive jaundice, tense ascites, lower extremity edema, anuria, fever, and hepatic encephalopathy that resulted in his death. ludwig van beethoven died on march 26, 1827, at 56 years of age, in the city of vienna. the autopsy report described an emaciated, cachectic body, particularly the lower limbs, with multiple petechiae, and a distended abdomen. the abdominal cavity was filled with a turbid, grayish-brown fluid. the liver was half its normal size, hard, bluish-green, and had a nodular surface, characteristic of macronodular cirrhosis. there was sludge in the interior of the gallbladder. the spleen was more than twice its normal size, hard, and blackish. both kidneys were pale, and when sectioned, each calyx was full of calcareous concretions, consistent with papillary necrosis or kidney stones. 9 the probable cause of beethoven's death was acuteon-chronic liver failure. that syndrome is characterized by acute decompensation manifested by ascites, hepatic encephalopathy, coagulation alterations, bacterial infection, multiple organ failure, and elevated short-term mortality in patients with underlying cirrhosis of the liver. 10 even though 40% of patients do not have a recognizable precipitating factor, in beethoven's case, it can be attributed to an infectious process due to the ascitic fistula and secondary peritonitis, severe alcoholic hepatitis, or circulatory dysfunction after the paracentesis. alcohol consumption appears to be the most probable cause of beethoven's cirrhosis of the liver, despite the macronodular appearance described in the autopsy. although that finding is in contrast to micronodular cirrhosis (laennec's cirrhosis) described in patients with alcoholic liver cirrhosis, other diagnoses that have been proposed for explaining cirrhosis of the liver are hemochromatosis and autoimmune liver disease with primary sclerosing cholangitis associated with probable idiopathic inflammatory bowel disease. however, there is not sufficient evidence to confirm any of them. ' we music lovers and professionals of medicine, ''the most humanistic of the sciences and the most scientific of the humanities'', 12 celebrate your 250 years of existence and are eternally grateful for your legacy as a pianist, violinist, organist, composer, conductor, professor, idealist, and as a man who overcame enormous adversities to give full expression to his genius. beethoven forever! none. beethoven's autopsia revisitad: a patólogos rounds a final note a modern case sheds light on a classical enigma: beethoven's deafness the deafness of ludwig van beethoven: an immunopathy cogan syndrome: a retrospective review of 60 patients throughout half century lead and the deafness of ludwig van beethoven el genio de bonn atormentado por sus enfermedades: historia médica historical hepatology: ludwig van beethoven beethoven's renal disease based on his autopsy: a case of papillary necrosis acute-on-chronic liver failure rialp: traducción de joaquín esteban perruca the virtues in medical practice valdovinos-díaz * departamento de gastroenterología, instituto nacional de ciencias médicas y nutrición salvador zubirán the authors declare that they have no conflict of interests. key: cord-327601-4uqgwlnx authors: bangash, mansoor n.; patel, jaimin m.; parekh, dhruv; murphy, nicholas; brown, rachel m.; elsharkawy, ahmed m.; mehta, gautam; armstrong, matthew j.; neil, desley title: sars-cov-2: is the liver merely a bystander to severe disease? date: 2020-06-02 journal: j hepatol doi: 10.1016/j.jhep.2020.05.035 sha: doc_id: 327601 cord_uid: 4uqgwlnx nan we read the recent article from wang et al with great interest. 1 their study shows sars-cov-2 positive patients with ≥1 week history of increased aminotransferases have worse acute pulmonary disease (radiological and physiological) than those without. they also report higher ferritin levels, higher proportions of patients with a low albumin and raised direct bilirubin, and histological features (albeit in only two patients) possibly in keeping with a viral-mediated liver injury in this group. considering that interleukin (il)-6 and c-reactive protein (crp) are similar between patients with normal and prolonged abnormal liver aminotransferases, the authors speculate that liver injury is a direct effect of sars-cov-2 viral hepatitis rather than an indirect immune mediated injury. the fact that increases in liver aminotransferases occur and tend to parallel the severity of pulmonary disease remains unquestioned 2 , however, whether the liver injury is a true viral hepatitis rather than a bystander to the multi-organ pathophysiology of critical illness requires further discussion. wang et al provide evidence for direct viral infection based on electron microscopy where they identified multiple intra-hepatocyte microvesicular structures with "crowns" as sars-cov-2 virions. however, normally occurring clathrin-coated vesicles have a similar appearance. additionally, the tissue is undergoing autolysis, as is usual for post-mortem tissue, and autolysed multi-vesicular bodies (mvbs) are seen in the images. it is therefore possible that the observed cytosolic microvesicles are the intraluminal vesicles of autolysed mvbs. in the context of systemic inflammation, hepatocytes are known to produce mvbs which release the contained vesicles as extracellular vesicles by exocytosis during non-apoptotic cell death (e.g. pyroptosis). 3 indeed, the authors demonstrate tunel-positive hepatocytes (not specific for apoptosis, but also positive in non-apoptotic cell death and autolysis 4 ) and elevated ldh levels (a marker of non-apoptotic cell death), supporting pyroptosis and autolysis as alternate explanations for these clinical and tissue findings, respectively. moreover, as the authors acknowledge, hepatocytes express little to no angiotensin converting enzyme-2 (ace2) receptors, the cellular entry point for sars-cov-2. taken together, and in the absence of sar-cov-2 in situ hybridisation, immunohistochemistry/immunoelectron microscopy or demonstration of sars-cov-2 rna or proteins within the liver, we believe the authors, as others, have mislabelled these electron microscopic structures as sars-cov-2 virions. 5 regarding the blood parameters in the study, aminotransferases (in particularly ast) are not specific to liver injury and are also released after acute muscle injury. the authors identify higher levels of creatinine kinase in patients with raised aminotransferases raising the possibility of a predominantly muscle rather than the liver source. acute and chronic infective illnesses drive catabolic processes that involve muscle (protein) breakdown. 6 in keeping with this, patients with severe pulmonary sars-cov-2 infection lose weight and we have found them to have a high incidence of critical illness neuromyopathy on recovery from their respiratory failure. notwithstanding this, the real elephant in the room is the greater degree of respiratory compromise that associates with only modest liver aminotransferase derangement and the complete lack of clinical correlation with clinically significant liver disease. parameters disturbed in severe acute liver failure are lactate, glucose and inr -these were all well preserved in the data presented by the authors. the patterns of direct bilirubin and albumin are therefore unlikely due to poor synthetic liver function. reductions in albumin more likely reflect increased systemic endothelial permeability and albumin loss from the circulation, something which commonly and rapidly occurs in acute systemic illnesses in patients without liver disease. 7 despite il-6 and crp being similar between patient groups, lymphocyte subset depletion, neutrophil counts, ferritin and markers of fibrinolysis are all significantly increased in patients with prolonged abnormal aminotransferases, clearly suggesting increased immune activation, as we have previously highlighted. 2 furthermore recent studies have confirmed increased netosis, a form of non-apoptotic and highly immunogenic cell death causing bystander damage and coagulation changes, accompanies disease severity. 8 immune-mediated bystander damage then remains a credible mechanism for liver enzyme release and has already been shown to be involved in chimeric antigen receptor t cell-mediated cytokine release syndrome. 9 in conclusion, we do not believe that the findings of wang et al conclusively demonstrate a direct cytotoxic effect of sars-cov-2 on the liver. based on the above perspectives, we feel that raised liver aminotransferases associated with sars-cov-2 positivity are more likely attributable to illness severity, in which host response and iatrogenic harm (i.e. drugs, ventilation) drive bystander liver injury, thus explaining its association with mortality and in an analogous fashion to patterns seen in sepsis. 10 we still encourage clinicians to remain vigilant for drug-induced liver injury, and for liver damage in high risk groups (i.e. drug/alcohol abusers, family history etc), but not to get overly distracted by raised liver aminotransferases in this context. sars-cov-2 infection of the liver directly contributes to hepatic impairment in patients with covid-19 covid-19 and the liver: little cause for concern lps induces active hmgb1 release from hepatocytes into exosomes through the coordinated activities of tlr4 and caspase-11/gsdmd signaling in situ detection of fragmented dna (tunel assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note visualization of putative coronavirus in kidney not the usual suspect: type i interferon-responsive t cells drive infectioninduced cachexia capillary leak syndrome: etiologies, pathophysiology, and management neutrophil extracellular traps in covid-19. jci insight gasdermin e-mediated target cell pyroptosis by car t cells triggers cytokine release syndrome sepsis-associated liver injury: incidence, classification and the clinical significance key: cord-328147-61gtx2h2 authors: lopez-mendez, ivan; aquino-matus, jorge; gall, sofia murua-beltrán; prieto-nava, jose d.; juarez-hernandez, eva; uribe, misael; castro-narro, graciela title: association of liver steatosis and fibrosis with clinical outcomes in patients with sars-cov-2 infection (covid-19) date: 2020-10-21 journal: ann hepatol doi: 10.1016/j.aohep.2020.09.015 sha: doc_id: 328147 cord_uid: 61gtx2h2 introduction and objectives: liver function tests (lft) abnormalities are reported in up to 50% of covid-19 patients, metabolic comorbidities are associated with poorer outcomes. the aim of the study is to determine prevalence of liver steatosis and fibrosis in patients with covid-19 and their association with clinical outcomes. material and methods: retrospective study in hospitalized covid-19 patients. risk for liver steatosis was estimated by hsi > 36, and risk for advanced liver fibrosis with apri > 1.0, nafld fs > 0.675 and/or fib-4 > 3.25. clinical outcomes were admission to intensive care unit (icu) and mortality. results: of 155 patients, 71.6% were male (n = 111), and 28.4% (n = 44) were obese. abnormal lft were present in 96.8% (n = 150), prevalence of steatosis was 42.6% (n = 66) and of significative liver fibrosis was 44.5% (n = 69). liver fibrosis by fib-4 was associated with risk of icu admission (or 1.74 [95%ci 1.74-2.68; p = 0.023]) and mortality (or 6.45 [95%ci 2.01-20.83, p = 0.002]), no independent associations were found. conclusions: the prevalence of steatosis and significant liver fibrosis was high in covid-19 patients but was not associated with clinical outcomes. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus emerged in wuhan, china, in december 2019; it is a highly transmittable pathogen that causes the coronavirus disease 19 . the two previous betacoronavirus epidemics of global concern where the severe acute respiratory syndrome coronavirus (sars-cov) the centers for disease control and prevention (cdc) revised and updated a summary of medical conditions associated with increased risk of severe illness derived from covid-19, which included among others, obesity (body mass index (bmi) >30 kg/m 2 ) and type 2 diabetes mellitus (t2dm). in mexico, 72.5% of the adult population is overweight and 9.4% have t2dm(4) additionally, the prevalence of hepatic steatosis in mexico ranges from 14 .4% to 62.9%, (5) and he prevalence of liver fibrosis has been reported in 8.1% (noninvasive assessment).(6) currently, mexico city is one of the most affected regions in the world with rising numbers of cases and deaths caused by covid-19, and we have very few data regarding gi symptoms and lft abnormalities and their prognostic value in mexican patients. j o u r n a l p r e -p r o o f we conducted a retrospective, cross sectional, descriptive study using electronic medical records of adult patients (>18 years old) with a positive rt-pcr sars-cov-2 test (genefinder tm covid-19 plus realamp kit, osang healthcare co., ltd. korea) in nasopharyngeal swab, admitted to medica sur clinic & foundation between march 14 th through june 5 th , 2020. patients with covid-19 diagnosis but without lft determination in the first 48 hours after admission were excluded. clinical and biochemical data were included in the analysis. the study was approved by the medica sur ethics committee (2020-ext-487). the presence of steatosis was determined by hepatic steatosis index (hsi) by the formula 8 the distribution of data was assessed by the kolmogorov-smirnov test. continuous data were presented as median an interquartile range; categorical data were presented as percentage and frequencies. the mann-whitney u test was used to evaluate differences between patients with and without steatosis, as well as liver fibrosis. the association of categorical data and clinical outcomes was analyzed by chi square. univariate and multivariate analyses were performed for associations between steatosis and liver fibrosis with intensive care unit j o u r n a l p r e -p r o o f (icu) admission and mortality. the p-values <0.05 were considered statistically significant. spss v.21 was used for statistical analysis. a total of 155 patients were included in the final analysis, from which 71.6% (n=111) were male; median of age and bmi were 51 [42-62] years and 27.9 [25.8-30.5 ] kg/m 2 , respectively. obesity (28.4%, n=44), hypertension (23.2%, n=36), and t2dm (15.5%, n=24) were the most frequent comorbidities. only 2 patients (1.3%) reported a preexisting liver disease. among biochemical parameters, the median of hemoglobin was 14.8 [13.7-15.9] g/dl, platelets 198 [162-254] x 10 9 /l, and leukocytes 7.1 [5.3-9.9] x 10 9 /l. demographic data, clinical characteristics, drug history, and biochemical parameters are shown in table 1 . patients had a median of 7 [5] [6] [7] [8] [9] [10] days with symptoms before admission, which included fever in 69.7% (n=108), cough in 65.2% (n=101), and dyspnea in 47.7% (n=74). median of hospital stay was 9 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] days and 34.2% (n=53) of the patients required admission to the icu, with a median stay of 13 [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] days. at least one gi symptom was reported in 36.1% (n=56) of the patients, which included diarrhea in 25.8% (n=40), nausea in 9% (n=14), and anorexia in 9% (n=14). regarding lft (table 2 ), a total of 96.8% (n=150) of patients presented at least one abnormality, and lactate dehydrogenase was the most frequent in 89.7% (n=139) of the cases. the prevalence of steatosis by hsi score >36 was 42.6% (n=66) and it was not associated with clinical outcomes. on the other hand, the prevalence of advanced liver fibrosis by any model was 44.5% (n=69), which is shown in figure 1 . the prevalence of t2dm was higher among patients with fibrosis (66.7% (n=16) vs. 33.3% (n=8), p=0.025), and mortality was also higher in these patients (81.8% (n=9) vs. 18.2% (n=2), p=0.012); however, no independent associations were found in multivariate analyses. differences in basal biochemical levels were found in patients with advanced liver fibrosis. mortality was 7.1% (n=11) and the associated factors were hypertension, basal total bilirubin, basal direct bilirubin, and liver fibrosis by any method, as well as by nafld fs and fib-4; ( as expected, due to the epidemiological context of mexico, in our study we found a high prevalence of metabolic comorbidities which included obesity, hypertension, and t2dm, which were associated with clinical outcomes in univariate analyses. obese patients face an increased risk for severe complications and mortality. they also represent a challenge for therapeutic maneuvers such as imaging diagnosis, intubation, mechanical ventilation, and pronation, among others.(11) a meta-analysis including 3,207 patients with covid-19 described that underlying chronic conditions such as hypertension, diabetes, and cardiovascular and respiratory diseases were higher in critical/non-surviving patients; clinical manifestations such as fever and dyspnea were also associated with the progression of the disease.(12) we found similar results in our study, with dyspnea as the most important associated symptom for icu admission with or 4.07 (ci95% 1.6-9.86). the pathogenesis of gi symptoms and liver injury in covid-19 is still a subject of research. although ace2 is not expressed in kupffer cells, hepatocytes, and endothelium of liver sinusoids,(15) expression of ace2 is induced by hypoxia in cultured hepatocytes (17) and it has been shown that covid-19 infection is associated with liver disease, ranging from a mild to severe damage, but it is usually transient.(2) currently, there is no consensus on the exact mechanism of liver injury, but it may be related to: 1) direct cytopathic damage, 2) systemic inflammatory response, 3) liver hypoxia and ischemia, 4) acute-on-chronic liver injury, or 5) drug-induced liver injury. (18) it has been recently demonstrated the presence of sars-cov-2 virions inside hepatocytes (19) and that death of cholangiocytes induced by sars-cov-2.(20) even though it has not been demonstrated in humans, hepatic steatosis in mice models increases hepatic ace2 mrna (21) , which could explain, at least in theory, the relationship between sars-cov-2 and liver injury in mafld patients. the liver harvests a great pool of macrophages and immune cells, that in addition to a particular susceptibility of hepatocytes to proinflammatory cytokines, the systemic inflammatory response can easily produce liver injury as a collateral damage. (19) the global prevalence of gi symptoms in covid-19 patients has been reported between 11.3% and 79.1%. (22) in our study, gi symptoms were reported in 36.1% (n=56), which is similar to another recent mexican study which reported a prevalence of 20.8%. (23) additionally, a meta-analysis which included 6,686 patients estimated that 15% of them had at least one gi symptom and that 10% only had gi symptoms upon presentation.(24) another systematic review and meta-analysis which included 12,797 patients reported that mortality in the group of patients with gi symptoms (0.4% [ic95%, 0-1.1%; i 2 =74%]) was similar (p=0.15) to overall mortality (2.1% [ic95%, 0.2-4.7%; i 2 =94%]). (25) abnormal lft were found in 96.8% (n=150) of our patients, which is higher that recent studies reporting a prevalence ranging from 16.1% to 76.3%, (3, 26) including 21.5% of liver injury prevalence during hospitalization.(3) our study did not find an association between abnormal lft and mortality, as a multicentric study in china that included 5,771 patients which found that abnormal ast was associated with increased mortality risk by any cause with an or 4.81 (ic95%, 3.38-6.86; p<0.001), which increased to or 14.87 (ic95%, 9.64-22.93; p<0.001) in the group with ast above 120 iu/l (27) . we described a prevalence of 42.6% (n=66) of steatosis, which was not associated with clinical outcomes. in contrast, a retrospective study (28) j o u r n a l p r e -p r o o f found a prevalence of mafld of 37.6% and higher risk of disease progression. since mafld is currently considered the liver manifestation of metabolic syndrome (29) these patients are considered chronically inflamed and this fact could contribute to the interplay in the cytokine storm described in covid-19, resulting in the progression of the disease, its complications, and fatal outcomes. (30) this explanation is supported by zheng et al's study (31) in china, in which 30% of the patients presented mafld, according to tomographic criteria, and found an increased 6-fold risk of severe covid-19 and obesity in mafld patients, in comparison to non-obese patients (or 6.32, 95%ci 1.16-34.54, p=0.033). the addition of metabolic comorbidities may increase the risk of serious complications in this group of patients, but further research is required. in our study, we found a pooled prevalence of advanced liver fibrosis of 44.5% (n=69) through any non-invasive liver assessment model, which was associated with hospital outcomes. this result is in concordance to the multicenter study by ibáñez-samaniego et al. (32) in spain, which included 160 patients and estimated a risk for advanced fibrosis in 28.1%. a fib-4 ≥2.67 score increased the risk of icu admission as an independent risk factor (or 3.41, 95%ci 1. 30-8.92 ). interestingly, in the multivariate analysis with exclusion of patients with mafld, fib-4≥2.67 remained as a risk factor for mechanical ventilation (or 3.25; 95% ci, 1.24-8.53). in our study, no independent associations were found for liver fibrosis and clinical outcomes. these associations are interesting but must be interpreted with caution, since most of the noninvasive predictive models include variables that may be affected by the covid-19 infection as well, resulting in overdiagnosis of liver fibrosis upon presentation. nonetheless, we consider that screening for liver fibrosis in patients with covid-19 would contribute to further risk stratification and to design follow-up strategies in surviving patients who were unaware of their liver diseases, especially in regions where obesity and other metabolic comorbidities are highly prevalent. our study only included two patients with preexisting chronic liver disease, but a previous report by an international registry (covid-hep.net and covidcirrhosis.org) that included 152 patients showed that 23.3% were admitted to the icu and 17.5% required mechanical ventilation, with a reported mortality of 39.8%. (33) a meta-analysis with 1,558 patients from reported important associations between mafld and covid-19 severity. in a retrospective study of 202 patients in china, mafld was associated with covid-19 progression had (or 6.4; 95% ic, 1.5-31.2). (36) further research, especially in the americas, is required to elucidate the relationship between liver disease and covid-19 regarding clinical outcomes. limitations of our study include the retrospective design, the assessment of liver fibrosis through non-invasive models, and the conduction of the study in a private hospital; additionally, as this is not a prospective design, decisions in management and timing of laboratory tests are based on each attending physician during hospital course. in covid-19 patients, the prevalence of liver steatosis and advanced liver fibrosis by noninvasive assessment prediction models was high and was not associated with clinical outcomes; 96.8% of covid-19 patients had at least one abnormal lft. the presence of metabolic comorbidities was associated with mortality and icu admission. the timely diagnosis of covid-19 patients, including those presenting gi symptoms and abnormal lft, is of utmost importance in the fight towards reducing mortality. j o u r n a l p r e -p r o o f clinical features of patients infected with 2019 novel coronavirus in wuhan gastrointestinal, hepatobiliary, and pancreatic manifestations of covid-19 covid-19: abnormal liver function tests obesity and public health in mexico: transforming the hegemonic food supply and demand pattern the mexican consensus on nonalcoholic fatty liver disease prevalence of liver fibrosis in an unselected general population with high prevalence of obesity and diabetes mellitus. time for screening? hepatic steatosis index: a simple screening tool reflecting nonalcoholic fatty liver disease. dig liver dis performance of the aspartate aminotransferase-to-platelet ratio index for the staging of hepatitis c-related fibrosis: an updated meta-analysis the nafld fibrosis score: a noninvasive system that identifies liver fibrosis in patients with nafld development of a simple noninvasive index to predict significant fibrosis in patients with hiv/hcv coinfection covid-19 infection: origin, transmission, and characteristics of human coronaviruses a new coronavirus associated with human respiratory disease in china tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis review article: gastrointestinal features in covid-19 and the possibility of faecal transmission chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2 what has the covid-19 pandemic taught us so far? addressing the problem from a hepatologist's perspective direct or collateral liver damage in sars-cov-2-infected patients recapitulation of sars-cov-2 infection and cholangiocyte damage with human liver ductal organoids pioglitazone upregulates hepatic angiotensin converting enzyme 2 expression in rats with steatohepatitis beware: gastrointestinal symptoms can be a manifestation of covid-19 solís-gonzález a. initial gastrointestinal manifestations in patients with sars-cov-2 in 112 patients from veracruz manifestations and prognosis of gastrointestinal and liver involvement in patients with covid-19: a systematic review and meta-analysis prevalence and mortality of covid-19 patients with gastrointestinal symptoms: a systematic review and metaanalysis liver injury in covid-19: management and challenges longitudinal association between markers of liver injury and mortality in covid-19 in china non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study a new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement covid-19 and nonalcoholic fatty liver disease: two intersecting pandemics letter to the editor: obesity as a risk factor for greater severity of covid-19 in patients with metabolic associated fatty liver disease elevation of liver fibrosis index fib-4 is associated with poor clinical outcomes in patients with covid-19 high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry does comorbidity increase the risk of patients with covid-19: evidence from meta-analysis clinical characteristics and outcomes of coronavirus disease among patients with preexisting liver disease in the united states: a multicenter research network study non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study key: cord-017603-wq4cgqs2 authors: shanmugam, naresh; dhawan, anil title: acute liver failure in children date: 2018-10-16 journal: pediatric hepatology and liver transplantation doi: 10.1007/978-3-319-96400-3_8 sha: doc_id: 17603 cord_uid: wq4cgqs2 “acute liver failure” (alf) and “fulminant liver failure” are terms used interchangeably to describe severe and sudden onset of liver cell dysfunction leading on to synthetic and detoxification failure across all age groups. considerable variations exist between alf in children and adults, in terms of aetiology and prognosis. encephalopathy is not essential to make a diagnosis of alf in children but when present has a bad prognosis. early recognition of alf and initiation of supportive management improve the outcome. liver transplantation remains the only definitive treatment when supportive medical management fails. trey and davidson coined the term "fulminant liver failure" 40 years ago to define onset of altered mental status within 8 weeks of initial symptoms of liver dysfunction in an otherwise healthy individual with no previous history of liver disease [1] . this definition was difficult to apply in children with alf as the disease process could start in utero and time quantification might not be possible and, also, encephalopa-thy might be difficult to diagnose. trying to address this issue, bhaduri and vergani defined alf in children as "a rare multisystem disorder in which severe impairment of liver function, with or without encephalopathy, occurs in association with hepatocellular necrosis in a patient with no recognized underlying chronic liver disease" [2] . this newer definition for children failed to differentiate between acute hepatitis and alf as "severe impairment of liver function" is very subjective and can vary from person to person. paediatric acute liver failure (palf) study group has come up with practical definition to select cases for their multicentre study. they used the following criteria to define acute liver failure (alf) in children: (1) hepatic-based coagulopathy defined as a prothrombin time (pt) ≥ 15 s or international normalized ratio (inr) ≥ 1.5 not corrected by vitamin k in the presence of clinical hepatic encephalopathy (he) or a pt ≥ 20 s or inr ≥ 2.0 regardless of the presence or absence of clinical hepatic encephalopathy (he), (2) biochemical evidence of acute liver injury and (3) no known evidence of chronic liver disease [3] . due to its simplicity and objectivity, palf definition is widely used in children. palf has used inr as a surrogate marker to denote overall liver synthetic inadequacy and an impending multiorgan failure. coagulopathy is not only a key criterion in diagnosing paediatric alf but also helps as prognostic marker. due to short half of several liver-based clotting factors, pt/inr functions as a dynamic marker of synthetic inadequacy due to loss of functioning hepatocytes in alf. factors ii, vii, ix and x depend on vitamin k for carboxylation of terminal glutamic acid residues to convert them into active form ( fig. 8.1 ). correction of coagulopathy by intravenous vitamin k differentiates between vitamin k deficiency due to decreased absorption and synthetic liver failure. isolated prolonged aptr is not due to liver disease, as factor vii in extrinsic pathway (fig. 8.1 ) has the shortest half-life of the • encephalopathy is not essential in the diagnosis of alf in children. • coagulopathy is being used as a prognostic marker in paediatric alf. • role of liver assist devices and hepatocyte transplant is still limited. • auxiliary liver transplantation if feasible should be offered where indicated as it provides a chance for native liver regeneration. vitamin k-dependent factors; therefore, it is the first factor depleted in alf and invariably affects inr. due to defective synthesis and impaired clearance of procoagulant/anticoagulant factors, inflammatory mediators, infection, etc., there could be a degree of intravascular coagulation (ic) that invariably exists in alf which can progress to fulminant disseminated intravascular coagulation. haemostasis in liver disease is best assessed using thromboelastography (teg). teg is a point-of-care assay using a specialized machine that assesses clot formation in whole blood, including plasmatic and cellular components. teg provides a graphical representation ( fig. 8.2 ) of assembly of a clot in whole blood and provides an assessment of overall haemostasis. the causes of alf vary with the age and geographical location. infectious aetiology predominates as a cause of alf in children in the developing countries, while drug-induced alf predominates in adults and is indeterminate in children in europe and north america [4] . [5] . though exact frequency of alf in the paediat-ric age group is unknown, overall annual incidence of alf in the usa was around 5.5 million population among all ages [6] . investigation of alf in children is outlined in table 8 .2. in developed countries drugs and toxins have become the most common identifiable cause of drug-induced acute liver failure in adults and children. drug-induced liver injury (dili) can be a dose-dependent response, an idiosyncratic reaction or a synergistic reaction when two medications are given together. it is essential to enquire about any indigenous/herbal medicine intake as some are potentially hepatotoxic [7] . acetaminophen is the most common drug associated with alf and is normally a dose-dependent hepatotoxic agent. acetaminophen is detoxified mainly by glucuronidation (40%), sulphation (20-40%) and n-hydroxylation (15%). a small fraction is metabolized via cytochrome p450 to yield n-acetyl-para-benzoquinoneimide (napqi), a toxic intermediate compound which irreversibly conjugates with the sulphhydryl group of glutathione and causes hepatocyte necrosis [8] . napqi forms acetaminophen-protein adducts, which acts as a specific biomarker for chronic acetaminophen-related toxicity. in acute acetaminophen poisoning, serum levels after 4 h of ingestion are useful in identifying high-risk patients. genetic polymorphism of cytochrome p450 isoenzymes could predispose affected people to acetaminophen toxicity. anti-tuberculosis drugs, particularly isoniazid, are associated with drug-induced alf. the mechanism of toxicity is similar to acetaminophen; oxidation via cytochrome p450 pathway results in toxic metabolites. the true incidence of idiosyncratic drug-induced liver injury (dili) is unknown; reports have suggested to up to 14 new cases/100,000/year [9] . around 8% of idiosyncratic dili developed alf [10] . dili is unpredictable, but genetic susceptibility of an individual to certain drugs and underlying mitochondrial cytopathies are proposed causes [11] . the councils for international organizations of medical sciences/ roussel uclaf causality assessment method (cioms/ rucam) scale is helpful in establishing causal relationship between offending drug and liver damage. using the scoring system, suspected drug could be categorized into "definite or highly probable" (score > 8), "probable" (score 6-8), "possible" (score 3-5), "unlikely" (score 1-2) and "excluded" (score ≤ 0) [12] . this scale is helpful in identifying druginduced hepatotoxicity even in newly marketed drugs and for a previously unreported older drug. chemotherapy drugs are known to produce veno-occlusive disease leading on to alf due to endothelial damage. few of the common drugs that cause alf are outlined in table 8 .2. water-borne viral hepatitis (hepatitis a and e) is the most common cause of alf in developing countries with poor sanitation facilities. following infection with hepatitis a virus, the risk of developing liver failure is 0.1-0.4%, and this further increases with underlying chronic liver disease. usually the disease runs a benign course with spontaneous recovery, but some might require liver transplantation [13] . with hepatitis e infection, the risk of developing alf in adults is 0.6-2.8% [14] . recent evidence suggests that case fatality due to hepatitis e-induced alf in pregnancy is similar to that of age-matched general population [15] . the alf due to hepatitis b virus (hbv) can occur at the time of acute infection, reactivation of chronic hbv infection or seroconversion from a hepatitis b e antigen-positive to a hepatitis b e antibody (hbeab)-positive state. superinfection or coinfection of hbv-infected patients with hepatitis delta virus (hdv) can cause liver failure. hepatitis c virus (hcv) infection has not been reported as a cause of alf, and herpes simplex virus can cause alf, of which herpes simplex virus 1 and 2 (hsv) is the predominant cause of viral-induced alf during the first month of life. babies presenting with fever, rash, lethargy, poor feeding and raised transaminase (in thousands) are usually suggestive of hsv hepatitis. disseminated neonatal herpes with liver failure carries high mortality. in stable neonates with alf due to hsv, liver transplantation has been successful. treatment with high-dose acyclovir should be initiated in all neonates with alf, until serology results are known. other members of herpes virus family such as cytomegalovirus, epstein-barr virus and varicella-zoster virus can cause alf. dengue virus causing alf is common in tropical countries, which was thought to be multifactorial due to direct viral injury, dysregulated immune response, hypoxic/ischemic injury secondary to shock, etc. [16] . neonatal haemochromatosis (nh) is the single most common cause of alf during the first month of life, due to massive iron deposition in the liver and extrahepatic tissues with sparing of the reticuloendothelial system. nh presents with jaundice, coagulopathy, moderately elevated alanine aminotransferase, high ferritin and raised iron saturation levels. the disease varies in severity; at one end of spectrum, it is associated with foetal death, while at the other end, spontaneous recovery is reported. the pattern of iron overloading is similar to hereditary haemochromatosis, but nh is entirely different condition affecting newborn, and so far no specific genetic mutation has been identified [17] . current hypothesis suggests nh to be an alloimmune process where maternal antibody is directed towards foetal liver cells resulting in hepatocyte loss [18, 19] . this hypothesis is supported by successful prevention of severe disease by antenatal and postnatal treatment with intravenous immunoglobulin. high serum ferritin is a non-specific marker and elevated in other causes of alf and so should not be used a marker for diagnosis. the diagnosis could be safely confirmed by labial salivary gland biopsy, showing extrahepatic iron deposits with reticuloendothelial system sparing [20] . metabolic disorders are important cause of alf in paediatric population particularly during infancy. galactosaemia, tyrosinaemia type i and fructosaemia are few of the metabolic disorders that could present as alf. tyrosinaemia type i is an inborn error of amino acid metabolism, due to absence of enzyme fumarylacetoacetase, the last enzyme in a series of five enzymes needed to break down tyrosine. this results in formation of intermediate compounds, maleylacetoacetic acid and fumarylacetoacetic acid, which is converted to succinylacetone, a toxin that damages the liver and kidneys. oral ntbc (nitro-4-trifluoromethylbenzoyl-1,3-cyclohexanedione) and phenylalanine-and tyrosine-free diet could help liver recovery, but some children might require lt. galactosaemia type 1 is autosomal recessive disorder with mutation in galactose-1-phosphate uridyl transferase (galt) gene located on chro-mosome 9p13. lactose-free diet should be started in any infant presenting with alf or hepatitis until the quantitative galt activity is available. galactose-free diet and supportive treatment may allow recovery of alf. rarely inborn errors of bile acid synthesis can present as alf. mitochondrial disorders are group of spontaneous or inherited disorders of mitochondrial proteins resulting in defective oxidative phosphorylation, fatty acid oxidation, urea cycle and other mitochondrial pathways [21] . deficiencies of complexes i, iii and iv, multiple complex deficiencies and mitochondrial dna (mtdna) depletion syndrome are associated with liver failure. diagnosis might be difficult due to particularly (mtdna) depletion syndrome where there is tissue-specific mitochondrial enzyme deficiency. these infants usually present with hypotonia, hypoglycaemia, feeding difficulties, seizures and deranged liver function. liver transplantation could be done in isolated liver-based mitochondrial disorders, and it is usually contradicted in multisystemic involvement [22] . sasaki et al. have reported 78% survival in a cohort of nine children with mitochondrial respiratory chain disorder, which included children with extrahepatic manifestation such as developmental delay and failure to thrive [23] . medium-chain acyl-coenzyme a dehydrogenases (mcad) are group of enzymes involved in β-oxidation of 6-12 carbon chain fatty acids in mitochondria. affected children could present with hypoketotic hypoglycaemia and recurrent liver failure, precipitated by otherwise minor illness. unless treated with dextrose supplementation, these episodes may quickly progress to coma and death. wilson's disease, an autosomal recessive disorder, could present as alf. the acute hepatic presentation is usually characterized by the presence of liver failure, coombs-negative haemolytic anaemia and low serum alkaline phosphatase. diagnosis might be difficult in acute presentation as blood test might show weakly positive autoantibodies, and tissue copper estimation might not be possible due to coagulopathy. new wilson index proposed by dhawan et al. used five parameters such as serum bilirubin, serum albumin, international normalized ratio, aspartate aminotransferase (ast) and white cell count (wcc) at presentation. based on serum levels, each parameter is graded from 0 to 4, with a total maximum score of 20. they identified a cutoff score of more than 11 for death without transplantation and proved to be 93% sensitive and 98% specific, with a positive predictive value of 88% [24] . haemophagocytic lymphohistiocytosis (hlh) is a type of haematological malignancy that could present as alf in children. hlh is due to paradoxical overactivation of natural killer cells and of cd8+ t-cell lymphocytes resulting in destruction of own haemopoietic cells. it could be familial (inherited) or acquired. familial hlh usually presents during infancy, while secondary hlh usually occurs after systemic infection or immunodeficiency, which can affect people at any age. familial hlh is an autosomal recessive disease resulting in reduced or defective production of cytoplasmic granules such as perforin in cytotoxic cells resulting in paradoxical overactivation. hlh presents with fever, cutaneous rash, hepatosplenomegaly, pancytopenia and, in severe cases, alf [25] . though rare, leukaemia or lymphoma could present with alf [26] . other causes: autoimmune hepatitis (aih), particularly type 2 (positive liver-kidney microsomal (lkm) antibody), can present with alf. alf due to aih with encephalopathy usually does not respond to any form of immunosuppression and needs urgent liver transplant [27] . in spite of extensive investigation, the diagnosis could not be found in some of the children (indeterminate). there is centre-to-centre variation in incidence of indeterminate alf, probably due to incomplete investigations, which has been highlighted by narkewicz et al. [28] . general investigation should include liver function tests, serum electrolytes, uric acid, lactate, cholesterol/triglyceride, amylase, serum amino acids, blood gas analysis, blood glucose levels, full blood count, blood grouping, coombs test coagulation studies (inr), urinary amino/organic acids and toxicology screen along with surveillance blood and urine cultures. in liver function tests, coagulation should be checked on regular basis that helps in monitoring the progression of disease. investigations to establish the underlying aetiology are listed in table 8 .2. detailed clinical history and physical examination give valuable clue of underlying diagnosis. this would provide guidance in choosing appropriate investigations. transplant-free survival is aetiology dependent. age of patient was not associated with outcome in adults [29] , while in children, neonates have worst prognosis (fig. 8.2) , probably due to predominance of certain aetiology in different age groups. prognostic scoring helps in predicting mortality and helps in listing appropriate patients for transplantation. for non-acetaminophen alf, several prognostic scoring systems are available for adults, but in children there are no universally accepted criteria for listing. to date, inr and factor v concentration remain the best indicators of mortality without transplantation in paediatric alf. bhaduri and mieli-vergani showed that the maximum inr reached during the course of illness was the most sensitive predictor of the outcome, with 73% of children with an inr less than 4 surviving compared with only 4 of 24 (16.6%) with an inr greater than 4 [2] . in children, a factor v concentration of less than 25% of normal suggests a poor outcome. a prognostic score incorporating serum bilirubin, serum albumin, international normalized ratio, aspartate aminotransferase (ast) and white cell count (wcc) is available predicting the outcome of decompensated wilson's disease [24] . in acetaminophen overdose adult criteria of inr > 6.5, creatinine > 300 μmol/l and hyperphosphatemia or metabolic acidosis arterial ph less than 7.3, after the second day of overdose in adequately hydrated patients, is used to list children for liver transplantation [30] . management of alf and its complications still remains a challenge. early diagnosis helps in initiation of investigation and safe transfer to a specialist centre. diagnostic algorithm for any child with abnormal liver function test is outlined in fig. 8 .3. all children with alf should be closely monitored in a quiet setting. vital parameters such as oxygen saturation, pulse, blood pressure and neurologic observations should be done on regular basis. prophylactic broad-spectrum antibiotics and antifungals should be started in all children, and acyclovir should be added in infants and neonates. hypoglycaemia should be avoided either by parenteral glucose or adequate enteral feeds. children with encephalopathy or an inr > 4 (regardless of encephalopathy) should be admitted to an intensive care unit for continuous monitoring. prophylactic histamine 2 blockers or proton-pump inhibitors should be started to all patients requiring mechanical ventilation [31] . coagulopathy is corrected only if the patient is already listed for transplant or prior to an invasive procedure. to correct coagulopathy, fresh frozen plasma could be given at a dose of 10 ml/kg and cryoprecipitate at 5 ml/kg (if fibrinogen is <1 g/l). factor vii concentrates improve the coagulopathy for a short period. platelet count should be maintained above 50 × 10 9 /dl, as thrombocytopenia is an important risk factor for haemorrhage. n-acetylcysteine (nac) is being increasingly used as a part of general supportive measure in non-acetaminopheninduced alf, as it enhances circulation and improves oxygen delivery. in a prospective, double-blind trial in adults with non-acetaminophen alf, nac usage is associated with significant improvement in transplant-free survival in patients with early (stage i-ii) coma [32] . a similar study in children failed to show any benefit, and paediatric acute liver failure study group does not recommend routine use of in non-acetaminophen-induced alf in children [33] . bowel cleansing agents and benzodiazepine antagonists are of no proven benefit [34] . elective intubation and mechanical ventilation should be considered for patients with grade 3/4 encephalopathy. apart from providing secure airway, sedation and controlled ventilation help in reducing sudden variation of intracranial pressure (icp). induction using suxamethonium and fentanyl and combination of morphine or fentanyl with a hypnotic such as midazolam for sedation is usually safe in children. normocapnia is to be maintained, as hypercapnia causes vasodilatation and increases cerebral congestion, while hypocapnia causes vasoconstriction and thus decreased blood flow to the brain. intravenous fluids should be restricted to two-thirds maintenance, with the idea of decreasing the possibility of development of cerebral oedema. ultrasonic cardiac output monitor (uscom), which is a non-invasive method to measure cardiac parameters, helps in decision-making regarding appropriate fluid regimens/inotropes even in small infants. in the presence of persistent hypotension, noradrenaline is the inotropic agent of choice. continuous filtration or dialysis systems should be considered when the urine output is less than 1 ml/kg/h to prevent acidosis and volume overload. the most serious complications of alf are cerebral oedema with resultant encephalopathy and intracranial hypertension, progressively leading on to brain herniation and death. systemic hypertension, bradycardia, hypertonia, hyperreflexia and in extreme cases decerebrate or decorticate posturing are clinical features of raised icp. ammonia-lowering measures such as dietary protein restriction, bowel decontamination or lactulose are of limited or no value in rapidly advancing encephalopathy. mannitol is an osmotic diuretic commonly used to treat intracranial hypertension. a rapid bolus of 0.5 g/ kg as a 20% solution over a 15-min period is recommended, and the dose can be repeated if the serum osmolarity is less than 320 mosm/l. hypertonic saline could be also used in emergency situation, where there is impending brainstem herniation. studies have shown mild cerebral hypothermia (32-35 °c) , sodium thiopental and hypernatremia (serum sodium > 145 mmol/l) improves cerebral perfusion. disease-specific management is outlined in table 8 .2. intravenous immunoglobulin (ivig) at a dose of 1 g/kg body weight given weekly from the 18th week until the end of gestation as antenatal prophylaxis to mothers whose previous pregnancy/child was affected with nh has been associated with milder phenotypic expression of the disease and 100% survival of babies [35] . evidence is accumulating towards the usefulness of high-dose ivig (1 g/kg), in combination with exchange transfusion resulting in significant decrease in the need for liver transplantation in nh. dietary intervention with restriction of phenylalanine and tyrosine together with oral medication, 2(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexenedione (ntbc), helps in normalization of liver function, but doesn't prevent long-term risk for development of hepatocellular carcinoma in children started beyond infancy. plasmapheresis is the removal or exchange of blood plasma. therapeutic plasmapheresis and therapeutic plasma exchange (tpe) are terms that are often used synonymously. tpe has been increasingly used over the past decade as a first-line and lifesaving treatment for various conditions classified by the american society for apheresis (asfa). in acute fulminant wilson's disease, it can rapidly remove significant amount of copper and, thereby, reduce haemolysis, prevent progression to renal failure and provide clinical stabilization. it has been reported to be used as a bridge to lt or can lead to elimination of the need for urgent lt. tpe is also helpful in stabilizing alf due to viral hepatitis, drug-induced hepatitis, etc. by removing albumin-bound toxins, large molecular weight toxins, aromatic amino acids, ammonia, endotoxin, indols, mercaptans, phenols, etc. simple liver assist devices detoxify blood by simple osmotic diffusion, while bioartificial liver support system which contains human or animal liver cells could perform complex synthetic function and detoxifying and detoxification. these devices have shown to decrease the toxins (ammonia, bilirubin, cytokines, etc.) but have no effect on mortality. successful use of these devices in children with alf as a bridge therapy, supporting liver function while the native liver regenerates, is not recommended outside research setting. liver transplant remains the only proven treatment that has improved the outcome of alf. appropriate patient selection and timing of transplantation are essential for graft and patient survival. several surgical techniques such split liver grafts, reduced grafts and auxiliary liver transplants are practiced, depending upon patient size, organ availability and surgical expertise available. auxiliary liver transplant is a surgical technique where the donor liver is placed alongside of native liver and the allograft supports the entire liver function, while the native liver regenerates. either left lateral segment or right lobe allograft could be used, based on recipient weight. once native liver regeneration is optimal [36] , then immunosuppression could be weaned and eventually stopped. in a series from king's college hospital, of the 20 children who received auxiliary liver transplantation for alf, immunosuppression was withdrawn successfully in 11 patients at a median time of 23 months after transplantation [37] . this would be an ideal option in alf due to indeterminate aetiology, as spontaneous regeneration of native liver remains a possibility. liver transplantation is indicated in alf due to liverbased disorders while contraindicated in haematological malignancies, uncontrolled sepsis, systemic mitochondrial/ metabolic disorders and severe respiratory failure (ards) [38] . relative contraindications are increasing inotropic requirements, infection under treatment, cerebral perfusion pressure of less than 40 mmhg for more than 2 h and a history of progressive or severe neurologic problems. hepatocyte transplantation, where hepatocytes are infused intraportally into the patient's liver, has been tried with variable success in certain liver-based metabolic disorders [39] . research is underway to use alginate-encapsulated hepatocytes, which could be injected intraperitoneally. this could act as a bridge until native liver regenerates. hepatocyte transplantation is not recommended outside research setting. improved intensive care management has greatly increased the alf survival. when compared to adult alf, the spectrum of underlying aetiology, management and outcome varies in paediatric alf. acyclovir should be started in all neonates with alf along with prophylactic antibiotics, until viral cultures are negative. liver transplantation is the only definitive treatment that improves survival in paediatric alf. wilson's disease and autoimmune liver disease presenting as alf usually do not respond to medical management and warrant liver transplantation. liver assist devices and hepatocyte transplantation are potential emerging therapies in paediatric alf. the management of fulminant hepatic failure fulminant hepatic failure: pediatric aspects. semin liver dis acute liver failure in children: the first 348 patients in the pediatric acute liver failure study group profile and outcome of first 109 cases of paediatric acute liver failure at a specialized paediatric liver unit in india neonatal liver failure: aetiologies and management-state of the art population-based surveillance for acute liver failure protective agents for acetaminophen overdose review article: drug-induced liver injury in clinical practice the characteristics and clinical outcome of drug-induced liver injury: a single-center experience drug-induced liver injury: a clinical update assessment of drug-induced hepatotoxicity in clinical practice: a challenge for gastroenterologists prognostic factors in paediatric acute liver failure global epidemiology and medical aspects of hepatitis e. forum (genova) a 20-year single-center experience with acute liver failure during pregnancy: is the prognosis really worse? dengue and its effects on liver neonatal hemochromatosis. genetic analysis of transferrin-receptor, h-apoferritin, and l-apoferritin loci and of the human leukocyte antigen class i region neonatal hemochromatosis: is it an alloimmune disease? novel mechanism of fetal hepatocyte injury in congenital alloimmune hepatitis involves the terminal complement cascade minor salivary gland biopsy in neonatal hemochromatosis disorders of the mitochondria liver transplantation for mitochondrial respiratory chain disorders: to be or not to be? liver transplantation for mitochondrial respiratory chain disorder: a single-center experience and excellent marker of differential diagnosis wilson's disease in children: 37-year experience and revised king's score for liver transplantation hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. pediatr blood cancer leukaemia presenting with fulminant hepatic failure in a child autoimmune paediatric liver disease pattern of diagnostic evaluation for the causes of pediatric acute liver failure: an opportunity for quality improvement results of a prospective study of acute liver failure at 17 tertiary care centers in the united states early indicators of prognosis in fulminant hepatic failure aasld position paper: the management of acute liver failure n-acetylcysteine on its way to a broader application in patients with acute liver failure intravenous n-acetylcysteine in pediatric patients with nonacetaminophen acute liver failure: a placebo-controlled clinical trial acute liver failure in children high-dose immunoglobulin during pregnancy for recurrent neonatal haemochromatosis clinical human hepatocyte transplantation: current status and challenges auxiliary liver transplantation for acute liver failure in children hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure cell therapy for liver disease: from liver transplantation to cell factory key: cord-318755-fip8wj6y authors: el kassas, mohamed; alboraie, mohamed; al balakosy, amira; abdeen, nermeen; afify, shimaa; abdalgaber, mohammad; sherief, ahmed f; madkour, ahmad; abdellah ahmed, mohamed; eltabbakh, mohamed; salaheldin, mohamed; wifi, mohamed-naguib title: liver transplantation in the era of covid-19 date: 2020-05-12 journal: arab j gastroenterol doi: 10.1016/j.ajg.2020.04.019 sha: doc_id: 318755 cord_uid: fip8wj6y liver transplantation is considered the ultimate solution for patients with end-stage chronic liver disease or acute liver failure. patients with liver transplant need special care starting from preoperative preparation, surgical intervention ending with postoperative care. transplanted patients have to receive immunosuppressive therapy to prevent rejection. such a state of immune suppression could predispose to different types of infections in liver transplant recipients. currently, the world is suffering a pandemic caused by a new strain of the coronavirus family called covid-19. certain infection control precautions are needed to protect immunocompromised and vulnerable patients, including liver transplant candidates and recipients from acquiring covid-19 infection. restricting non-transplant elective surgical procedures, managing transplant patients in separate outpatient clinics, and in-patient wards can prevent transmission of infection both to patients and healthcare workers. telemedicine can help in the triage of patients to screen for symptoms of covid-19 before their regular appointment. management of immunosuppressive therapy and drug-drug interactions in liver transplant recipients infected with covid-19 should be cautiously practiced to prevent rejection and effectively treat the underlying infection. in this report, we are trying to summarize available evidence about different aspects of the management of liver transplant candidates and recipients in the era of covid-19. liver transplantation (ddlt) represented 20%. fifty-six percent of the reported cases were in egypt [8] . based on previous observations for sars and other related viruses, a theoretical risk of liver damage exists with covid-19 infection [9, 10] . however, available data only reported hepatic dysfunction in the form of abnormal levels of liver aminotransferases and slightly elevated bilirubin levels, mainly in critically ill patients [11] . on the other hand, reports during an influenza outbreak in germany in winter 2017/2018 showed increased organ failure scores of patients with liver cirrhosis where 5 out of 11 patients with liver cirrhosis developed acute liver failure during influenza infection [12] . no data available on the impact of covid-19 on decompensated liver disease patients awaiting ltx, but because of the known immunocompromised state of these patients, adequate protective measures should be maintained. although healthcare facilities are overwhelmed with management of covid-19 patients & health resources are being rapidly consumed, the american association for the study of liver diseases (aasld), recommended against postponing transplantation. moreover, they advised each program to consider its capability regarding intensive care unit (icu) beds, ventilators availability, and blood donation [10] . prioritization of transplant candidates is another problem that may face clinicians due to limited resources during the pandemic, as well as the exclusion of donors infected with covid-19 [10] . immunosuppression in the post-transplant recipients may be protective against cytokine storm induced by covid-19, which is responsible for the severe illness on the one hand. however, and on the other hand, recipients on immunosuppression may have more intense and prolonged shedding of the virus, increasing the risk of transmission to contacts, including healthcare workers [13] . this could emphasize the crucial role of implementing infection control measures to avoid losing candidates on the ltx waiting list because of the closed transplantation centers [14] . international societies like world health organization (who) and centre for disease control and prevention (cdc) are always confirming the necessity to use personal protection equipment (ppe) in addition to the restriction of outpatient and elective procedures as preventive measures against covid-19 [15] . limitations of aerosol-generating procedures like suction, endotracheal intubation, and advanced endoscopy are of major concern due to the fear of the possibility of disease transmission. further restrictions to prevent other routes of infections like feco-oral transmission, included colorectal surgeries and colonoscopies. currently, many interventional surgical societies, anesthesia, endoscopy, radiology, and intensive care have placed their statements, guidelines, and recommendations to adjust their practice to the current epidemic [16] . different reasons rationalized the delay or even cancellation of non-emergency procedures as they would consume ppe tools which are currently running short supply worldwide. the second reason that such elective procedures are postponed or canceled is to prevent unnecessary infections to medical staff and caregivers, which may be transmitted from asymptomatic covid-19 patients or their companions. also, they consider such procedures a further burden and workload on an already exhausted medical system. finally, occupying the operative theatres with such cases would warranty the need for mechanical ventilators that might be more beneficial and valuable if they are directed to rescue a covid-19 patient's life [17] . meticulous evaluation should be done before deciding for the priority of the procedure through detailed history taking, one by one consultation, temperature measurement, hand hygiene, and reporting of any suspected case of covid-19 (even if afebrile), and finally cleaning and disinfection protocols of premises. repeated physical examination and temperature measurement along with the revision of chest imaging like a computed tomography scan or a chest radiograph, and if covid-19 is suspected or confirmed, all non-emergency procedures would be delayed or canceled [18] . because of the rapidly changing situation of covid-19 infection worldwide, indications of ltx will need to be updated according to the emerging data. bearing in mind that any liver transplant related activity not only involves the donor and the recipient, but it involves many individuals, including doctors, paramedical staff, nurses, and health care workers. taking into consideration that there is a risk of the donor to recipient transmission of covid-19, from both deceased donors and living donors. the risk of donor-derived infection would depend upon donor exposure, infectivity in the incubation period, degree and duration of viremia, and viability of the virus within blood or specific organ compartments [13] . traditionally, the aasld/ast guidelines outline four major types of indications for ltx in the united states: acute liver failure, complicated cirrhosis, metabolic liver diseases, and systemic complications of chronic liver disease [19] . acute liver failure is the most important indication for emergency ltx. common causes of acute liver failure include acetaminophen overdose, acute viral hepatitis, drug-induced liver injury, mushroom poisoning, autoimmune hepatitis, wilson's disease, acute ischemic hepatitis (shock liver), and acute fatty liver of pregnancy [19] [20] [21] [22] . similarly, patients with cirrhosis and type 1 hepatorenal syndrome have a median survival of fewer than two weeks and should be urgently referred to a transplant center for expedited transplant evaluation, as should patients with other evidence for rapid hepatic decompensation [19, 23] . elective ltx is indicated in cases with cirrhosis associated with deteriorating hepatic synthetic function, renal function, and related complications like hcc [19, [24] [25] [26] [27] [28] [29] [30] [31] . to minimize the risk of covid-19 exposure in the hospital setting, ltx should be done judiciously as per the following criteria. in essence, non-urgent transplants should be postponed, and acute liver failure can be done according to the standard indications, acute on chronic liver failure decisions for transplantation should be based on individual center's experience [32] . despite being potentially transmitted by organ donation, recent recommendations suggest that ltx could be performed during the covid-19 pandemic [10] . as the organism is predominantly found in respiratory secretions, lungs are considered high-risk organs for transmission of covid-19infection if the donor is infected. however, all other organs could be considered at risk as the virus was isolated from blood in nearly 15% of cases [33] . these observations are supported by evidence from the sars epidemic in 2003 in which autopsy results could demonstrate the virus in almost all body organs [34] . two types of ltx techniques are available; the first is the conventional or standard method, which involves implantation of whole liver grafts ddlt, and this type is the most widely used in the usa and europe [19] . the other type is ldlt, and this is the only option available for patients living in some countries like egypt as cadaveric organ transplant is still banned there, and that is why some patients travel abroad seeking organ transplant elsewhere [35] . at present, the organ procurement organizations (opos) and hospitals having ldlt programs should screen potential donors for exposure and clinical symptoms suggestive of covid-19, and as more information builds up, the ideal approach would hopefully be developed [36] . although personal contact is the basis of the doctor-patient relationship, which allows rapid and accurate assessment of the patient's condition, unfortunately, it exposes physicians to the risk of infection and becomes a source of the virus spread. therefore, it is considered wise to substitute direct contact with patients with more distant interactions with the utilization of available technology. the same approach could also be applied to the post-operative period for the donor in the case of ldlt, aiming to be discharged as soon as possible from the hospital [37] . with the increasing number of infected cases and mortality caused by covid-19, a recent discussion for using organs from patients infected with covid-19 has led to several points of debate. for instance, from the clinical point of view, it could potentially be a life-saving procedure, especially that delays might result in more deterioration for the patient's need for the organ and even increase the risk of covid-19 exposure. on the other hand, it may transmit infection, which would even be more severe in transplant recipients, especially in the absence of effective therapy. financially speaking, it can be a possible source of income for hospitals despite reimbursements remain uncertain. it might be argued from the legal point that organs with hcv have already been transplanted after getting informed consent from patients. nevertheless, this disease has a different risk profile compared to hcv. as regards the ethical perspective, this procedure respects the wishes of donors and their families and ensures the autonomy of patients willing to do so. however, patients could be overwhelmed with responsibility for informed consent in the presence of little guidance [38] . the emergence of the covid-19 pandemic has posed extensive threats and problems to all the healthcare facilities, including ltx centers [39] . these effects are not only confined to donor or recipient issues but also extends to involve many other problems in the availability of healthcare resources [13] . we are faced with enormous challenges owing to the high communicability and virulence of the virus, the risk of introducing immunosuppressive therapies during this pandemic, and our utmost need for all the health care utilities. on the other hand, we do have a very long list of miserable patients waiting for ltx, which is the only available treatment option for this difficult to treat a group of patients, so judicious decisions and strict precautions became now mandatory [40] . streptococcal pneumonia and influenza vaccines are strongly recommended to all recipients prior to ltx, together with strict prophylaxis against complications of cirrhosis to reduce the number of hospital admissions [41] . it is also recommended to test for covid-19 in patients with acute decompensation or acute on top of chronic liver failure (aclf) [39] . for those on ltx lists, it is recommended to test both donors and recipients for covid-19 before ltx, putting into consideration that negative results do not totally exclude the infection. alternatively, computed tomography (ct) of the chest can be considered [42] . pre-procedure consent should include the potential hazard for the acquisition of nosocomial covid-19infection [39] . accepting only grafts with a low risk of delayed graft function to reduce complications and minimize the length of postoperative stay is also recommended [10] . diminishing exposure of health care workers as much as possible, through using online clinics and phone calls as a substitute to primary clinics can prevent unnecessary risk of infection. doctors may also talk to all patients by phone before their visits to rule out any possibility of covid-19 infection. deferring optional visits and restricting it only to urgent ones also help to prevent nosocomial infections [37] . modifications of the outpatient transplant clinics by widening the patients' waiting areas and following strict infection control precautions is of utmost importance [43] . workforce affairs are crucial; any member of the transplantation team suffering from any symptom suggestive of covid-19infection should be absent from work and self-isolate him/herself for fourteen days if the exposure occurred. moreover, a rapid test for the transplantation team is highly recommended [44] . post-operative care for ltx recipients during the pandemic of covid-19 is challenging. to guarantee the maximum benefits for both the patient and the graft, a multidisciplinary management team is usually involved in postoperative care of ltx recipients. such management includes infection control with extra care directed at preventing postoperative infections, including covid-19 infection [40] . liver transplant recipients should be admitted to separate wards where there is complete separation from covid-19 admission wards, along with the strict implementation of standard disinfection measures. it is recommended to limit surgical and medical rounds, requests for the image, and blood tests to the least required number [10] . follow up of liver transplant recipients is usually performed in tertiary referral hospitals, where covid-19 hotspots may be present [39] . it is thus recommended to limit in-person outpatient visits even in areas without significant covid-19 community spread [45] . hospital admission of liver transplant recipients should be considered only for patients suffering from major complications like rejection, decompensation, or vascular complications. during in-person outpatient follow up visits, liver transplant recipients should be evaluating in dedicated hepatology/liver transplant clinics away from clinics where confirmed or suspected cases of covid-19 are evaluated [10] . once the patient is clinically stable, it is recommended to perform his routine laboratory investigations, including drug levels at primary care facilities [39] . telemedicine can mitigate exposure of both patients and healthcare workers to covid-19 as it allows better physician-patient communication. development of covid-19 symptoms (fever, cough, shortness of breath, sore throat, diarrhea, the new loss of sense of taste or smell, contact with known covid-19 patients, history of recent travel) in a liver transplant recipient should prompt urgent referral for evaluation along with hepatic symptomatology surveillance and drug compliance assurance [37, 46] . epidemics can lead to a significant increase in demand for icu beds, so reducing the available beds. as an example, the sars outbreak in toronto led to closures of 35 icu beds for ten days, which represented 38 % of the tertiary-care university medical-surgical beds of icu in toronto [47] . many reasons represent a challenge in caring for patients with covid-19 with a high risk of exposure for icu staff. covid-19 is highly contagious, along with more than one route of transmission.the high exposure dose, long contact hours with cases, some procedures such as noninvasive ventilation, and the length of icu stay are representing the challenges for icu staff during the epidemic [48] . moreover, patients and staff from each sector of the icu should use different routes inside the icu to decrease the risk of infection [51] . another way to minimize the possibility of icu admissions is by applying the fast track protocols which can be applied safely for selected patients undergoing either living or ddlt [52] . the definition of fast-tracking in liver transplant still lacks consensus among different centers, ranging from early postoperative extubating in the operating room once the surgery is finished, to strategies that minimize postoperative ventilation time.generally, this term is reserved for early extubation, recovery in a post-anesthesia care unit (pacu), and direct transfer to the surgical ward avoiding an icu stay [53, 54] . patients who underwent ltx are usually poly medicated using many drug classes. the most important are immunosuppressive drugs. as most anti-covid-19 agents are investigational, drug-drug interactions are very critical in this situation in those fragile patients. possible drug-drug interactions between sars-cov-2 antiviral drugs and commonly used immunosuppressants for liver transplant recipients are presented in table 1 [55] [56] [57] . possible interactions with other drugs are summarized below: chloroquine and hydroxychloroquine act either as viral entry blockers or as immunomodulators. despite encouraging preliminary reports, side effects, and interactions with other medications are well known [55] . chloroquine has potential interactions with commonly used drugs like ampicillin, amlodipine, azithromycin, propranolol, and antacids [56] . chloroquine has significant drug interaction with ciclosporin as it increases levels of cyclosporin by decreasing its metabolism. also, chloroquine increases levels of tacrolimus by the same mechanism but to a lesser extent [56]. remdesivir is a nucleotide analog with broad-spectrum antiviral activity against single-stranded rna viruses [55] . it is an investigational drug that appears safe and may not affect other medications; however, remdesivir concentration can be affected by enzyme inducers like clarithromycin, rifampin, phenytoin, and phenobarbital [57] . it is also an investigational drug with antiviral activities through its selective inhibition of viral rna-dependent rna polymerase [55] . favipiravir increase concentration of pioglitazone, rosiglitazone, paracetamol, oseltamivir, and hormonal replacement therapy; however, no significant interactions with immunosuppressive medications nor steroid [57] . a fixed-dose combination for the prevention and treatment of hiv infection. the cytochrome p450 inhibitory effects of ritonavir prolong the half-life of lopinavir and extend its protease inhibitory action but, on the other hand, increases its liability for many drug interactions [55] . this combination is not recommended to be co-administer with many steroids forms, simvastatin, atorvastatin, domperidone, and sirolimus. there is also a potential interaction with ciclosporin, mycophenolate, tacrolimus, which makes its use in the setting of ltx is questionable. however, a recent case report confirmed successful covid-19 treatment by lopinavir/ritonavir in liver transplant recipients [58] . lopinavir/ritonavir can increase chloroquine and hydroxychloroquine concentrations. also, lopinavir/ritonavir induces qt interval prolongation that may potentiate chloroquine and hydroxychloroquine toxicity [57] . tocilizumab is a monoclonal antibody that targets the interleukin-6 receptor, which is the possible mediators of covid-19 induced inflammation and cytokine storms [55] . it has potential possibly minor interaction with ciclosporin, tacrolimus, and sirolimus. tocilizumab can reduce concentrations of calcineurin inhibitors with necessary drug level monitoring. its use with chloroquine and hydroxychloroquine may have additive toxicity. also, it may potentiate hematological toxicity of ribavirin and interferon-beta if used together as tocilizumab has a myelosuppressive effect [57] . ribavirin is an old antiviral drug used in the treatment of hcv for years, and that had a role in the treatment of sars. as it causes dose-dependent hemolytic anemia, ribavirin may potentiate the hematological toxicity of interferon-beta and tocilizumab. ribavirin had no significant interaction with immunosuppressive drugs [57] . one of the lessons learned from mers-cov infections is that host inflammatory responses play a major role in disease progression. this was the base of using interferon-beta in mers-cov and covid-19 infections [55] . in the setting of ltx, interferon-beta had no interactions with immunosuppressive drugs or steroids. nevertheless, it induces myelosuppression, so it should not be combined with tocilizumab. also, potential interaction with chloroquine and hydroxychloroquine may increase its toxicity [57] . patients with advanced liver disease and those after ltx represent vulnerable patient cohorts with an increased risk of infection and/or a severe course of covid-19 because of the immunosuppressed state they have [59] . as the innate immune response associated with increased serum interleukin-6 (il-6), interleukin-8 (il-8), and tumor necrosis factor-alpha (tnf-î±) levels may be the main driver for pulmonary injury due to covid-19, immunosuppression may be protective [33, 60] . coronaviruses have not shown to cause more severe disease in immunosuppressed patients. more interestingly, reviewing the mortality and morbidity reports published on coronaviruses outbreaks such sars that emerged in 2002, mers, and more recently covid-19, no fatalities were reported in patients undergoing transplantations, receiving chemotherapies or other immunosuppressive treatments [61] . risk factors for poor outcome include older age (post-transplant recipients aged >60 years old) male sex and presence of comorbidities (obesity, diabetes, heart disease, lung disease, kidney disease) [10] . available data on coronavirus before and during outbreaks suggest that immunosuppressed patients are not at increased risk of severe pulmonary disease compared to the general population; however, immunosuppression may prolong viral shedding in post-transplant patients with covid-19 if they are already infected [36, 60] . immunosuppressive therapy should be started in patients with liver disease with or without covid-19 who have strong indications for treatment (e.g., autoimmune hepatitis, liver transplant patients, and in cases of graft rejection). this should be done without compromising their transplant management as reducing the dosage or stopping immunosuppressants as this may precipitate acute rejection in a liver transplant recipient, so there is no need to reduce or stop immunosuppression for asymptomatic post-transplant patients without known covid-19 [10] . the reduction should only be considered under special circumstances (e.g., medication-induced lymphopenia, or bacterial/fungal superinfection in case of severe covid-19) after consultation of a specialist [62] . the potential role of corticosteroids for the prevention of progression of mild covid-19 to severe pneumonia is unknown. who recommends avoiding corticosteroids for the treatment of covid-19 unless indicated for another therapeutic purpose [63] . in patients with confirmed covid-19, we should minimize the dosage of prednisone, maintaining a sufficient dosage to avoid adrenal insufficiency [39] . similarly, it is also advised to reduce daily calcineurin inhibitor dosage; consider decreasing tacrolimus/cyclosporine by 50%, stop mycophenolate (cellcept/myfortic) and azathioprine especially in the setting of lymphopenia, fever, or worsening pneumonia attributed to covid-19. in the case of ground-glass opacities, pneumonia switching mammalian target of rapamycin (mtor) to calcineurin inhibitors (cni, e.g., tacrolimus) should be done given the possibility of pneumonitis with mtor; otherwise, we should stop all immunosuppression [59] . for outpatients on belatacept, switching to tacrolimus or cyclosporine should be considered after 28 days from the last dose, to avoid clinic visit. for inpatients on belatacept, we should not administer any further belatacept. twenty-eight days after the last dose, adding low dose cni should be considered. for cni intolerant patients, increasing daily prednisone dose from 5 mg to 7.5-10 mg daily might be considered. low dose prednisone (5 mg) in all patients who were receiving it before hospitalization can be maintained [59] . world 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the covid-19 pandemic covid-19 drug interactions successful treatment of severe covid-19 pneumonia in a liver transplant recipient massachusetts general hospital covid-19 treatment guidance coronaviruses and immunosuppressed patients. the facts during the third epidemic middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission successful recovery of covid-19 pneumonia in a renal transplant recipient with long-term immunosuppression clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance (ncov)-infection-is-suspected accessed online key: cord-332827-gll4nqdd authors: peixe, paula; calinas, filipe; tato marinho, rui title: hepatology in the covid era: another c virus, again challenging the liver date: 2020-04-30 journal: ge port j gastroenterol doi: 10.1159/000508116 sha: doc_id: 332827 cord_uid: gll4nqdd nan our way of life has changed! the world is changing dramatically. read the story! [1] . since the beginning of 2020, the worldwide spread of a new virus has made us rethink and reinvent new ways of living and caring for patients. doctors no longer have "the magic touch." in late 2019, a new type of severe pneumonia associated with a new type of coronavirus was first detected in the chinese province of hubei (people's republic of china). coronaviruses have existed for a long time, conditioning diseases in several animal species, but until 2003 (sars [severe acute respiratory syndrome]) there was no report of transmission to humans. the new coronavirus, initially called 2019n-cov and now sars-cov2, is the seventh coronavirus to infect humans, as identified by the chinese health authorities. it emerged as a human-transmitted zoonosis originating from the wild animal market in the city of wuhan. viruses with similar characteristics have been identified in bats, snakes, and pangolins, with an homology of genomic features showing an 89 and 82% nuclear acid sequence similarity with bat sars-covzxc21 and human sars-cov (wuhan patients) but the true transmitter of the disease has not yet been found [2] . the disease is called covid-19 (corona virus disease 2019, i.e., the year of the first case and virus identification). from the point of view of transmission, it occurs among humans through contamination by particles expelled through the airways (by coughing, sneezing, and talking) but also by fomites and interpersonal contact. aerosols and fecal-oral transmission are also considered potential sources of contamination. in addition to this community transmission, nosocomial transmission is important, since many symptomatic patients use health services where they can infect other people, particularly 2 doi: 10.1159/000508116 health professionals, when not adequately protected. the risk of infection for health professionals is 3 times more than in the general population [3] . the spread of covid-19 occurred in the beginning by contact with the original cases identified in wuhan but then, within < 30 days, it went from reaching a city to the whole country. the chinese new year season had a decisive impact as it corresponds to a period of the year when thousands of people are travelling. public transport, always full, favored the transmission of the infection at this time [4] . due to the ease of travel nowadays, cases quickly spread to all countries in the world. around 40 million flights worldwide each year is a terrific number for the capacity of disease transmission. it was possible to designate the transmission chain by identifying index cases. however, in each country, transmission started to occur locally and the links were lost. by march 2020, covid-19 had become widespread in all countries and the world health organization declared it a pandemic. today, about 2,400,000 cases and 165,000 deaths were reported worldwide (https://www.worldometers.info/coronavirus/ [accessed 2020abril19 18: 00]). understanding the pathophysiology of viral infection and the relationship between virus and host has become a matter of urgency. the scientific community has made an intense effort to identify these relationships, but most of the issues are still open. it was possible to identify the virus receptor in humans, namely, the angiotensin-converting enzyme 2 (ace2), a transcellular protein to which sars-cov2 binds. the cellular serine protease, transmembrane protease serine 2 (tmprss2), which cleaves the s protein of human coronaviruses onto the cell membrane, is critical for the fusion of viral cells and the cellular membranes in the host. the coexpression of these molecules seems to be crucial to the infection. ace2 is widely expressed in the human body, i.e., in the lungs, heart, vasculature, kidneys, brain, intestines, and testes. it is also expressed in hepatocytes and in cholangiocytes. the same is true for tmprss2 [5] [6] [7] . the ubiquity of distribution of ace2 and tmprss2 may explain the systemic involvement of the sars-cov2 infection, however, the disease activity is more significant at the respiratory level due to the expression of the molecules in these organs. patients may remain asymptomatic, without knowing exactly the percentage of infected people who do not have manifestations of the disease [8] . respiratory conditions manifesting as fever (41%), cough (56%), headache (25%), muscle pain (29%), and generalized weakness (24%) can have a mild and moderate evolution. severe cases with respiratory insufficiency eventually require ventilatory support (and, in some cases, extracorporeal life support); this has occurred in portugal in 17% of cases according to the records [9, 10]. gastrointestinal (gi) manifestation in covid-19 patients who report at least one gi tract symptom (nausea, vomiting, or diarrhea) varies from 5 to 11.4%. jin et al. [11] reported a rate of chronic liver disease of 10.81% in patients with gi symptoms, significantly higher than those without these symptoms (2.95%) (p = 0.004). they also reported that the rate of covid-19 patients with severe disease is significantly higher in those with gi symptoms (22.97 vs. 8.14%, p < 0.001). the impact of covid-19 on the liver is unclear. the analysis of this relationship must be done by examining 2 distinct aspects, namely liver damage caused by the new coronavirus/covid-19 and the impact of preexisting liver disease on covid-19. as already mentioned, the molecules involved in infectivity of the virus are also present in the cells of the liver (hepatocytes and cholangiocytes), so the liver is damaged by sars-cov2, as occurs with the other coronaviruses. the initial description of hepatic involvement emerged from the analysis of the first 99 cases, 43 of whom presented with an elevation of liver enzymes, alanine aminotransferase in 28% of patients, aspartate aminotransferase in 35%, and total bilirubin in 18%. the incidence of elevated serum liver biochemistry in hospitalized patients with covid-19 ranges from 14 to 53%. in a recent review, changes in liver test values were recorded in about 46% of patients at diagnosis (table 1) and more frequently during hospitalization (table 2 ) [12] . these changes were mild to moderate. only 1 patient had a significant hepatocellular lesion. there was no identification of a cholestatic pattern or liver failure [13] . zhan et al. (unpubl. data from their own center), reported the presence of elevated γ-glutamyl transferase (ggt) in 30 (54%) of 56 patients with covid-19 during hospitalization. such data have not been previously recorded [14] . ge recently, an association between the severity of co-vid-19 and the alteration of liver tests has been described, showing that patients with more severe or critical disease have significantly higher aminotransferase and bilirubin values than cases of mild-to-moderate disease. hypoalbuminemia and low platelets are also associated with more serious disease, but may be related to covid-19 instead of previous liver disease. following autopsy studies, liver changes included dark red hepatomegaly, hepatocyte degeneration with focal lobular necrosis and infiltration by neutrophils, infiltration by lymphocytes and monocytes in the portal spaces, and sinusoidal congestion with microthrombosis. despite these findings, no lesions of the bile ducts or liver failure characteristics were identified [15] . multiple mechanisms involved in liver injury have been described: immune-mediated damage, direct cytotoxicity, anoxia due to respiratory failure, drug-induced liver injury, and the reactivation of preexisting liver disease [16] . after recovery, liver enzyme values return to normal and no evolutive disease seems to occur. chronic liver disease has a significant burden worldwide that can be assessed by the joint contribution of cirrhosis (11th) and liver cancer (16th) as the cause of 3.5% of all deaths [17] . in published series, liver disease was not identified as a risk factor for sars-cov2 infection [11] [12] [13] [14] [15] . it is assumed that risk factors for the general population are also risk factors for patients with liver disease. however, covid-19 may indeed be more severe in liver patients. hepatic disease is associated with multiple factors including viral diseases, alcoholic disease, non-alcoholic fatty liver disease (nafld), and autoimmune pathology (particularly autoimmune hepatitis and primary biliary cholangitis and other cholangiopathies). special reference must be made to cirrhosis, hepatocellular carcinoma (hcc), and transplantation. despite this significant burden, the impact of cov-id-19 on liver disease and liver disease on covid-19 has not been evaluated and studies are needed to better understand the impact of these associations. despite this, some situations deserve a comment for the lessons that its analysis can give. the covid-19 pandemic started in china and rapidly spread to nearby countries. southeast asia has a high prevalence of hepatitis b virus (hbv) infection. in the study by guan et al. [15] 23 patients (n = 1,099) had hbv, according to positive tests for hbv surface antigen (hbs ag) with or without elevated liver enzymes. of these 23 patients, 22 had nonsevere disease (2.4 vs. 0.6%). no warning signs were triggered by the association of these infections. no reference has yet been made to hepatitis c virus (hcv) in the literature nor any comment has come from the usa. in the usa, the country with the largest number of covid-19 cases and with a relevant expression of hepatitis c, no statement has been issued by the centers of disease control and prevention or any group under surveillance, e.g., the veterans cohort. at this stage, it is urgent to modify the strategy regarding the approach to hcv, simplifying the necessary assessments for the institution of treatment and its availability. approximately 2 billion adults are obese or overweight and over 400 million have diabetes. these are risk factors for nafld and hcc [17] . diabetes, hypertension, and obesity associated with nafld are also dealt, alanine aminotransferase; ast, aspartate transaminase; ggt, γ-glutamyl-transpeptidase; alp, alkaline phosphatase; tbil, total bilirubin [12] . scribed as factors for a poor prognosis associated with covid-19. a preliminary report on nafld in patients with co-vid-19 calls attention to an increased risk of disease progression [6.6% (5/126) vs. 44.7% (34/76) p < 0.0001], a higher probability of abnormal liver function on admission and at discharge [70% (53/76) vs. 11.1% (14/126) p < 0.0001], and a longer time for viral clearance (17.5 ± 5.2 days vs. 12.1 ± 4.4 days p < 0.0001), when compared with non-nafld individuals [18] . the authors state that nafld patients also had a higher risk of progression to severe covid-19 and present an increased viral clearance time. with the global prevalence of nafld on the increase, it is worth noting that a significant number of patients may be at risk of developing severe covid-19. immune-mediated liver diseases, particularly autoimmune hepatitis, have not been mentioned as risk factors for covid-19, but the immunosuppressive treatment required has triggered fears about the risk of infection in patients. the analysis of previous outbreaks of human infection by coronaviruses (sars 2002 and mers 2012) did not record an increase in cases in patients under immunosuppression, or that when infected, the disease was more severe. recent data from the red zones in china and bergamo (italy) have shown neither an increase in the number of cases nor in their severity in immunosuppressed patients. even when infected, as long as there were no other risk factors, these patients had a mild or moderate course of covid-19 [19] . no analysis of the different types of drugs used in immunosuppression in liver diseases has been carried out. a warning has been issued about the use of corticosteroids in the context of sars-cov2 infection, where the most seriously ill patients most likely need corticosteroid therapy. the use of these drugs is associated with increased mortality from coronavirus pneumonia. no assessment has been made in patients with liver disease who were previously on stable treatment with corticosteroids and were infected; however, it is assumed that low doses have no impact on covid-19 (as described for patients in the wuhan observational study) [20, 21] . lleo et al. [22] drew attention to two relevant points: balancing the need to maintain treatment to prevent a serious episode that may require therapeutic measures and hospitalization (with an increased risk of nosocomial infection) and assessing the need for certain procedures, e.g., postponing liver biopsy until a period of less epidemic risk. there has been an expectation of canalicular lesions being caused by sars-cov2 due to the presence of ace2 in cholangiocytes. however, recent research has shown that ace2 has a canalicular specificity, which could explain the scarce documentation of the cholestatic lesions and the low number of cases in which ggt changes have been reported [13, 14] . the impact of sars-cov2 infection on cholestatic diseases such as primary biliary cholangitis or sclerosing cholangitis is unknown; further studies are needed to understand whether there is any clinical relevance [23, 24] . changes in liver tests, often associated with coagulation disorder and hypoalbuminemia, are commonly seen in patients with severe covid-19. these changes fluctuate according to the severity of the respiratory disease (pneumonia) and are not due to the direct effect of the virus on the liver. this may explain why there was no worse prognosis in a group of patients with cirrhosis and sars-cov2 infection. in a review of 7 published studies, it was possible to identify 42 patients with cirrhosis with mortality ranging from 0-2% [13] . despite this strange benign report, doctors should consider patients with chronic liver disease, liver cirrhosis, or liver cancer, to be a risk group as they have underlying immune dysfunction and a more reserved prognosis than other patients that go on to develop acute respiratory failure syndrome. for these reasons, patients with chronic liver disease need particular attention and have different specificities from other patients, especially if they are elderly and/or have comorbidities. data on sars-cov2 infection in cirrhotic and hcc patients are scarce. extensive records and targeted studies are needed to explore multiple open-ended questions such as the severity and mortality of covid-19 and episodes of acute-on-chronic or decompensation associated with the presence of this disease (ascites, hepatic encephalopathy, digestive bleeding, kidney dysfunction, and the risk of infection) or the response to treatment [25, 26] . regarding transplantation and cancer patients, there are also no warnings [16] . however, it is not yet possible to say whether transplantation-associated immunosuppression can alter the predisposition for the acquisition of sars-cov2 infection or how covid-19 evolves in these patients. a small published series showed a mortality rate of 27.8% (5/18) in a mixed group of solid organ-transplanted patients (liver recipients (33.3% [6/18] ) infected with sars-cov2 [27] . cancer patients undergoing chemotherapy may be at increased risk for illness and serious illness, especially if they have other risk factors. however, it is not known whether patients with liver cancer are at the same risk. there are still no data on decompensated cirrhosis, patients on the transplantation ge port j gastroenterol doi: 10.1159/000508116 list and, patients in the immediate posttransplant period or with rejection. decisions about transplantation should be considered on a case-by-case basis at this stage of the pandemic [28] . another concern is for patients with liver disease (either advanced or decompensated), namely, the use of drugs used off-label which have not been studied for use for the current indication or in fragile populations such as liver patients. multiple drugs have been referenced and are under study, but currently there is no known effective treatment [29] . during the covid-19 pandemic, recommendations have been issued to mitigate the transmission of sars-cov2, voluntarily isolation has been promoted, and frequenting public places has been advised against. patients associate hospitals and clinics with the risk of transmitting the virus. many patients have conditions that remain unchecked. however, it is necessary for patients to maintain regular follow-up and for situations that require intervention to be detected in a timely manner. on the other hand, the risk-versus-benefit of some interventions must also be weighed up and those without a negative impact should be postponed. a careful assessment will need to be made of the risk of episodes requiring hospitalization and the consequences associated with it or the risk of increased mortality if patients do not receive appropriate treatment or necessary treatment is delayed. scientific societies for the study of the liver, alone or in association, have been publishing guidelines for the approach and management of liver patients. basically, we can divide patients into outpatients and those patients who require hospitalization. stable outpatients with compensated liver disease should be supported in order to maintain the prescribed medication and recognize early warning signs and promote health literacy. teleconsultations are encouraged and consideration is given to carrying out diagnostic and follow-up exams. it is proposed that the blood samples and ultrasound examinations should, if possible, be postponed. if they are deemed necessary, they should be carried out in laboratories nearby avoiding the patient's movement, so as to minimize contacts and the risk of sars-cov2 transmission. patients must comply with all the protection and risk minimization rules proposed for the general population. the maintenance of surveillance of hcc has been discussed. in high-risk patients, this must be maintained so that lesions that could have an early diagnosis are not detected too late. patients with decompensated liver disease should minimize their contact with health-care personnel and, if possible, should not be admitted to hospital. if a patient (without covid-19) does have to be admitted, he or she must stay in a "clean" area in the hospital and the hospitalization time must be minimized (e.g., ambulatory paracentesis and outpatients albumin infusions can be performed). endoscopic exams, due to the extensive production of aerosols involved, are a high risk for the spread of sars-cov2, so they should only be performed in urgent cases. in the context of hepatology, the 2 main indications are upper gi bleeding, with particular attention to any related to portal hypertension or bile duct obstruction. the strategy for eradicating esophageal varices after an episode of rupture is not defined and should be considered on a case basis. endoscopic examinations for the stratification of portal hypertension should be postponed. it may be decided to start nonselective β-blockers, according to the baveno vi criteria, without endoscopy. liver biopsy should be deferred when not essential. follow-up of liver patients will be increased when collaboration with primary care doctors is possible [28, 29] . however, cirrhosis is a disease with inexorable evolution, so the need to focus all efforts on responding to the pandemic has consequences for these patients in the short, medium, and long term. in the short term, patient follow-up can be modified, i.e., seeing that there are fewer appointments and assessments and, potentially, crucial decisions can be delayed, such as the introduction of prophylactic therapy for the rupture of esophageal varices or the indication of resection or transplantation in cases of malignant liver nodules. a reduction of around 25% of donors has already been identified in this first quarter of the pandemic, and also a reduction in the number of liver transplants. in the medium term, when the social-distancing measures are withdrawn or reduced, it is expected that 3 problems will arise. the first is the identification of seriously ill patients and those who may have far exceeded the optimal time point for treatment decisions about a biologically more aggressive disease which evolves without adequate surveillance during the entire phase of mitigation. second, during the pandemic, there has been an increase in the consumption of alcohol and opioids. more people may develop liver disease and will appear as new patients, 6 doi: 10.1159/000508116 thus increasing the disease burden [30, 31] . the third problem relates to institutions and health professionals, who, also exhausted, will have to respond to an excessive number of patients in a short time, and their capacity to respond will likely be exceeded [32] . in the long term, irregular follow-up will be reflected in the health metrics, with an increase in the number of decompensations, hcc, patients on transplantation lists, and mortality [33] . modulation studies of sars-cov2 infection suggest that the pandemic will not end abruptly, but that there will be periods of covid-19 activity in the coming years [34] . we must prepare ourselves clinically and socially for the outcomes for liver patients as a consequence of changes in their follow-up due to covid-19. there is a huge gap in knowledge regarding sars-cov2/covid-19 and the relationship of both the virus and the disease to liver disease, concerning its different etiological aspects and different clinical forms. it is extremely important to acquire this knowledge in the present moment and for the future. the associação portuguesa para o estudo do fígado (apef; portuguese association for the study of the liver) supports all initiatives that favor the acquisition of this knowledge. this work does not require evaluation by an ethics committee. the authors declare that they have complied with the principles of ethics and the absence of plagiarism. severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms death from covid-19 of 23 health care workers in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention call for papers: the pathophysiology of covid-19 and sars-cov-2 infection letter to the editor: angiotensin-converting enzyme 2: an ally or a trojan horse? implications to sars-cov-2-related cardiovascular complications covid-19 and the gastrointestinal tract: more than meets the eye. gut universal screening for sars-cov-2 in women admitted for delivery for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care (cpam). a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms characteristics of liver tests in covid-19 patients epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study liver injury in co-vid-19: management and challenges china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china co-vid-19 and liver disease. liver int burden of liver diseases in the world implication of non-alcoholic fatty liver diseases (nafld) in patients with covid-19: a preliminary analysis coronaviruses and immunosuppressed patients. the facts during the third epidemic the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis risk factors for severity and mortality in adult covid-19 inpatients in wuhan highlights for management of patients with autoimmune liver disease during covid-19 pandemia liver injury during highly pathogenic human coronavirus infections. liver int characteristics and mechanism of liver injury in 2019 coronavirus disease parekh dcovid-19 and the liver: little cause for concern risk factors for severity and mortality in adult covid-19 inpatients in wuhan covid-19 in solid organ transplant recipients: a single-center case series from spain clinical best practice advice for hepatology and liver transplant providers during the covid-19 pandemic: aasld expert panel consensus statement care of patients with liver disease during the cov-id-19 pandemic: easl-escmid position paper alcohol use and misuse during the covid-19 pandemic: a potential public health crisis? lancet public health editorial: challenges to opioid use disorders during covid-19 a shift on the front line covid-19 pandemic will have a long-lasting impact on the quality of cirrhosis care projecting the transmission dynamics of sars-cov-2 through the postpandemic period the authors declare no conflicts of interest of any kind regarding this manuscript. there was no funding. all authors participated in the conception, writing and revision of the manuscript. the final version was approved by the authors. key: cord-014770-cgtzlra1 authors: brandt, lawrence j.; farmer, richard d.; achord, james l.; friedman, lawrence s.; powers, cynthia i.; dorman, nancy j.; sullivan, donna c.; el-newihi, hussein m.; bank, simmy; ahn, john; devault, kenneth; thomas, eapen title: book reviews date: 1995 journal: dig dis sci doi: 10.1007/bf02065002 sha: doc_id: 14770 cord_uid: cgtzlra1 nan ad astra per aspera: this, the motto of kansas, teaches us that we achieved great things only by encountering and overcoming adversity. i felt as if i were achieving great things merely by successfully working my way through several of the chapters in this tome. pto, siology of the gastrointestinal tract is an awe-inspiring product--as it should be, and it was a daunting experience for this clinician to browse through it but, as one of my former professors said during an introductory seminar in medicine, "if you understand all you read, you are reading the wrong books." this is the right book! it is the right book for a library or research laboratory; it is the right book for a physiologist or an academically oriented clinician. anyone who has an interest in a particular problem in physiology will most likely find the answer herein; it may not, however, be readily apparent. thus, for example, if one wanted to know the recent advances made in understanding hunger and appetite control, the information is found under "gastric storage, satiety"--not an imponderable listing, but not too "user friendly" either. certain citations are missing or given short shrift. the physiology of the gastrointestinal tract in aging is covered in just 10 pages while aids is not discussed at all, despite severe associated derangements in the intestinal immune system. the gastric circulation is discussed fully, but blood flow of the colon is not mentioned and intestinal blood flow is treated in only nine lines within the enteric neuropeptide chapter. liver disease is covered only in two chapters on bile acids and the enterohepatic circulation, both by the same expert author (alan f. hofmann): one from the hepatobiliary perspective and one from the intestinal viewpoint. perhaps here then is my most significant critique. while i enjoyed studying the text, and learned a great great deal from each and every section that i read, i found little that was directly applicable to my everyday duties. rather, i felt as if i were on vacation in a land that spoke a vaguely familiar language, but not one with which i grew up. the growth of gastroenterologic knowledge during the 20th century. by joseph b. kirsner. lea & febiger, philadelphia, 1994, 522 pp., $75.00. as one approaches a book of this type and title, it might appear to be boring and anachronistic, especially in light of the rapidly paced, high-tech nature of the specialty of gastroenterology at the present time. happily, this is not so. the book is moderately sized and reflects very nicely the devotion of the authors and their enthusiasm for the exciting and evolving knowledge in gastroenterology during the 20th century. there are 31 chapters, each directed toward the historical background, incidence, and epidemiology and an emphasis on changes that have occurred in this aspect of the specialty during the past century. perhaps the initial response by a reader is of the usefulness of this book as background material in case one must give a talk relating to one of the subjects covered in the book. in addition to a very nice historical review of the subject covered, there are photographs and historical anecdotes relating to the specific field or disease. it is obviously not possible to review each contribution, but a number of highlights can be emphasized. in the section "history of acid, peptic disease," basil hirschowitz takes us from bismuth to billroth to black and back to bismuth. he provides a sharply focused review of peptic ulcer disease, including a review of the natural history and current status. likewise, the section "esophageal malignancies and premalignant conditions" is well written and well referenced and goes through the evolution of the concept of premalignant conditions. likewise, dr. kirsner, himself, describes the historical antecedent of inflammatory bowel disease including the contributions of crohn, bargen, and the evolution of our understanding of the diseases. it does seem, however, that the chapters, "gastrointestinal defenses against injury and inflammation" and "immunology of the gastrointestinal tract," should have been incorporated or integrated more along with the inflammatory bowel disease chapter. the chapter by dr. dienstag, "viral hepatitis," and by dr. lieber, "pathogenesis of cirrhosis," linked well with each other. chapters on important diseases and symptoms, appendicitis, diverticular disease, intestinal gas, and irritable bowel syndrome, were all well developed and likewise provide good background particularly if one were to lecture on one of these topics. one of the most interesting review chapters was by dr. starzl--"contribution of transplantation'--as this would certainly appear to be a subject for the immediate future and this serves as a very nice background for it. on the other hand, the chapters on gastrointestinal endoscopy and gastrointestinal radiology appear to be somewhat dated, undoubtedly because of their rapid and continuing evolution. nevertheless, the text is well written, well edited, and lends itself nicely to the reading of an individual chapter at a specific time (rather than reading the book from cover to cover, unless one's curiosity is piqued). in addition to the obvious historical aspects, which are covered very nicely, one can see somewhat into the future, by persons who are expert, knowledgeable and experienced, and it wilt certainly be of interest to see whether the predictions come to pass. the book seems to be particularly valuable for gastroenterologists in a teaching situation and also for internists, surgeons, and nurses who work in gastroenterology to illustrate how events of the past have led to the evolution and development of current activities and how these might lay the cornerstone for the future. for this reason, a hospital library would very likely wish to have a copy of this book as well. washington, dc this relatively small book is well presented. the organization is good and the imaging illustrations appear to me to be excellent, in contrast to the few histology illustrations, which are not good. there are 18 international contributors to nine chapters. eleven of the 18 contributors are radiologists experienced in ultrasound. the first five chapters are each written by radiologists specializing in ultrasound and are typically descriptive. the clinical correlations make up the differential diagnosis of the observations, which are extensive and well presented. the experienced clinician (and pathologist) will find fault with some of the statements made concerning clinical manifestations and presentations of disease but that would miss the more important points in the presentations. those doing diagnostic ultrasound will, i think, find these five chapters on the cutting edge of diagnostic techniques. the interest of the gastroenterologist lies in the last four chapters. these are written by gastroenterologists who are developing the techniques of intraluminal endoscopic ultrasonography, a topic that has captured the attention of a large number of clinicians. the presentation of this diagnostic method is, of course, descriptive but is appropriately conservative. nevertheless, it conveys the excitement and the promise of this new diagnostic tool. those gastroenterologists who are interested in getting into the field will profit from careful perusal of these four chapters. the cost of the book is a bit steep but is reasonable in view of the approximately 240 figures, an expensive process. i can recommend it to those who are interested in the field and suggest that it should be in institutional medical libraries for those who simply want to learn more about gastrointestinal ultrasound. jackson, mississippi in many ways, alcoholic liver disease is the final frontier of liver transplantation. with an estimated one to two million persons having alcoholic cirrhosis in the united states and a scarcity of donor livers limiting the number of liver transplants to about 3000 annually, the potential for patients with alcoholic liver disease to overwhelm the system is substantial. until recently, a lid was kept on the problem by the perception that results of liver transplantation in patients with alcoholic liver disease were significantly worse than those for patients with nonalcoholic liver disease. however, this conclusion has proved to be erroneous, and several centers have recently reported comparable results in carefully selected patients with alcoholic liver disease and those with nonalcoholic liver disease undergoing liver transplantation. this monograph by dr. lucey and his colleagues is therefore timely. drawing on their interest in and experience with liver transplantation in patients with alcoholic liver disease at the university of michigan and reviewing the literature carefully and critically, they have put together an elegant summary of the state of the art. in seven concise, beautifully orga-nized, and clearly written chapters, they explore important aspects of alcoholic liver disease as it relates to liver transplantation, including diagnostic difficulties, psychiatric assessment, medical assessment, peritransplant management, psychiatric follow-up, and the ethics of liver transplantation in alcoholics. by focusing on the issue at hand and ignoring related but peripheral matters that can be found in standard textbooks, such as the pathophysiology of alcoholic liver disease, the authors have produced a tightly knit and practical guidebook that can be read easily in an evening and that will be of enormous use to anyone with an interest in alcoholism, liver disease, or liver transplantation. a particular strength of this book are chapters by dr. beresford, a psychiatrist, on the diagnosis of alcoholism and the psychiatric assessment of alcoholic candidates for liver transplantation. with a scope that is worldly and perceptive, dr. beresford provides insight into the complexities and frailties of human behavior that underlie alcoholism. sprinkling his discussion with numerous enlightening case studies and clinical vignettes, he expertly discusses the difference between alcohol abuse and alcohol dependence, positive and negative risk factors for abstinence, the value of administering a pledge to an alcoholic, difficulties in confirming abstinence, and the important distinction between denial and ambivalence in the alcoholic's perception of his dependence. these discussions will be particularly enlightening to physicians who focus primarily on the medical aspects of alcoholic liver disease. a chapter by dr. lucey provides similarly perceptive insights into the medical aspects of liver transplantation in the alcoholic. he points out, for example, that, despite initial concerns, alcoholic cardiomyopathy has proven to be a less significant problem in patients undergoing liver transplantation than anticipated. in some cases, one may quibble with the opinions expressed by the authors. for example, prophylactic variceal band ligation in patients with varices that have not bled is dismissed, perhaps too hastily, as ineffective by extrapolation of studies of prophylactic variceal sclerotherapy. nevertheless, one must admire the forthrightness of the authors and their willingness to take a stand and defend it on virtually all relevant issues. their recommendations are often empirical, though always reasoned. the problem, of course, is that the data base is quite limited. even with their particular interest in alcoholic liver disease, they report on only 98 transplants in 90 alcoholics, and their principal study on assessing candidacy for liver transplantation is based on only 99 alcoholics, of whom 54 were deemed unsuitable for liver transplantation and in many cases lost to follow-up. still, this book is a superb introduction to a topic that is at the forefront of liver transplantation. moreover, the issue of liver transplantation in alcoholics is likely to become more contentious with time as greater constraints are placed on health-care expenditures. fittingly, the book concludes with a thoughtful consideration of the ethical aspects of liver transplantation in the alcoholic patient by drs. martin benjamin and jeremiah turcotte, who point out that it is no longer justifiable to exclude alcoholics from liver transplantation in the alcoholic will require considerable more study, debate, and thought for years to come. this book is an excellent start. lawrence s. friedman, md in their foreword to the 5th edition of margulis and burhenne's alimentary tract radiolog}" drs. alexander r. margulis and h. joachim burhenne state that their philosophy in the development of their textbook has been to create a resource that will stand as the "international cutting edge final word on alimentary tract radiology." this latest edition of the most comprehensive general textbook on radiology of the gastrointestinal system succeeds in its mission and proves itself a fitting successor to its four predecessors. the editors of the 5th edition, drs. patrick c. freeny and giles w. stevenson, have enlisted the assistance of 152 contributors from 11 countries in north america, europe, and asia. these men and women bring to their task a breadth of knowledge and experience in the field of gastrointestinal imaging that is remarkable in its scope. the textbook consists of 2154 pages organized into two volumes with 16 major subdivisions and 112 chapters. volume 1 presents sections on the history of alimentary tract, radiology, general anatomy and physiology, the hollow viscera, and miscellaneous disorders of the gastrointestinal tract, including infections and infestations, gastrointestinal manifestations of the acquired immunodeficiency syndrome, and the diagnosis and therapy of gastrointestinal hemorrhage. volume 2 deals with the solid viscera, the biliary tract, the peritoneum and retroperitoneum, the pediatric alimentary tract, interventional abdominal radiology, the acute abdomen, and abdominal trauma. the appropriate imaging procedures for the diseases specific to each organ--including plain films, fluoroscopically guided contrast examinations, ct, mri, ultrasonography, scintigraphy, arteriography, and endosonography--are discussed and illustrated. among the interventional techniques that are covered are: extracorporeal shock-wave lithotripsy; endoscopic intervention in calculus disease, chronic pancreatitis, and biliary duct neoplasm; percutaneous intervention in benign and malignant biliary disease; percutaneous biopsy of abdominal masses; percutaneous abscess drainage; percutaneous fluoroscopically guided gastrostomy; and therapeutic embolization for spontaneous gastrointestinal hemorrhage and hemorrhage after trauma. the text is supplemented with 46055 well-chosen illustrations of very good to exceptional quality. the index is 74 pages long and has detailed crossreferencing. this excellent textbook sets the standard against which other references devoted to radiology of the entire alimentary tract must be measured. it should be in the library of all gastrointestinal radiologists, all general radiologists performing gastrointestinal procedures, and all interventional radiologists. it is highly recommended to gastroenterologists. the book is expensive, but its cost is comparable to other radiology texts of similar magnitude. because of its expense, however, and the presumption, stated in the editors' preface, that a major revision will be required in four or five years, residents and fellows are advised to postpone purchase of this textbook until the completion of their training. this tenth monograph in the infectious disease and therapy series details scientific advanced concerning the etiology, epidemiology, pathophysiology, molecular biology, and immunology of viral agents causing diarrheal illness. the volume combines some basic reviews, such as chapters on the pathophysiology of viral diarrhea and gastrointestinal tract immunology, with detailed exploration of more focused topics, such as rotavirus proteins. this hardbound book is handsomely illustrated in black and white with excellent quality print. there are excellent photomicrographs of gastrointestinal tract histopathology and superb electron micrographs of various viruses which, in addition to some of the protein profiles and dna studies, are the outstanding features of this book. as seems inevitable in books authored by multiple contributors, the chapters varying quality and detail. particularly praiseworthy are the chapters on the natural history of human rotavirus infections and the rotavirus proteins, the latter providing fine detail with methodical approach and the most current references in the book. the chapter on enteric adenoviruses is also very current and covered many aspects of viral molecular biology including protein profiles, restriction fragment length polymorphisms, amino acid sequence comparisons, and genetic maps. the norwalk and norwalk-like viruses chapter provides an excellent synthesis of the literature with references dating into 1993 and contains a notably well-presented discussion of acquired immunity to these agents. rotavirus enthusiasts will be pleased to find at least 43% of the text devoted to this viral agent, including discussions of the natural history of human infections, viral proteins, detection assays, animal and non-group a rotaviruses, and vaccine-related issues. the section on jennerian approaches to vaccine development deals exclusively with rotaviruses. the chapter is well written but too brief and may surprise some readers by its failure to even mention live polio vaccines as an example and/or prototype to round out this introductory chapter. subsequent chapters cover current rotavirus vaccines that use animal viruses as possible human vaccine candidates. these chapters were informative, particularly the more up-to-date discussion of rhesus rotavirus-based human vaccines. in addition to rotaviruses, other viruses highlighted include norwalk agents, enteric adenoviruses, caliciviruses, toroviruses, and coronaviruses (human and animal). while interesting and one of the more clinically applicable sections of the book, the inclusion of a chapter on bacterial diarrhea in a volume dedicated to viral gastroenteritis is somewhat puzzling. similarly, the final chapter, which deals with the treatment of (predominantly bacterial) diarrheal diseases, may strike some readers as more of an afterthought than an integral part of a text focused on viral infections of the gastrointestinal tract. while this book has certain outstanding features, there are some shortcomings which warrant mention, most notably a paucity of references beyond 1990 in some chapters and the failure to recognize viral pathogens which, through gastrointestinal tract infection, cause significant disease other than diarrhea. some readers may be disappointed to find the enteroviruses (eg, poliovirus, echovirus, coxsackievirus) simply listed in a single table with no further recognition or discussion. hepatitis a virus is neglected altogether. human immunodeficiency virus receives short shrift in a single paragraph discussing immunology of the gastrointestinal tract. in all fairness, these omissions likely reflect a deliberate focus on viral gastroenteritis rather than an oversight implied by the broader scope of the book's title. perhaps a more descriptive title would be "viral gastroenteritis." the book is weighted toward animal models and should appeal to those interested in research concerning diarrheal disease caused by viral agents. clinical applications are limited, but the volume could be a valuable reference resource for physician-scientists with a strong interest in viral molecular biology. this atlas is written by a panel of national and international experts in the field of gastrointestinal motility. the book is divided into two sections. the first deals with the basic physiology and the available technology to study gut motility. this section reviews thoroughly various manometric devices as well as the different tests available for the clinician to study gut motility. these include the radiopaque markers and hydrogen breath tests used for large and small bowel transit measurement, respectively. scintigraphy, ambulatory ph monitoring, and ultrasound of the anal sphincter are also covered. the second section of the book deals with the clinical use and application of these tests in the clinical practice of gastroenterology. the effects of sleep and stress are detailed in two chapters. the final chapter in the book is devoted to the therapeutic aspects of gastrointestinal motility, including biofeedback training for fecal incontinence. this atlas is well balanced with enough text and extensive illustrations to help the reader understand the various technological aspects of studying gut motility. the numerous photographs of actual tracings and machines as well the different schematic repre-book reviews sentations made this valuable reference quite appealing and use friendly. this is a major strength in a book that deals with several topics not frequently covered in the basic training of a gastroenterologist. each chapter is preceded by a table of contents, which facilitates looking up a certain point. the bibliography that follows each chapter is both comprehensive and largely up to date. the size of the atlas is appealing, making it useful as a quick reference to the various tests described. as more tests of gastrointestinal motility are moving out of the confines of research laboratories and into our daily practice of gastroenterology, clinicians will find this atlas a helpful source to understand both the technological aspects of each test and their applications. gastroenterology fellows in training will also appreciate the wealth of diagrams and motility tracings in this atlas. technicians working in gastrointestinal motility laboratories will find this atlas invaluable for their daily practice. endoscopy is becoming only one of several tools made available for the clinician to tackle the complexity of gastrointestinal diseases, and this atlas is a valuable addition to the library of every gastroenterologist. hussein m. el-newihi, md jackson, mississippi clinical gastroenterology, 4th ed. edited by howard b. spiro. mcgraw-hill, new york, 1993 , 1235 pp., hardbound, $141.00. since the first edition of dr. howard spiro's, clinical gastroenterology, generations of physicians (today referred to as health-care workers) have learned from it the pearls and realities of gastroenterologic practice. as more editions were forthcoming, more and more pages illustrated the seemingly unsoundable depths of dr. spiro's erudition and fatherly advice. and now, dabit qui didit, for like the professional fundraiser's maxim, "he who has given, will give." once again, dr. spire has reached down into the charitable pockets of his experience and come up with a hearty 1200 plus pages of instinct, anecdote, and guidance, laced with a healthy dose of physiology, sophisticated diagnostics, and current nostrums--all the while, cautioning us "to mediate between the images and the patient" and "to listen to the story." hence, the beauty of this book. despite dr. spiro's need to enlist the aid of nine colleagues to write about half of the text, it still is flavored with redux of spiro. the book reads like the lyric of a storyteller whose main character is still the patient--with the patient's narrative woven into a clinical tapestry rich with pearls of wisdom and edged with science. ten parts, 57 sections, 332 divisions, countless subsections (at least for this review) and 1235 pages make up the volume. it is comprehensive, including the hollow viscera as well as the solid organs, adult diseases and congenital/metabolic disorders. the book does not pretend to be encyclopedic, and thus differs from the multivolume text in gastroenterology; nor is it a reference work written in the third person singular to maintain scientific objectivity. rather, it is to a large extent a personal approach to the practice of gastroenterologic medicine by a master--nuts and bolts with some chrome plating. illustrations are mainly roentgenographs, with black and white pathologic specimens. endoscopic photos are sequestered in a section inserted halfway through the volume. the latter galley is colorful and runs the gamut of common pathology seen throughout the gut. unfortunately, almost half of the photos are oriented upside down, not diminishing their teaching value, but distracting the purist's eye. reviewers are expected to criticize, for that is part of the job description. yes, the text paper is somewhat thinner than the weight i enjoy feeling as i languish over the text; yes, i wish there were specific sections on 1-1. pylori, so i did not have to search frantically in different sections to get a complete discussion of the organism; i would have liked a comprehensive discussion of aids and the gastrointestinal tract, rather than a passing reference to microsporidium, one of the most common causes of infectious diarrhea in these patients; and i wish there were a softening of such absolutisms as "cmv esophagitis does not occur in an immunocompetent host" or that "mesenteric angiography is of value (in bowel infarction) only to exclude the possibility that the superior mesenteric artery needs to be bypassed or cleaned out..." these and other minor carpings notwithstanding, i accept some "dogma"--even if i believe it to be wrong--from this great educator. at $141.00, the book is somewhat pricey, but i would pay that for the wisdom in the introduction alone. a great book for house staff, fellows, and young attending physicians who were not fortunate enough to grow up with the first three editions and those who rely too much on the data rather than on the patient's story. new york, 1994, hardbound, 205 pp., $49.50 . in a number of ways research with human subjects is more difficult than animal or basic research. if the project is well constructed, it usually takes two to five years to gather the data, perform a careful analysis, write the paper and, finally, see it in print. while molecular biology and other basic research methods have provided understanding of physiology and pathophysiologic processes, therapeutic clinical research must provide the final step in human applications. funding for clinical research has received short shrift in recent years. in some part, this is probably related to a dearth of well-trained md researchers. this small offering is based on seminars conducted at the university of texas southwestern medical school in dallas. its aim is to provide the basic principles of clinical research for those interested in the field. it addresses guidelines on possible conflicts of interest, informed consent, protocol preparation, and research grant applications. especially valuable are chapters 5 and 6, which cover various study designs and appropriate statistical approaches. these two chapters alone, comprising only 67 pages, justify the price. this book fills a need in all advanced training programs. it is intended to provide the basic needs and, if carefully read, serves its purpose admirably. in addition, it can serve every postgraduate as a source of reference when analyzing the clinical research published by others. i recommend it highly; for $49.50 it's a real bargain. it is a mere 15 years that the first textbook on the exocrine pancreas, by howat and arles, was published. since then, numerous books, monographs, proceedings of meetings and two international journals devoted solely to the pancreas have appeared. the present book, renamed simply the pancreas, from its former title, the exocrine pancreas, is clearly the most comprehensive text on the organ currently available. the addition of a chapter on endocrine-exocrine relationships of the pancreas in the new volume has all but perfected the book, making it impressive in content and range of subjects covered as well as size. whereas most previous texts have tended to have a clinical bent, the sections in this book relating to the biology of the gland, experimental models, and endocrine-exocrine relationships comprise 729 of the 1137 pages. these include the gross and fine developmental and comparative anatomy of the gland cell and molecular biology, gene expression of protein processing receptors, and electrophysiology of the acinar cells. hormonal and neural control of enzyme, lithostatine, and mucous secretion are all considered in detail, while experimental procedures encompass not only animal models but the cell and molecular biology of cell injury. current concepts of the enteroinsular axis, insulin-acinar relationships, â�¢ and the endocrine changes encountered in protein insufficiency are fully explored. the clinical sections, while more standard fare, have been revised and updated, and one would be hard pressed to find any subject of clinical pancreatology that has not been covered. one omission that may be mentioned is that it contains very little information regarding the use of endoscopic ultrasound in diagnosing pancreatic lesions, one of the rapidly growing areas in pancreatology today. both the medical and surgical aspects of pancreatic cancer as well as endocrine tumors are fully considered, and the penultimate chapter on congenital and hereditary diseases are particularly informative as they are rarely considered in detail in more recent books on the pancreas. needless to say, the editors have chosen authors who are household names in pancreatology with lives steeped in the biochemical or clinical aspects of the gland, and the list of contributors, of which there are 83, is as impressive as the book itself. despite the formidable scientific content of many chapters, the book is highly readable but has geared itself more to scientists and pancreatologists than general readership. it is just this wide net of content and expertise that makes it virtually essential in every library and in gastroenterological research and clinical departments. the quality of the text, the reproduction of the x-rays, figures, and tables are excellent, with ample bibliography and indexing. unfortunately, as the book became heftier, so did the price, which hopefully will not compromise access to it. simmy this book is designed as a review of the motor disorders of the gastrointestinal tract. it is divided into two sections. the first section consists of a series of chapters by an impressive collection of experts on gastrointestinal motility disorders, basic biochemistry mechanisms, and an overview of neuropeptides and pharmacology. the sequence of chapters then begins in the oropharynx and extends through to the rectum including discussion of pain management as well as psychiatric therapy for symptoms from gastrointestinal motor disorders. the second part of the book is a transcript of a series of interactive sessions. these sessions were conducted at the 1992 aga postgraduate course on gastrointestinal motility. the data were obtained using a computerized audience interactive system. the audience response and transcription of the panel discussions are provided. the chapters are well organized and presented in a logical sequence. they seem to follow the lecture format from the aga postgraduate course. the first three chapters discuss motility in general along with neuroregulation. this is, by necessity, an overview of those areas and adequate supplemental readings are suggested. a brief section on pharmacology then follows. once again, adequate references are provided for those who desire more in-depth study, the next several chapters begin at the oropharynx and proceed through the gastrointestinal tract, presenting a discussion of the motility of each area. the second half of the book is devoted to the question and answer sections from the postgraduate course. the panel discussions are interesting, although frequently not in-depth. it would have, perhaps, been better to have the moderator summarize the comments in more of a generalized statement. the editors and the american gastroenterological association should be congratulated for compiling the topics from their postgraduate course into this very useful book. it is a much more usable reference than the lecture handout guideline that was presented at the time of the course. one hopes similar efforts are forthcoming from subsequent aga postgraduate courses. i would recommend this book to gastroenterologists seeking an overview of motility and to gastroenterological trainees to provide a core of knowledge to assist in their training to assist in their as well as prepare for their board examination. the price is listed at $74.95 which is somewhat high for a 247-page paperback book but can be justified by the expertise of the individual chapter authors acute pancreatitis: diagnosis and therapy. edited by e. l. bradley. raven press, new york, 1994, 294 pp., $131.50 .this excellent book is a summary of the atlanta international symposium, the presentations of 40 experts who assembled in atlanta, georgia, in the fall of 1992 to discuss acute pancreatitis and its complications. these contributors represented six medical disciplines and 15 countries. about half were pancreatic surgeons. the book is organized based on the atlanta classification and divided into sections, such as severe acute pancreatitis, necrotizing pancreatitis, pancreatic abscess, acute pancreatic fluid collection and pseudocysts, and mild acute pancreatitis. each section has multiple authors. professor bradley has most succinctly written the last section, which is the summary.since each chapter is written by an international expert, there is variability in style and quality. dogmatic and controversial opinions are evident. the majority of authors are surgeons. these merely reflect the fact that it is basically a symposium proceedings.many chapters are excellent. these include the chapter on the atlanta classification by bradley, pathology of severe acute pancreatitis by kloppel, and contrast-enhanced ct by balthazar. internists and generalists would benefit from following the concepts put forward by barkin's group about medical therapy of severe acute pancreatitis. the whole section on the important topic of pancreatic abscess is outstanding. in general, the figures and tables are clear and of high quality.this is a fine monograph on an important topic. it should be of value to generalists as well as specialist in fields such as medicine, surgery, radiology, and emergency medicine--indeed all who encounter patients with acute pancreatitis. it covers all the relevant areas related to acute pancreatitis and so should be purchased by all libraries as a single authoritative reference source.eapen thomas, md johnson city, tennessee key: cord-290412-m6fesoyb authors: zhao, chang-qing; zhou, yang; ping, jian; xu, lie-ming title: traditional chinese medicine for treatment of liver diseases: progress, challenges and opportunities date: 2014-09-30 journal: journal of integrative medicine doi: 10.1016/s2095-4964(14)60039-x sha: doc_id: 290412 cord_uid: m6fesoyb abstract traditional chinese medicine (tcm) is commonly used in treating liver diseases worldwide, especially in china. the advantages of using tcm for treatment of liver diseases include: protecting hepatocytes, inhibiting hepatic inflammation and antifibrosis in the liver. in this article, we introduce tcm herbal preparations from the chinese materia medica (such as fuzheng huayu) that are typically used for the treatment of liver diseases. literature surrounding the mechanisms of tcm therapy for treatment of liver diseases is presented and discussed. we propose that side effects of herbal compounds are often under-appreciated, and that more care should be taken in the prescription of potentially hepatotoxic medicines. further, to deepen the understanding of tcm mechanisms, new techniques and methodologies must be developed. future studies will lead to the enhancement of clinical outcomes of tcm. as complementary and alternative therapies, tcms will play an expanding role in the future of liver disease treatment. in china, traditional chinese medicine (tcm), especially traditional chinese patent medicine, has been, and continues to be widely used to treat various diseases. even during the severe acute respiratory syndrome (sars) outbreak in 2003, tcm, combined with western medicines were used to control and eventually halt the spread of the disease. compared with western medicine alone, patients receiving treatment with western medicine and tcm had reduced hospital stays, pneumonia duration and mortality. early tcm treatment can also decrease glucocorticoid dosage needed in the treatment of sars [1] . before western medicine was introduced into china, the chinese health care system mainly depended on tcm. although tcm does not treat specific conditions, it treats patterns of illness such as those associated with infectious diseases [2] [3] [4] , cardiovascular and cerebrovascular diseases [5] [6] [7] [8] [9] [10] [11] , respiratory diseases [12] [13] [14] , digestive diseases [14] [15] [16] , urinary diseases [17] [18] [19] , reproductive diseases [20] and blood system diseases [21] , as well as fractures [22] , trauma [23] , ear, nose and throat diseases [24, 25] , skin diseases [26] and mental disorders [27] . tcm can improve the clinical symptoms, reverse some pathological changes and restore traditional chinese medicine for treatment of liver diseases: progress, challenges and opportunities the body's normal physiological function. since western medicine was introduced into china in the 16th century ce, most diseases listed above are treated mainly with western medicinal interventions. gradually, tcm has become an alternative medicine rather than mainstream medicine. even so, tcm therapy still has its advantages in some medical fields where western medicine has not been as effective, such as in liver diseases. this review will introduce tcm in the treatment of liver diseases. liver diseases are mainly classified into viral hepatitis, nonalcoholic fatty liver, alcoholic liver disease, autoimmune liver disease, schistosomiasis liver disease, drug-induced liver injury, hereditary liver disease, liver cirrhosis due to various causes and diverse liver tumors. tcm is widely applied in the treatment of liver diseases in china by both chinese medicine doctors and western medicine doctors because its ability to protect hepatocytes, inhibit hepatic inflammation and reduce fibrosis in the liver. in recent years, the application of tcm in liver cancer treatment has been increasingly widespread [28, 29] . it has been confirmed that tcm can not only reduce the toxic side effects of chemotherapy or radiotherapy, but also inhibit tumor growth and increase survival of patients with tumors [30] . although tcm has many uses in treating liver diseases, it cannot replace other treatment methods such as antiviral drugs, hormones, schistosomicide, surgical operation and transplantation. tcm can be applied to treat diseases in one of the two ways: treatment based on disease differentiation or syndrome differentiation [31] . for the disease differentiation approach, western medicine methods are typically employed to diagnose specific liver diseases. subsequently the appropriate tcm formula or patent drug is selected to treat the disease according to tcm's characteristics and advantages. the evaluation of the curative effect is based on the recovery of liver function or improvement in pathological changes. in the syndrome differentiation approach, tcm diagnosis of a patient's symptoms and signs is used to determine to which syndromes the patient belongs. the appropriate tcm formula or patent drug is then chosen to treat that tcm syndrome. the evaluation of the curative effect depends on the relief or elimination of the symptoms and signs. it is believed that the combination of two kinds of therapies can obtain greater curative effects for liver diseases [32] . 3 chinese materia medica is frequently used to treat liver diseases in acute stage of liver diseases, liver inflammation is prominent. materials listed in the chinese materia medica, especially those for heat-clearing and detoxifying, are often applied to protect the liver, inhibit inflammation, decrease activity of serum transaminase and reduce serum bilirubin [32] . meanwhile according to the symptoms and signs of each patient, a matching therapy, such as adjusting yin and yang, invigorating qi and blood, soothing the liver, regulating qi, clearing heat and removing dampness, is also applied. in the chronic stage of liver diseases, the symptoms of the disease are more complicated. treating the source of the disease (i.e., the virus) is one important and necessary approach. western medicine does well in inhibiting the viruses that cause hepatitis b (hbv) and hepatitis c (hcv). tcm has little effect in inhibiting the virus, but works well to protect liver function, inhibit inflammation, decrease activity of serum transaminase, reduce serum bilirubin, lower lipid levels [33] , promote diuresis [34] and relax the bowels [35] . depending on the stage of liver disease progression, or different syndrome classifications, tcm can be used to adjust yin and yang, invigorate qi and blood, sooth liver, regulate qi, clear heat and remove dampness [36] [37] [38] . zhang et al [39] summarized and provided a critical meta-analysis of randomized controlled trials (rcts) of tcm formulations for the treatment of chronic hepatitis b (chb) that were reported in china from 1998 to 2008. the results showed that (i) tcms (tcm formulations alone or in combination with interferon (ifn) or lamivudine (lam)) had a greater beneficial effect than ifn (p=0.000 3) and slightly better effect than lam (p=0.01) on normalization of serum alanine aminotransferase; (ii) tcms had a similar beneficial effect on antiviral activity when used in conjunction with inf or lam for chb, which was evidenced by the reduction of serum hbeag and hbv dna; (iii) treatment with tcms in conjunction with inf or lam resulted in improved liver function. when the liver tissue is damaged, its repair is accompanied by the formation of an extracellular matrix, also known as fibrosis. fibrosis is the common pathological process of many liver diseases, and is also reversible. antifibrotic effects are an important component in the treatment of various chronic liver diseases [40] . one famous hepatologist, professor hans popper, once said, "anyone who can stop or delay liver fibrosis would be able to cure most chronic liver diseases" [41] . the focus of western medicine scientific and medical research has been on discovering targets for antifibrotic therapy, and developing customized multi-drug regimens [42] . according to the tcm theory, diseases of liver zang will transmit to the spleen zang, thus in the course of treatment, the spleen zang should be strengthened before it is impaired. if the liver disease has been long-standing, kidney yin should be evaluated during the treatment because the liver zang and kidney zang are derived from the same source. in clinical practice, symptoms and signs of spleen-qi deficiency journal of integrative medicine www.jcimjournal.com/jim are frequently seen in patients with liver diseases. they always complain of fatigue, pain or weakness in the legs, abdominal fullness, right upper quadrant discomfort or pain, loose stool, pale tongue or swollen tongue (teeth-marked tongue) with whitish fur and weak pulse. patients with chronic liver disease additionally present symptoms and signs of kidney-yin deficiency, such as dry mouth, internal-heat, red (or red and dry) and uncoated tongue, and weak pulse. according to the theory of syndrome differentiation therapy, methods of invigorating spleen-qi and nourishing kidney-yin should be used to treat liver diseases. in tcm, the pathogenesis of liver diseases does not necessarily relate to the liver zang, it can also be related to the spleen and kidney. table 1 shows tcm functions matched with herbs and dosages commonly used in the treatment of liver diseases. herbs with different functions are selected according to syndrome differentiation. dosages within the recommended range are determined by the severity of the symptoms and signs. several patent drugs (chinese herbal formulas) for treatment clinical observations showed that fzhyc can effectively improve liver function and decrease the expression of fibrosis biomarkers such as serum hyaluronic acid, collagen type iv, procollagen type iii and laminin, in chronic liver disease patients with fibrosis or cirrhosis [43, 44] . fzhyc can also regulate immune function [45] , balance amino acid [46] and endocrine [47] metabolism and reduce portal hypertension [48] . the results of several multicenter rcts have confirmed that tcm can reverse liver fibrosis [49] [50] [51] . fzhyc was used to treat liver fibrosis in patients with chb for six months. fifty patients in the trial group were treated with fzhyc. a control group of 43 patients was treated with heluo shugan capsule, another chinese patent medicine. all patients received liver biopsies pre-and post-treatment. pathology results showed that in the treatment group, the stage of fibrosis was decreased by one or more in 52% of the patients; the remaining 48% of the patients in the trial group had no changes. however, in the control group only 23.3% patients had a decrease in fibrosis stage, 55.8% patients had no change and 20.9% of the patients had an increase in fibrosis stage [49] . in another study, fzhyc reversed the fibrosis at a rate of 57.9% early cirrhotic patients with chb [52] . to prevent esophageal variceal bleeding in cirrhotic patients, a multicenter randomized and placebo-controlled trial was carried out. the probability of survival in the fzhyc group was higher than that in the propranolol group (90.22% vs 70.92%, p=0.044 9). compared to the propranolol group the probability of esophageal variceal bleeding in the fzhyc group was significantly reduced (43.0% vs 23.9%, p=0.013 1). when the two treatments were taken together there was an even lower probability of bleeding which was also significantly lower than the propranolol alone (12.4% vs 43.0% p=0.008 6). in patients with small esophageal varices, treatment with fzhyc reduced the size of the varices. its effects may be related to the prevention of hepatic fibrosis, amelioration of liver function, and the decrease of et-1 concentration in the blood plasma [53] . a meta-analysis was conducted to evaluate the efficacy and safety of fzhyc combined with nucleoside antiviral drugs in treating fibrotic patients with chb. the analysis included seventeen rcts, with a total of 1 320 patients with chb, of which 636 were in control groups and 684 in trial groups. the meta-analysis showed that there was no significant improvement in serum hbeag level and hbv-dna copies. however, there was a statistically significant improvement in liver fibrosis and liver function after treatment for 24 or 48 weeks [54] . for the past six decades, many researchers have carried out extensive research to explore the mechanism of tcm in the treatment of liver diseases. it was found that tcm can improve hepatic microcirculation, scavenge oxygen free radicals, resist lipid peroxidation, promote bilirubin metabolism, accelerate synthesis of liver glycogen and protein, and increase the content of liver microsomal cytochrome p-450 [55] . these effects lead to a decrease in hepatocyte necrosis, inhibition of apoptosis and promotion of the hepatocyte regeneration [56] . we searched the literature for studies exploring the mechanism of fzhyc's antifibrotic activity. it has been shown that fzhyc can protect hepatocytes, resist lipid peroxidation and inhibit some cytokines [57, 58] . fzhyc can also inhibit the activation and proliferation of hepatic stellate cells (hscs), which play an important role in hepatic fibrogenesis and fibrosis [58] . fzhyc can also promote apoptosis of activated hscs, inhibit synthesis and secretion of collagen, inhibit angiogenesis and promote degradation of collagen [59] . studies showed that fzhyc achieved its antifibrotic activity through multiple signal pathways and targets [57, 58, [60] [61] [62] [63] , such as transforming growth factor β-1 (tgf-β1), smads, insulin-like growth factors-1 (igf-1), phosphatidylinositol 3-kinase (pi3k), extracellular signal-regulated kinase (erk), p38 mitogen-activated protein kinase (p38 mapk), rhoa/rho-associated coiled-coil forming protein kinase (rock) and renin-angiotensin system (ras) signaling pathways [64, 65] . the elevation of portal vein pressure is a common symptom of liver cirrhosis. the portal vein pressure is positively correlated with endothelin-1 (et-1) concentration in the liver tissue during the process of liver cirrhosis [66] . fzhyc can dramatically decrease cirrhosis-induced elevation of portal vein pressure by reducing et-1 levels in the liver tissue [67] . due to multiple ingredients in the chinese formula, the compound could not be used to incubate cells directly in vitro. only individual components or ingredients of herbal medicine are suitable for in-vitro research. to explore the underlying mechanisms of fzhyc's antifibrosis activity, we looked in the literature for research on salvianolic-acid b (sa-journal of integrative medicine www.jcimjournal.com/jim b), a component of danshen that is the main constituent herb of fzhyc. studies show that the effects of sa-b are just like fzhyc in the treatment of hepatic fibrosis and cirrhosis [64, 65, 68] . sa-b dramatically decreased the fibrosis level of rats with fibrotic liver, and also markedly decrease cirrhosis-induced elevated portal vein pressure and liver et-1 levels [16] . sa-b achieves its antihepatic fibrosis effect by inhibiting the erk and the p38 mapk pathways of tgf-b1 in hscs. it inhibits the erk pathway by inhibiting phosphorylation of mek. sa-b inhibits the p38 mapk pathway by blocking phosphorylation of mkk3/6 and inhibiting expression of myocyte enhancer factor 2 (mef2, a transcription factor), in hscs with or without tgf-b1 stimulation [69] . further, sa-b inhibits the crosstalk of the samd signaling pathway to the erk signaling pathway [63] . sa-b also significantly reduces et-1-activated hsc contractility by inhibiting rhoa/rock ii activation and the downstream mypt1 phosphorylation at thr696 [64] . although some positive effects of tcm in liver diseases are reported, we should also note the possibility of liver damage induced by some herbs during treatment. the perspective that herbs, as natural medicine, have no side effects is outdated and wrong. some chinese herbal medicines, which were used to treat liver diseases, have been reported to have hepatotoxicity in high doses or even in standard doses, such as heshouwu (polygonum multiflorum [70] . this potential toxicity should serve as a reminder that clinical practitioners should administer chinese medicine with as much caution as western pharmaceutical drugs. it is very important to take precautions against drug-induced liver injury when selecting herbs and their doses. generally speaking, applying herbs in accordance with the pharmacopeia is quite safe. for example, an aqueous solution of zhizi (gardenia jasminoides) extract, which has been reported to have hepatotoxicity, has no significant side effects on mice liver at medium dose (equivalent to 18 g dose for adults) and low dose (equivalent to 9 g dose for adults). the medium and low doses of zhizi extract did not obviously affect structure of the liver tissue or damage hepatocytes. we suggest that zhizi is not suitable for chronic use at a high dose, but short-term use at a modest dose (9 g per day) is still safe [71] . further, by appropriate preparation of herbal products, such as heshouwu, the hepatotoxicity can be reduced [72] . it is also important to note that in chinese medicine, herbs with similar names cannot be used interchangeably. for instance, using tusanqi (gynura segetum), which has severe hepatotoxicity, as a substitute for shensanqi (panax notoginseng) is dangerous, and they come from very different plants. 6 new techniques and methodologies are needed for studying tcm although tcm therapy is effective in the treatment of liver diseases, more research is required to understand the underlying mechanisms of action. at present, the pharmacological studies of chinese medicinal formulae are carried out extensively in vivo, however, the induced animal models cannot be relied on to fully mimic clinical pathogenesis of human patients. studies that examine the mechanisms behind tcm can only be conducted in vitro, are ill suited to the complex formulations of herbs used in chinese medicinal remedies and are restricted by existing research techniques and methodology [73] . current research technology is not designed to evaluate responses from multi-dimensional variables, like the herbal formulations used in tcm. this may be one of the reasons that the curative effects of tcm have been slow to receive approval among western medicinal practitioners. new research techniques and methodologies should be developed to evaluate the curative effects of tcm and to elucidate its mechanisms [74, 75] . we believe that as techniques and methodologies evolve to address the complex nature of tcm herbal formulations, a more mechanistic understanding of the use of tcm in treating liver diseases will emerge. these studies will thus lead to the improvement of clinical results and refinement of the contemporary practice of tcm. as a complementary and alternative therapy for the treatment of liver diseases, tcm is a powerful but underused tool in the present, and has great potential for future use. the authors have no conflicts of interest to declare. journal of integrative medicine (jim) is an international, peer-reviewed, pubmed-indexed journal, publishing papers on all aspects of integrative medicine, such as acupuncture and traditional chinese medicine, ayurvedic medicine, herbal medicine, homeopathy, nutrition, chiropractic, mind-body medicine, taichi, qigong, meditation, and any other modalities of complementary and alternative medicine (cam). article types include reviews, systematic reviews and meta-analyses, randomized controlled and pragmatic trials, translational and patient-centered effectiveness outcome studies, case series and reports, clinical trial protocols, preclinical and basic science studies, papers on methodology and cam history or education, editorials, global views, commentaries, short communications, book reviews, conference proceedings, and letters to the editor. • no submission and page charges • quick decision and online first publication for information on manuscript preparation and submission, please visit jim website. send your postal address by e-mail to jcim@163.com, we will send you a complimentary print issue upon receipt. effect of glucocorticoid with traditional chinese medicine in severe acute aespiratory syndrome (sars) interaction of a traditional chinese medicine (phy906) and cpt-11 on the inflammatory process in the tumor microenvironment artemisinin: discovery from the chinese herbal garden anti-infective and cytotoxic properties of bupleurum marginatum a review of the pharmacological mechanism of traditional chinese medicine in the intervention of coronary heart disease and stroke puerarin: a review of pharmacological effects potential benefits of chinese herbal medicine for elderly patients with cardiovascular diseases danqi pill protects against heart failure through the arachidonic acid metabolism pathway by attenuating different cyclooxygenases and leukotrienes b4 chinese medicine shensongyangxin is effective for patients with bradycardia: results of a randomized, double-blind, placebo-controlled multicenter trial effects of chinese herbal medicine yiqi huaju formula on hypertensive patients with metabolic syndrome: a randomized, placebo-controlled trial favorable circulatory system outcomes as adjuvant traditional chinese medicine (tcm) treatment for cerebrovascular diseases in taiwan randomized controlled multicenter clinical trial for integrated treatment of community-acquired pneumonia based on traditional chinese medicine syndrome differentiation prevention of allergic airway hyperresponsiveness and remodeling in mice by astragaliradix antiasthmatic decoction the anti-asthma herbal medicine ashmi acutely inhibits airway smooth muscle contraction via prostaglandin e2 activation of ep2/ep4 receptors clinical effects of the method for warming the middle-jiao and strengthening the spleen on gastric mucosa repair in chronic gastritis patients modified da-cheng-qi decoction reduces intra-abdominal hypertension in severe acute pancreatitis: a pilot study optimized project of traditional chinese medicine in treating chronic kidney disease stage 3: a multicenter double-blinded randomized controlled trial efficacy and safety of traditional chinese medicine (shenqi particle) for patients with idiopathic membranous nephropathy: a multicenter randomized controlled clinical trial tripterygium wilfordii hook f (a traditional chinese medicine) for primary nephrotic syndrome clinical effect of jiutengzhuyu tablets on promoting blood circulation in women with oviducal obstruction study on the efficacy and safety of xueyou mixture in treating hemophilia beneficial effects of traditional chinese medicine on the treatment of osteoporosis on ovariectomised rat models a traditional chinese medicine therapy warming meridians to nourish blood in treating chronic pain due to soft tissue injury of the neck and shoulder: a randomized controlled trial trial of chinese medicine wu-ling-san for acute low-tone hearing loss mechanism of traditional chinese medicine in the treatment of allergic rhinitis identifying core herbal treatments for urticaria using taiwan's nationwide prescription database effect of yi-gan san on psychiatric symptoms and sleep structure at patients with behavioral and psychological symptoms of dementia traditional herbal medicine in preventing recurrence after resection of small hepatocellular carcinoma: a multicenter randomized controlled trial treatment of middle/late stage primary hepatic carcinoma by chinese medicine comprehensive therapy: a prospective randomized controlled study clinical observation on the treatment of middle-late stage liver carcinoma by combined therapy of hepato-arterial chemo-embolising and chinese drugs for strengthening pi and regulating qi studies on treatment of fatty liver with traditional chinese medicine experience of treatment for posthepatitic cirrhosis based on tcm syndrome differentiation clinical study on effect of jiangan jiangzhi pill in treating nonalcoholic fatty liver diseases effects of baogan lishui decoction on patients with ascites due to cirrhosis: a report of 84 cases. guang ming zhong yi protective effect of compound tongfu granule on intestinal barrier in patients with cirrhosis of decompensation stage effects of method of regulating qi to dissipate blood stasis and phlegm with chinese herbs on expression of srebp-1c/fas in rats liver tissue with non-alcoholic steatohepatitis a prospective cohort study on the influence of high doses of herbs for clearing heat and resolving stasis on survival rates in patients with hepatitis b-related acute-on-chronic liver failure clinical observation of clearing heat and removing dampness, cooling blood and detoxicating therapy for acute on chronic liver failure in hepatitis b. beijing zhong yi yao contemporary clinical research of traditional chinese medicines for chronic hepatitis b in china: an analytical review liver cirrhosis procollagen iii levels in serum: do they provide additional information in liver disease? reversal of hepatic fibrosis -fact or fantasy? effect of fuzheng huayu 319 recipe on serological parameters of fibrosis in treating chronic hepatitis b. zhongguo zhong xi yi jie he za zhi multicenter clinical study about the action of fuzheng huayu capsule against liver fibrosis with chronic hepatitis b effect of ganping capsule on posthepatitic cirrhosis in adjusting abnormal immune function influence of fuzheng huayu capsules on abnormal amino acids spectrum in chronic liver diseases effect of fuzhenhuayu recipe in adjusting endocrine disorders in cirrhotic patient clinical research of ganping capsule against liver fibrosis in patients with chronic hepatitis b multicenter clinical study on fuzhenghuayu capsule against liver fibrosis due to chronic hepatitis b capsule oxymatrine in the treatment of liver fibrosis in patients with chronic virus hepatitis: a randomized, double blind clinical study on treatment of alcoholic liver disease by qinggan huoxue recipe results set of traditional chinese medicine basic theory research in 973 program a randomized controlled study of fuzheng huayu capsule for prevention of esophageal variceal bleeding in patients with liver cirrhosis meta-analysis on fuzhenghuayu capsule combined with nucleoside antiviral drug for chronic hepatitis b clinical frequently used chinese crude drugs with hepatoprotective effect research progress on mechanism of clinical hepatoprotective drugs effect of fuzheng huayu formula and its actions against liver fibrosis role of the igf-1/pi3k pathway and the molecular mechanism of fuzhenghuayu therapy in a spontaneous recovery rat model of liver fibrosis effects of fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells, hepatocytes and fibroblasts in rats therapeutic efficacy of traditional chinese medicine 319 recipe on hepatic fibrosis induced by carbon tetrachloride in rats curative effects of fuzheng huayu capsules on hepatic fibrosis and the functional mechanisms: a review role of jak/stat pathway in ccl 4 -induced rat liver fibrosis model and molecular action mechanism of fuzheng huayu recipe in treatment of liver fibrosis salvianolic acid b inhibits erk and p38 mapk signaling in tgf-β1-stimulated human hepatic stellate cell line (lx-2) via distinct pathways. evid based complement alternat med salvianolic acid b lowers portal pressure in cirrhotic rats and attenuates contraction of rat hepatic stellate cells by inhibiting rhoa signaling pathway salvianolic acid b attenuates rat hepatic fibrosis via downregulating angiotensin ii signaling therapeutic potential of targeting the renin angiotensin system in portal hypertension traditional chinese medicine can improve liver microcirculation and reduce portal hypertension in liver cirrhosis salvianolic acid b inhibits hepatic stellate cell activation through transforming growth factor β-1 signal transduction pathway in vivo and in vitro effect of salvianolic-acid b on inhibiting mapk signaling induced by transforming growth factor-β1 in activated rat hepatic stellate cells facing the hepatotoxicity of chinese herbal medicine observation of treatment effect and hepatic toxicity of gardenia in mouse with acute liver injury effective components, toxic effects and research advances of present situation of modernization on compound prescriptions. zhongguo shi yan fang ji xue za zhi research status, problems and thinking of multi-component of chinese medicine. zhong yi xue bao discussion on research and development models in innovative chinese material medica key: cord-305956-l02xdq87 authors: alqahtani, saleh a; schattenberg, jörn m title: liver injury in covid-19: the current evidence date: 2020-05-26 journal: united european gastroenterol j doi: 10.1177/2050640620924157 sha: doc_id: 305956 cord_uid: l02xdq87 patients with novel coronavirus disease 2019 (covid-19) experience various degrees of liver function abnormalities. liver injury requires extensive work-up and continuous surveillance and can be multifactorial and heterogeneous in nature. in the context of covid-19, clinicians will have to determine whether liver injury is related to an underlying liver disease, drugs used for the treatment of covid-19, direct effect of the virus, or a complicated disease course. recent studies proposed several theories on potential mechanisms of liver injury in these patients. this review summarizes current evidence related to hepatobiliary complications in covid-19, provides an overview of the available case series and critically elucidates the proposed mechanisms and provides recommendations for clinicians. in the current pandemic coronavirus disease (covid19) , almost every country in the world has now registered covid-19 cases, and the confirmed cases have exceeded one million to date. while initial clinical studies, especially from china, the usa and italy, have highlighted the dominant clinical symptoms including fever, cough, fatigue and shortness of breath, the later research unveiled shreds of evidence on the extrapulmonary manifestations of the disease. these reports highlighted that beyond severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a complicated course of the disease or even viral infection itself can lead to involvement of other organs and multiorgan failure. the liver is the primary organ for detoxification and metabolism, and maintaining an optimal function is imperative to engage all available therapeutic modalities in the treatment of covid-19. abnormal liver function requires clinical evaluation, continuous surveillance and, potentially, specific therapy. to support clinical decision making and optimize the outcome in the treatment of covid-19, it will be crucial to clearly understand the possible mechanisms involved in liver injury. the current review summarizes the pathophysiology and potentially specific role of covid-19 in liver disease based on the available data and case series published, ahead of print and non-peer-reviewed preprints as of 2 april. the search strategy is detailed in the supplementary material online. emerging data from small clinical case studies have proposed that liver injury in covid-19 is frequently seen, but the extent and underlying mechanisms remain undetermined. figure 1 summarizes the pathophysiological findings, which are discussed below. the liver exerts a crucial function in host defense against microbes and is involved in most systemic infections as it receives both the portal and systemic circulation. certain viruses exert a direct cytopathic effect on hepatocytes and cholangiocytes although, in most cases, the pathogenesis seems multifactorial. yang et al. reported that sars-cov could cause direct cytopathic liver injury rather than inducing cellular stress from low oxygen supplies or cytokines as seen in sepsis. 1 autopsy studies in patients revealed that sars-cov was detectable in 41% of the liver tissue, with a maximum viral load of 1.6 â 10 6 copies/g of tissue. 2 the pathological findings of liver biopsy specimens from sars patients showed hepatocellular necrosis, mitoses, cellular infiltration and fatty degeneration. in a recent autopsy analysis of liver tissue from a patient with covid-19, moderate microvesicular steatosis and mild inflammation in the lobular and portal area was observed. however, this pattern of histological injury is not specific for one etiology but can also be observed during sepsis or drug-induced liver injury (dili). 3 similar to sars-cov, sars-cov-2 uses the angiotensin-2 converting enzyme (ace2) receptor protein to attack the host system. 4 the cell entry receptor, ace2, is widely expressed across the human body, including the lungs (type ii alveolar cells), gastrointestinal tract (esophageal epithelial cells and absorptive enterocytes of ileum and colon), hepatobiliary system (hepatocytes and cholangiocytes), cardiovascular system (myocardial cells), the renal system (proximal tubule cells and urothelial bladder cells) and the pancreas. 5 recent studies have observed that ace2 expression in the cell clusters of cholangiocytes was significantly higher than that in the hepatocytes population (59.7% vs. 2.6%). 6 the authors conclude that sars-cov-2 may directly bind to ace2 positive cholangiocytes, but not hepatocytes, to exert a cytopathic effect. cholangiocytes are involved in many aspects of liver physiology, including regeneration and adaptive immune response mechanisms, and the disruption of cholangiocyte function can cause hepatobiliary damage. this is supported by cholestatic markers, including gamma-glutamyl transferase (ggt), that can be found in some, but not all, case series of covid-19. [7] [8] [9] notably, a recent review reported unpublished data with ggt elevations in 54% of cases. 8 in a human organoid model of liver ductal organoids, permissiveness to sars-cov-2 infection was observed. here viral infection impaired the barrier and bile acid transporting functions of cholangiocytes through dysregulation of genes involved in tight junction formation and bile acid transportation, supporting the susceptibility of cholangiocytes in sars-cov-2-related liver injury. 10 activation of the immune system in covid-19 dysregulation of the innate immune response can be one aspect of liver injury in covid-19. patients with covid-19 exhibit marked activation of inflammatory markers, including abnormal levels of c-reactive protein (crp), lymphocytes, neutrophils and cytokines, in particular interleukin-6 (il-6). 8, [11] [12] [13] these mechanisms may contribute to pulmonary and extrapulmonary injuries 12, 14 and the control of cytokine dysregulation at an early stage could be beneficial to curb the disease progression. 15 hepatic inflammation involving activation of innate immune cells and the release of cytokines is a well-established driver of liver injury from various causes. 16 in some of the available case series of covid-19, a correlation between lymphopenia and liver injury was observed and crp !20 mg/l and a lymphocyte count <1.1 â 10 9 /l were independent risk factors for liver injury. notably, lymphopenia in covid-19 studies was reportedly observed in 63% to 70.3% of patients and those with lower lymphocyte counts more susceptible to fatal outcomes. 11 more than 20 publications to date reported abnormal levels of aminotransferases in patients with covid-19. [7] [8] [9] [11] [12] [13] [17] [18] [19] 21, [23] [24] [25] [26] [27] [29] [30] [31] [32] [33] 35, 36 a recent systematic review and meta-analysis on lft abnormalities provided a pooled elevation of aspartate aminotransferase (ast) in 33.3% and alanine aminotransferase (alt) in 24.1% of cases. 39 various investigators across different studies reported a correlation between the severity of covid-19 and the degree of liver dysfunction. 8, 11, 25 in one retrospective study, one patient experienced severe hepatitis with alt of 7 on the other hand, mild and moderate cases experienced only discrete abnormal lft values. these reports support the concept that the disease severity and an older age predispose to more severe liver injury from covid-19. based on these case series, patients with severe covid-19 and pre-existing liver conditions 8but also elderly patients 11 -should undergo surveillance and individually tailored therapeutic approaches for potential liver injury. a recently published article by bangash et al. argues after careful review of seven relevant studies that elevated alt and ast may not necessarily be of hepatic origin alone. the authors have given a timely reminder that it is common for other respiratory viruses to create similar lft elevations 28 and thus more prospective data related to the clinical relevance covid-19 and liver injury is required. the previous pathogenic coronaviruses, such as sars-cov and the middle east respiratory syndrome coronavirus (mers-cov), were also reported to manifest with elevated levels of alt and ast. 39 more generally, non-hepatotropic viral infections may affect the liver and induce hepatitis or fulminant acute liver failure. however, in the majority of cases, recovery from viral illness is often sufficient to resolve liver injury. 40 like in sars-cov and mers-cov infections, abnormal levels of albumin and lactate dehydrogenase (ldh) were also reported in sars-cov-2 infection, with the maximum of 98% and 76% of the patients affected as reported in the study by chen et al. 17 it is important to remember that ldh and ast elevation could be from muscle damage and not necessarily reflect liver injury. the current armory of therapeutic agents explored against sars-cov-2 includes several antiviral agents, supportive therapy and trials of alternative medicines in many regions of the world. given the fact that the liver is involved in the metabolism of many drugs, including nucleoside analogs and protease inhibitors that are currently used to treat covid-19, hepatotoxicity from these drugs can arise. a recent randomized controlled trial of lopinavir and ritonavir in severe covid-19 reported that elevated levels of ast, alt and total bilirubin occurred as adverse effects in a few patients. 41 another case series from wuhan reported that 55.4% of patients experienced liver injuries after treatment with lopinavir and ritonavir. 26 fan et al. published a retrospective study on covid-19 and observed that the utilization rate of this drug combination was significantly higher in patients with abnormal lfts compared with patients without lft elevations (56.1% vs. 25%, p ¼ 0.009). in this study, 47.3% of the discharged patients showed elevated lfts at baseline, and 23.7% developed abnormalities during hospitalization, suggesting emerging liver injury from drugs or during the course of the infection. importantly, lft elevation during the hospital stay was associated with prolonged length of hospitalization. 7 chloroquine, an old drug with a potential of repositioning for new treatment indications, has recently been tried in patients infected with sars-cov-2. after a profound success in inhibiting viral replication in vitro, concurrent clinical trials (>20) on chloroquine conducted at 10 hospitals across china have demonstrated superior efficacy in viral control. 42 the pharmacodynamic activity of this drug in covid-19 may involve the arresting of cytokine storms or the activation of cd8þ cells or by preventing endocytosismediated uptake of the virus. 43 importantly, hepatotoxicity related to chloroquine or hydroxychloroquine has rarely been reported. in severe cases of covid-19 with cytokine release, tocilizumab, an il-6 antagonist, which is humanized igg1 monoclonal antibody to the il-6 receptor, has been used as a potential therapy for sars-cov-2. in previous clinical trials for other indications tocilizumab was reported to cause mild elevations of lfts which were usually transient and commonly resolved within 2-6 weeks from exposure. 37 remdesivir is an experimental antiviral nucleotide analog with broad activity against coronaviruses 44 that is currently being trialed for sars-cov-2 infection. safety data from ongoing studies will guide on its use in patients, but so far no reports of liver toxicity have emerged. patients with pre-existing liver disease scare data has been published for covid-19 infection in patients with pre-existing liver disease. experience from previous episodes of coronavirus infection can guide on the extent of hepatic involvement and on the management of patients with pre-existing liver disease. in sars, the highest mortality rates were observed in the elderly and adults with underlying liver disease. 45 therefore, it has to be expected that the patients with covid-19 are also more vulnerable to hepatic injury. 9 in a case series from the zhejiang province, a prevalence of 11% of underlying liver disease was reported. about half of them experienced symptoms for more than 10 days after the illness onset. 21 in another study from wuhan, 9% of patients had the underlying liver disease of cirrhosis or hepatitis. 23 li et al., who investigated risk factors involved with hepatic injury, stated that two patients had presented with alcoholic liver disease at baseline. one of them had a moderate elevation of alt (120 u/l) within a week of hospitalization, while the other showed no such abnormalities. 11 in the initial cohort described from china, 2.7% exhibited hepatitis b virus infection with no mention of worsening outcomes. 26 therefore, the association of the pre-existing liver conditions with disease prognosis and outcomes in covid-19 will have to be evaluated by comprehensive data registries which recently started enrolling patients (e.g. covid-hep registry and secure-cirrhosis registry). management of post liver transplant recipients during the covid-19 pandemic presents a special challenge for clinicians because of the limited data available and the crucial need to continue immunosuppressive drugs in these patients, which puts them at risk for more severe courses of covid-19 infection and possible prolonged viral shedding. case reports from china did not reveal an increased mortality in organ transplant recipients. qin et al. reported the first case of sars-cov-2 infection in a patient with hepatocellular carcinoma who underwent liver transplantation. 29 lowering immunosuppression to the most acceptable level appears reasonable in infected liver transplant patients, in particular, in the setting of lymphopenia or clinical worsening of infection. 46 in addition clinicians have to be aware of drug-drug interactions in the transplant setting. in particular immunosuppressive drugs and ritonavir-boosted antiviral therapies exhibit relevant interactions through cyp34a which lead to increased levels of calcineurin and mtor inhibitors. accordingly, chloroquine-based regimes or remdesivir (compassionate use program only) appear to be safe, while boosted protease inhibitors should be avoided (see table 2 ). additionally, preventive strategies in those vulnerable patients include early and prolonged screening with polymerase chain reaction-based testing for patients with early symptoms, a contact history or infection. personal liver function abnormalities -predominantly ast elevation -in covid-19 appear to be frequent but not severe in most cases. direct viral hepatotoxicity, dili, 'bystander effects' during a systemic viral infection and potentially sepsis, or exacerbation of an underlying liver disease have to be considered. ex vivo studies offer that sars-cov-2 can selectively target the liver, in particular cholangiocytes through ace2, and thus hepatobiliary injury appears plausible. irrespective of the mechanisms involved in the hepatic injury of patients with covid-19, activation of the immune-mediated pathway seems to be critical. special high risk populations require close monitoring. these include the elderly population, patients with endstage liver disease and liver transplant recipients. symptomatic treatment with acetaminophen and avoidance of non-steroidal anti-inflammatory drugs in cirrhosis is recommended. cautious use of antiviral agents in patients with decompensated liver disease and drug-drug interactions in post liver transplant patients has to be considered. as emphasized by a recent position paper of the european study of liver disease, elective procedures and routine tests should be postponed according to the risk-benefit at the given time. on the other hand, emergency medical care needs to be done with appropriate measures to prevent infection. 46 declaration of conflicting interests sa has nothing to declare. jms has acted independently of this study as a consultant to boehringer ingelheim, galmed, genfit, gilead sciences, intercept pharmaceuticals, novartis, roche, siemens healthineers, and has received research funding from gilead sciences. clinical characteristics and mechanism of liver damage in patients with severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus pathological findings of covid-19 associated with acute respiratory distress syndrome sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor highly ace2 expression in pancreas may cause pancreas damage after sars-cov-2 infection specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection clinical features of covid-19 related liver damage liver injury in covid-19: management and challenges liver injury during highly pathogenic human coronavirus infections. liver int. epub ahead of print 14 recapitulation of sars-cov-2 infection and cholangiocyte damage with human liver organoids risk factors related to hepatic injury in patients with corona virus disease 2019 clinical features of patients infected with 2019 novel coronavirus in wuhan, china a 55-day-old female infant infected with covid 19: presenting with pneumonia, liver injury, and heart damage covid-19 infection: the perspectives on immune responses covid-19: consider cytokine storm syndromes and immunosuppression innate immune cell trafficking and function during sterile inflammation of the liver epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of 82 death cases with covid-19 epidemiological and clinical features of the 2019 novel coronavirus outbreak in china clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical characteristics of 138 hospitalized patients with 2019 novel coronavirusinfected pneumonia in wuhan, china radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study clinical characteristics of 36 non-survivors with covid-19 in wuhan clinical characteristics of coronavirus disease 2019 in china covid-19 in a designated infectious diseases hospital outside hubei province clinical features and laboratory inspection of novel coronavirus pneumonia (covid-19 covid-19 and the liver: little cause for concern perioperative presentation of covid-19 disease in a liver transplant recipient. hepatology 2020. epub ahead of print 27 clinical characteristics of non-icu hospitalized patients with coronavirus disease 2019 and liver injury. a retrospective study liver impairment in covid-19 patients: a retrospective analysis of 115 cases from a single center in wuhan city, china. liver int 2020. epub ahead of print 2 the clinical characteristics of pneumonia patients coinfected with 2019 novel coronavirus and influenza virus in wuhan a comparative study on the clinical features of covid-19 pneumonia to other pneumonias covid-19 and liver dysfunction: current insights and emergent therapeutic strategies clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia. radiology epub ahead of print bethesda (md): national institute of diabetes and digestive and kidney diseases clinical, laboratory and imaging features of covid-19: a systematic review and metaanalysis clinical manifestations, laboratory findings, and treatment outcomes of sars patients systemic viral infections and collateral damage in the liver a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies insights from nanomedicine into chloroquine efficacy against covid-19 prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper the authors received no financial support for the research, authorship, and/or publication of this article. j€ orn m schattenberg https://orcid.org/0000-0002-4224-4703 key: cord-291851-xesef17i authors: wong, yu-jun; tan, malcolm; zheng, qishi; li, weiquan; kumar, rahul; fock, kwong-ming; teo, eng-kiong; ang, tiing-leong title: a systematic review and meta-analysis of the covid-19 associated liver injury date: 2020-08-31 journal: ann hepatol doi: 10.1016/j.aohep.2020.08.064 sha: doc_id: 291851 cord_uid: xesef17i introduction and objectives: the novel coronavirus disease 2019 (covid-19) has affected more than 5 million people globally. data on the prevalence and degree of covid-19 associated liver injury among patients with covid-19 remain limited. we conducted a systematic review and meta-analysis to assess the prevalence and degree of liver injury between patients with severe and non-severe covid-19. methods: we performed a systematic search of three electronic databases (pubmed/medline, embase and cochrane library), from inception to 24(th) april 2020. we included all adult human studies (>20 subjects) regardless of language, region or publication date or status. we assessed the pooled odds ratio (or), mean difference (md) and 95% confidence interval (95%ci) using the random-effects model. results: among 1543 citations, there were 24 studies (5961 subjects) which fulfilled our inclusion criteria. the pooled odds ratio for elevated alt (or = 2.5, 95%ci: 1.6-3.7, i(2) = 57%), ast (or = 3.4, 95%ci: 2.3-5.0, i(2) = 56%), hyperbilirubinemia (or = 1.7, 95%ci: 1.2-2.5, i(2) = 0%) and hypoalbuminemia (or = 7.1, 95%ci: 2.1-24.1, i(2) = 71%) were higher subjects in critical covid-19. conclusion: covid-19 associated liver injury is more common in severe covid-19 than non-severe covid-19. physicians should be aware of possible progression to severe disease in subjects with covid-19-associated liver injury. the novel coronavirus disease 2019 has affected more than 5 million patients globally, causing more than 300,000 death to date [1] . the symptoms of covid-19 range from mild respiratory symptoms to acute respiratory distress syndrome with multiorgan failure and death, particularly among the elderly with multiple comorbidities. extra-pulmonary symptoms such as covid-19 associated liver injury has been reported [2] [3] [4] . covid-19 associated liver injury is defined as any liver damage in patients with covid-19, with or without the pre-existing liver disease [5] . the possible mechanisms of covid-19 associated liver injury include immune-mediated damage and ischemic hepatitis secondary systemic inflammatory response syndrome in severe covid-19, drug-induced liver injury, as well as reactivation of underlying chronic liver disease [6] . besides, the direct virus-induced cytopathic effect has also been postulated as a result of viral replication within the infected hepatocytes [7] . the data on the prevalence and severity of covid-19 associated liver injury are conflicting. while growing evidence suggested a higher incidence of liver injury among severe covid-19, such findings are not consistent [8] [9] [10] . recent position statement highlighted our gaps in understanding this novel disease [11] [12] [13] . in particular, patients with non-alcoholic fatty liver disease may have co-existing comorbidities which put them at a higher risk of severe covid-19 [11] . furthermore, covid-19 associated liver injury is associated with prolonged hospitalization [6] . given this premise, it is of scientific interest to expand our understanding of the clinical outcome of patients with covid-19 associated liver injury. however, the severity of covid-19 patients was often poorly defined by including subjects ranging from tachypnea to intubation or death. as the literature in covid-19 is expanding exponentially, keeping up-to-date with scientific progress has become increasingly j o u r n a l p r e -p r o o f challenging for physicians. therefore, we systematically reviewed and summarized the existing literature on covid-19 associated liver injury among adult patients. our meta-analysis aims to compare the risks and clinical outcomes of covid-19 associated liver injury among adults with severe and non-severe covid-19. the objective of this meta-analysis is to compare the risk and clinical outcome of covid-19 associated liver injury between covid-19 patients with severe and non-severe covid-19. we included all adults with covid-19 associated liver disease, regardless of their underlying chronic liver disease or the severity of covid-19. only studies that report outcome data between severe and non-severe covid-19 were included, regardless of the pattern or the severity of the liver injury. our primary outcome was the pooled risk of serum alanine aminotransferase (alt) elevation in adult patients with severe and non-severe covid-19. our secondary outcomes were the pooled risk of the following parameters in adult patients with severe and non-severe covid-19: (1) serum aspartate aminotransferase (ast) elevation, (2) hyperbilirubinemia and (3) hypoalbuminemia. we also assessed the pooled mean difference (md) of gamma-glutamyl transferase (ggt) from the included studies. we defined the elevation of serum alt or ast as levels beyond 40 u/l. we defined hyperbilirubinemia as total bilirubin level higher than 17mmol/l. we defined hypoalbuminemia as serum albumin level below 40g/l. we conducted a comprehensive search of three electronic databases, pubmed/medline, embase and cochrane library (earliest inception to 24 th april 2020). keywords used in the search included a combination of "coronavirus disease 2019", "covid19", "sars-cov-2" and "coronavirus infection/complications [mesh]". we restricted our search to adult human studies. an experienced medical librarian helped with our literature search. in addition, we manually searched all references of selected articles for additional relevant articles. we used prisma (preferred reporting items for systematic reviews and meta-analyses) to report all studies identified using a pre-defined search protocol detailed in appendix 1. in this meta-analysis, we included all studies that met the following inclusion criteria: (1) population: adult patients infected with the covid-19, (2) reported outcome data on liver enzymes derangement (3) reported outcome data on the risk or severity of liver injury between severe and non-severe covid-19. all studies, j o u r n a l p r e -p r o o f regardless of language, geography, publication dates or publication status are included so long as they provided data relevant to this analysis. our exclusion criteria were: 1) any study with subjects < 21 years old, 2) case report or case series with less than 20 subjects, 3) review articles, editorial and guidelines. severe covid-19 was defined based on the definition used in the respective study. we further defined subjects as "critical covid-19" based on the need for admission to the intensive care unit, mechanical ventilation, or death. three authors (mt, jl, rk) independently reviewed all titles and abstracts of the studies identified in the primary search. studies that were duplicates or did not address the research question based on pre-defined criteria were excluded. we subsequently reviewed the full texts of all remaining articles to determine if they contained relevant information. any discrepancy in the article selection was resolved by consensus with a co-author (wyj). we extracted data on the demographic of study populations (age, gender, sample size, the proportion of subjects with baseline chronic liver disease and the use of lopinavir/ritonavir) as well as the pattern of covid-19 associated liver injury (alt, ast, bilirubin, albumin and ggt) from all included studies. the data from each study were independently extracted by into a standardized form by two authors (wyj, rk). we contact the corresponding author through email for any missing data. we did not encounter multiple reports from the same author or study population in this meta-analysis. all included studies were independently reviewed by 2 authors (mt, jl) to assess for quality and risk of bias using the subjects were followed-up = 0.5 points, < 50% subjects were followed-up = 0 point). the final decision on the overall risk of bias was made through discussion. all differences were resolved by discussions with third author (wyj). we considered studies with a score of ≥6, 3-4, < 3 as high-quality, medium-quality and low-quality, respectively. we used review manager software version 5.3 (the nordic cochrane centre, the cochrane collaboration, 2014) to perform our meta-analysis to estimate the pooled odds ratio (or), mean difference (md), and 95% confidence interval (95%ci). we used the random-effects model and validated our results using sensitivity analysis and heterogeneity assessment across the included studies. a p-value of less than 0.05 was considered to be statistically significant. the statistical heterogeneity was evaluated using the i 2 statistics. we defined substantial heterogeneity across study as low, moderate, substantial and considerable with a i 2 value of <30%, 31% to 60%, 61% to 74% and > 75% [15] . we performed subgroup analysis among subjects with critical covid-19 (defined as subjects needing admission to intensive care unit, mechanical ventilation, or death) to minimize the impact of heterogeneity in the definition of severe covid-19 on our results. as casecontrol studies are generally considered to have a higher risk of bias and more susceptible to selection and recall bias [16] , we performed subgroup analysis based on study design (cohort study versus case-control study). from an initial total of 1543 citations identified by using our search strategy, we identified a total of 112 studies. of these, 89 were excluded for the following reasons: no outcome data reported (n=44), case report or case series fewer than 20 subjects (n=25), review article (n=18) and pediatric study (n=2). we manually search the references of all the included studies and included one additional study [10] . a total of 24 studies met our inclusion criteria [10, . (figure 1 ). the characteristics of all included studies are presented in table 1 . most studies were retrospective (15 case-control studies and 8 cohort studies) in nature except for one prospective cohort study [24] . seven studies reported multicenter data, while the remaining reported single-center data. all studies were from asia. the definition of severe covid-19 was heterogeneous across all the included studies. the commonest definition of severe covid-19 was based on clinical criteria (n=15), followed by death (n=5) and icu admission (n=3). all studies were published as a full manuscript. overall, seven studies were considered to be of high quality, while the rest were considered to be medium quality (supplementary table 1) . twenty-four studies, with a total of 5952 patients (4024 in the severe group and 1928 in the non-severe group), were included in the final analysis [10, . the patients' characteristics were detailed in table 1 . the median age of subjects ranged from 38 to 71 years-old. the proportion of male ranged from 43% to 81%. the proportion of subjects with admission to an intensive care unit ranged from 1% to 100%. the prevalence of baseline chronic liver disease was low (ranged from 1% to 11%) [17-18, 20, 22-26, 28-33, 36, 38] . the liver injury occurred in 5%-76% of subjects with covid-19 [17-20, 22-26, 31-39] . j o u r n a l p r e -p r o o f twenty-three studies (5900 subjects) reported outcome data on alt [10, [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [35] [36] [37] [38] [39] . the mean level of alt is higher in severe covid-19 when compared to nonsevere group (31.9 u/l vs 23.5 u/l, 95%ci= 5.3-12.8, p<0.001), with high heterogeneity (i 2 =99%, p<0.001) (supplementary table 2) . the pooled risk of alt elevation was higher in severe covid-19 with moderate heterogeneity (or=2.8, 95%ci: 1.8-4.3, i 2 =63%) ( table 2) . when the stratified analysis was performed within the subgroup of patients with critical covid-19, the pooled risk of alt elevation remained higher among subjects with critical covid-19, with less heterogeneity (or=2.5, 95%ci=1.6-3.7, i 2 =57%) (figure 2) . twenty-one (5292 subjects) reported outcome data on ast [10, 17, [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [35] [36] [37] [38] . the mean level of ast is higher in severe covid-19 when compared to non-severe group (35.1 u/l vs 26.6 u/l, 95%ci= 8.7-14.2, p<0.001), with high heterogeneity (i 2 =99%, p<0.001) (supplementary table 2 ). the pooled risk of ast elevation was higher in severe covid-19 with moderate heterogeneity (or=3.4 95%ci: 2.3-5.1, table 2) . stratified analysis among subjects with critical covid-19 showed a higher pooled risk of ast elevation, with less heterogeneity (or=3.4, 95%ci: 2.3-5.0, i 2 =56%) (figure 3) . one study reported a higher risk of ast elevation in nonsurvivor (or=5.9, 95%ci=3.4-10.5, p<0.001) ( table 2) . [20] thirteen studies (3540 subjects) reported outcome data on serum bilirubin [17, 19-20, 23-26, 29, 32, 30, 36-38] . the mean level of bilirubin is higher in severe covid-19 j o u r n a l p r e -p r o o f when compared to non-severe group (12.2mmol/l vs 10.5 mmol/l, 95%ci= 1.5-3.5, p<0.001), with high heterogeneity (i 2 =98%, p<0.001) (supplementary table 2 ). the pooled risk of hyperbilirubinemia (serum bilirubin > 17mmol/l) is higher in subjects with severe (or=1.9, 95%ci: 1.1-3.1, i 2 =30%) ( table 2 ) and critical covid-19 (or=1.7, 95%ci: 1.2-2.5, i 2 =0%) (figure 4) . thirteen studies (3404 subjects) reported outcome data on serum albumin [19-20, 24-29, 32, 36-37, 39] . the mean level of albumin is lower in severe covid-19 when compared to non-severe group (37.2 g/l vs 40.1 g/l, 95%ci= -6.0, -2.9, p<0.001), with high heterogeneity (i 2 =100%, p<0.001) (supplementary table 2 ). the pooled risk of hypoalbuminemia (serum albumin <40g/l) was higher in subjects with severe covid-19 (or=8.8, 95%ci: 4.1-19.0, i 2 =46%) and critical covid-19 (or=7.1, 95%ci: 2.1-24.1, i 2 =71%) ( table 2) . three studies (699 subjects) reported outcome data on serum ggt [17, 20, 37] . there is a trend towards higher ggt is higher in severe covid-19 (68.5 u/l vs 36.8 u/l, p=0.07) and critical covid-19 (56.9 u/l vs 28.5 u/l, p<0.001), with high heterogeneity (i 2 =96% and 100% respectively, p<0.001) (supplementary table 2 ). the mean difference of ggt did not significantly differ in subjects with severe or critical covid-19 as compared to non-severe/non-critical covid-19 patients ( table 2 ). we performed a subgroup analysis based on the types of studies and the proportion of subjects with lopinavir/ritonavir usage. we performed a subgroup analysis based on study design by comparing a cohort study and case-control study. we found that the effect size for the casecontrol study was bigger as compared to cohort study for alt elevation, ast elevation, hyperbilirubinemia and hypoalbuminemia ( table 2) . the proportion of subjects using lopinavir/ritonavir (ranged from 14% to 100%) was reported in 12 studies [17-18, 20, 25-29, 32-34, 38] . we arbitrarily defined high lopinavir/ritonavir usage as a study with ≥ 80% of their subjects treated with lopinavir/ritonavir. among the four studies with high lopinavir/ritonavir usage [17, 25, 29, 33] , we observed a higher pooled odds risk for alt and ggt elevation ( table 2) table 4 ). we performed a sensitivity analysis to assess whether an individual study had a dominant effect on the overall pooled results. this was performed by serially removing one study at a time while repeating the analysis. the pooled risk of alt elevation ranged from 2.2 to 2.8, which is still within our reported 95%ci. besides, we also performed repeated analysis using the fixed-effect model. using the fixedeffect model, the level of ggt is higher in severe covid-19 as compared to nonsevere group (md=14.6, 95%ci: 14.1-17.2, i 2 =96%). all the remaining findings were similar in the sensitivity analysis (supplementary table 2 ). we assessed heterogeneity based on the percentages of i 2 for each reported outcome. we consider the heterogeneity on the observed risk and severity of covid-19 associated liver injury as moderate and substantial, respectively. this heterogeneity likely the result of a different definition of severe disease used in various studies. there was no evidence of publication bias based on visual inspection of the funnel plot ( figure 5) . covid-19 associated liver injury is defined as any liver damage in patients covid-19 patients, with or without the pre-existing liver disease [5] . while the exact j o u r n a l p r e -p r o o f mechanism remains unknown, several potential mechanisms have been postulated [6] [7] [8] [9] . severe systemic inflammatory response during severe covid-19 can lead to immune-mediated damage or ischemic hepatitis from the severe systemic inflammatory response. current treatment options such as lopinavir/ritonavir, hydroxychloroquine and remdesivir are potentially hepatotoxic and may cause druginduced liver injury (dili) [11] . other immunosuppressive alternative such as tocilizumab or steroid can result in reactivation of underlying chronic hepatitis b. while direct cytotoxicity due to viral replication within infected hepatocytes has been postulated, viral inclusions for sars-cov-2 has not been found in the liver [40] . the current data on the severity and prevalence of covid-19 associated liver injury remains conflicting due to the heterogeneity in study populations. to address these issues, we systematically reviewed the current literature on liver injury in covid-19 and performed a meta-analysis on the severity and risk of covid-19 associated liver injury in these patients. at the point when this meta-analysis was conducted, most studies did not specifically report the prevalence of covid-19 patients with baseline chronic liver disease. none of the included studies reported outcomes of covid-19 patients based on their underlying liver disease. as all included studies reported a low incidence of chronic liver disease (cld), our findings will be more reflective of the general population. our findings were consistent with current literature that adult patients with severe covid-19 have a higher risk of liver injury. most liver injuries were mild. to date, there is only one reported case of death from liver failure in covid-19 patients without pre-existing liver disease. in a recently published international registry of covid-19 among patients with chronic liver disease, only 12% of death was j o u r n a l p r e -p r o o f attributed to liver disease [41] . that having said, the mortality risk among patients with the pre-existing liver disease did increase with the severity of underlying liver disease (13% in chronic liver disease, 24% in child-pugh's a cirrhosis, 43% in child-pugh's b cirrhosis and 63% in child-pugh c cirrhosis) [41] . based on our analysis, the pooled or of alt elevation was 2.5, ast elevation was 3.4, and hyperbilirubinemia was 1.7 among covid-19 patients who were critically ill. we found that indirect markers for liver injury, such as hypoalbuminemia increased by seven-fold in patients with severe covid-19. while we are mindful that hypoalbuminemia could be confounded by factors such as systemic inflammation, malnutrition and the timing of presentation to hospital, similar findings were reported in other studies [42] [43] . therefore, physicians should be alert about potential clinical deterioration (intubation, icu admission and death) when covid-19 associated liver injury was observed. in this meta-analysis, the pooled risk of liver injury was higher among studies with high usage of lopinavir/ritonavir. in a recent retrospective study involving 417 covid-19 patients [17] , lopinavir/ritonavir was associated with 7-fold higher risk of liver injury. in this meta-analysis, we found a higher level of ggt and alt elevation in studies with high lopinavir/ritonavir usage, suggesting that lopinavir/ritonavir might be hepatotoxic ( table 2) . although lopinavir/ritonavir did not significantly increase the risk of liver injury in a recent open-labelled randomized trial, this trial excluded subjects with abnormal baseline liver functions. we are unable to exclude preferential treatment of severe covid-19 patients using lopinavir/ritonavir from this meta-analysis. based on our findings, physicians may consider monitor for liver injury when lopinavir/ritonavir is being used for covid-19 patients. this meta-analysis adds value to the current literature by summarizing the prevalence and pattern of liver injury in adults with severe covid-19. prior to this, the prevalence of covid-19 associated liver injury was confounded by the heterogeneity of study population, and study design as pertinent information such as the proportion of subjects' baseline chronic liver disease or icu admission were often not reported [5] . in this meta-analysis, we found that the severity of most covid-19 associated liver injury was considered mild, suggesting liver failure is uncommon among critical covid-19. nevertheless, physicians should be mindful of potential deterioration among covid-19 patients liver injury as it is associated with icu admission, mechanical ventilation and death. there are several strengths in our meta-analysis. to our best knowledge, this is the most comprehensive description of the liver manifestation of covid-19 in comparison. earlier meta-analysis had either included fewer studies evaluating liver manifestation among covid-19 patients [43] [44] [45] [46] [47] [48] [49] or included a wide range of "severe" covid-19 patients ranging from the presence of tachypnoea to death [43] [44] [45] [46] . in this meta-analysis, we conducted a systematic search of the literature using a predefined inclusion and exclusion criteria; including a large number of studies to allow assessment of publication bias and subgroup analysis, detailed extraction of data on study outcomes; subgroup analysis to define a more homogenous study population j o u r n a l p r e -p r o o f for sensitivity analysis; rigorous evaluation of study quality; and the use of various statistical methods to evaluate the validity of our findings. our meta-analysis was limited by the heterogeneity in the study population of included studies. we attempted to mitigate this limitation by performing subgroup analysis based on the characteristics of each study. most of the included studies were limited by their retrospective nature of study design and small sample size. because of this, we cannot estimate the incidence and comparative risk of druginduced liver injury across different treatment with high confidence. while liver injury is more likely to occur in adults with critical covid-19, we cannot be certain of its prognostic value as most of the studies were retrospective in nature. in conclusion, covid-19 associated liver injury is generally mild. as liver injuries are more common in patients with severe covid-19, physicians should be mindful about the potential risk of clinical deterioration in these patients. characteristics of and important lessons from the covid-19) outbreak in china: summary of a report of 72314 cases from chinese centre of disease control and prevention liver injury during highly pathogenic human coronavirus infections epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study covid-19 and liver disease clinical features of covid-19-related liver damage. biorxiv liver injury in covid-19: management and challenges pathological findings of covid-19 associated with acute respiratory distress syndrome clinical characteristics of 82 death cases with covid-19 clinical features and laboratory inspection of novel coronavirus pneumonia (covid19) in xiangyang hubei aasld clinical insights for hepatology 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china clinical features of patients infected with 2019 novel coronavirus in wuhan, china epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms analysis of epidemiological and clinical features in older patients with corona virus disease 2019 (covid-19) out of wuhan analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease clinical characteristics of refractory covid-19 pneumonia in wuhan clinical features and treatment of covid-19 patients in northeast chongqing clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of 69 cases with coronavirus disease 2019 in wuhan, china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical findings in a group of patients infected with the 2019 novel coronavirus 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disease 2019 and prevalence of chronic liver disease: a meta-analysis liver injury is associated with severe coronavirus disease 2019 (covid-19) infection: a systematic review and metaanalysis of retrospective studies aga institute rapid review of the gastrointestinal and liver manifestations of covid-19, meta-analysis of international data, and recommendations for the consultative management of patients with covid-19 and liver dysfunction: a systematic review and meta-analysis of retrospective studies markers of liver injury and clinical outcomes in covid-19 patients: a systematic review and meta-analysis liver profile in covid-19: a meta-analysis manifestations and prognosis of gastrointestinal and liver involvement in patients with covid-19: a systematic review and meta-analysis covid-19" and hasabstract[text] and humans[mesh])) or ("coronavirus disease 2019" and hasabstract[text] and humans[mesh])) or ("sars-cov-2" and hasabstract[text] and humans[mesh]) filters: abstract; humans 565 #5 search "covid19" filters: abstract abbreviations: *mean (sd); nr=not reported; cld = chronic liver disease; ct = computer tomography; nr=not reported j o u r n a l p r e -p r o o f key: cord-262152-gdnc51m5 authors: chaibi, sayma; boussier, jeremy; hajj, weam el; abitbol, yael; taieb, sarah; horaist, clemence; jouannaud, vincent; wang, pascal; piquet, jacques; maurer, cyril; lahmek, pierre; nahon, stéphane title: liver function test abnormalities are associated with a poorer prognosis in covid-19 patients: results of a french cohort date: 2020-10-19 journal: clin res hepatol gastroenterol doi: 10.1016/j.clinre.2020.10.002 sha: doc_id: 262152 cord_uid: gdnc51m5 aim: to assess the impact of liver function test (lft) abnormalities on the prognosis of patients with coronavirus disease 2019 (covid-19) in a french cohort of hospitalized patients. patients and method: from march 13 to april 22, 2020, we collected on a computerized and anonymized database, medical records, laboratory data and clinical outcomes of patients hospitalized for confirmed cases of covid-19 infection (rt–pcr and/or ct-scan). patients were followed up until april 22 2020 or until death or discharge. we have considered for statistical analysis, lft abnormalities with levels greater than two times the upper limit of normal. composite endpoint included admission to icu, mechanical ventilation, severe radiologic injury and death to define disease severity. results: among 281 patients (median age 60 years) with covid-19, 102 (36.3%) had abnormal lft. hypertension (45.6%) and diabetes (29.5%) were the main comorbidities. 20.2% were taken liver-toxic drugs at the admission and 27.4% were given drugs known to induce hepatic cytolysis during hospitalization. patients with elevated levels of alt or ast were significantly more severe with a higher rate of admission to icu (40.0% vs 6.0%, p < 0.0001), and global mortality (26.7% vs 12.1%, p = 0.03). in multivariate analysis, obesity and cytolytic profil were associated with the composite endpoint (respectively 2.37 [1.21; 4.64], p = 0.01 and or 6.20, 95% confidence interval [1.84, 20.95], p-value 0.003) conclusion: most of liver injuries are mild and transient during covid-19. lft abnormalities are associated with a poorer prognosis and could be a relevant biomarker for early detection of severe infection. coronavirus disease 2019 (covid-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). it was first identified in december 2019 in wuhan (china) and has resulted in a pandemic, with 1,771,514 confirmed cases worldwide as of april 12, 2020. the most common symptoms are fever, dry cough, shortness of breath, myalgia/fatigue. potential complications include pneumonia, acute respiratory distress syndrome, multi-organ failure and death. 1 2 potential gastrointestinal manifestations of j o u r n a l p r e -p r o o f have been reported, including nausea, vomiting, diarrhea and abnormal liver function test (lft). 1 3 one study has shown that 14-53% patients displayed abnormal levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) during disease progression, 4 and that liver abnormalities could be used to stratify patients' risk and monitoring illness severity. 4 notably, these results were obtained in the chinese population, and there is to date no data concerning clinical and liver function biological characteristics of european patients infected with sars-cov-2. in this study, we retrospectively investigated the changes in lfts in in-hospital infected patients from a single center in montfermeil, france. we aimed to assess the impact of liver function parameters on the clinical outcome of patients with covid-19. from march 13 2020 to april 22, 2020, a total of 281 consecutive patients were hospitalized and treated for covid-19 infection in montfermeil hospital, located in the suburb of paris (france). the study was performed in compliance with french legislation regarding noninterventional studies. the study protocol was submitted to the comité de protection des personnes (cpp) sud-est 4 and was in compliance with ethical consideration. it was also approved by cer-u paris (université paris descartes, 13 march 2020). the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the institution's human research committee. patients (or their family representative) were provided with written information and asked to give their oral consent before inclusion. we collected on a computerized and anonymized database patients'medical records who were hospitalized for confirmed cases of covid-19 infection. patients were followed-up until april 22, 2020 or until death or discharge. biological tests were performed in montfermeil hospital biomedical laboratory. blood tests were performed at the investigator's discretion. radiologic assessment was based on computed tomography (ct). images were interpreted by experienced radiologists. the presence of radiologic abnormalities was determined on the basis of medical charts. severity was defined as the extent of damaged lung tissue (<10%; 10-25%; 25-50 %; 50-70 %; >70 %). inclusion criteria were confirmed cases of covid-19 between march 13, 2020 and april 22, 2020 for all in-hospital patients over 18 years of age. diagnosis was based on positive realtime reverse-transcriptase-pcr (rt-pcr) and/or typical aspect on non-contrast lung ctscan, as defined by the radiological society of north america expert consensus statement. 5 confirmed cases were defined as a positive result on high-throughput sequencing or rt-pcr assay of nasal and pharyngeal swabs, on the basis of the world health organization (who) j o u r n a l p r e -p r o o f guidance. patients whose molecular biology testing for the covid-19 virus was inconclusive but who had typical injuries on lung ct scan were also included. we collected clinical data on admission such as sex, age, body mass index (bmi) and comorbidities with a special attention to underlying liver disease. drugs taken before and during hospitalization were also collected, especially those with liver toxicity. we additionnaly recorded the use of combined hydroxychloroquin-azithromycin administration. liver function was assessed through monitoring of alanine aminotransferase (alt), aspartate aminotransferase (ast), gamma-glutamyl transferase (ggt) and alkaline phosphatase (alp). if more than one sample was collected, the highest value of each analyte was used. abnormal liver function was defined as values greater than 2 times the upper limit of normal (uln). cytolytic profile refers to abnormal ast or alt levels, and cholestatic profile refers to abnormal ggt levels. patients with ast, alt, alp or ggt levels greater than 5 times the uln were analyzed separately. prothrombin, a reflect of liver function and coagulation, and crp level at the day of admission, a marker of inflammatory response, were also recorded. severity was determined through a composite outcome considering : (i) death, (ii) lung damaged extent >50% on ct-scan, (iii) respiratory failure requiring mechanical ventilation and (iv) admission to an intensive care unit (icu). criteria for intensive care admission in were: respiratory rate (rr) > 30, requirement of oxygen flow > 4 l/min in order to maintain an oxygen saturation > 96%, a heart rate (hr) > 90, systolic blood pressure < 90 mmhg, consciousness disorders, respiratory failure and/or signs of shock. summary statistics were medians and interquartile ranges for quantitative variables and percentages for categorical variables. univariate analyses were performed using t-tests for quantitative variables and chi-squared test of independence or fisher's exact tests for qualitative variables. cyctolytic profile and variables with a p-value ≤ 0.20 in the univariate analyses were included in the multivariate model. a stepwise logistic regression analysis j o u r n a l p r e -p r o o f was performed to identify factors associated with cytolytic profile. the following variables were considered for multivariable logistic regression: gender, age, obesity, diabetes, hypertension, combine therapy (hydroxychloroquine-azithromycin) and hepatic cytolysis. the odds ratio (or) and 95% confidence interval (ci) were computed for each independent factor. p-values < 0.05 were considered statistically significant. all statistical analyses was performed using soft ware spss (version 18.0 ). results 358 patients were hospitalized for presumed covid-19 infection from march 13 to april 22, 2020. 77 patients were excluded for negative pcr and no typical injuries on ct scan, when available. clinical and biological characteristics of the patients are shown in table 1 and table 2 . median age was 60 years (interquartile range (iqr), 46.5-73.5). most patients were either overweight (39.5%) or obese (34.3%). in the overall cohort, hypertension (45.6%) and diabetes (29.5%) were the main coexisting comorbidities. no patients were being treated with non-steroidal anti-inflammatory drugs, 20.2% with liver-toxic drugs at the admission and 27.4% with therapy known to induce hepatic cytolysis during hospitalization (e.g., hydroxychloroquin and tocilizumab). sixty point nine percent of confirmed covid-19 cases had a positive rt-pcr test from nasal and pharyngeal swabs. of 248 ct scans performed during the study period, 4.0% revealed >70% of lung damage. the median length of stay was 7.0 days (iqr 0-39) and 11.0 days only in icu. in total, 60.1% of patients suffered from severe disease, defined as icu admission (15.3%), respiratory failure requiring mechanical ventilation (10.7%), >50 % of lung damage on ct scan (20.9%) or death (16.0%). of the 281 patients included, 102 (36.3 %) had liver dysfunctions. high level of ggt was the most common perturbation (25.3%), followed by elevated ast (24.3%) and alt (12.8%) levels ( table 1 ). only a minority of patients (6.4%) had perturbations above 5 times the uln. factors associated to cytolytic injury were male (73.7% vs 44.7%, p < 0.0001), liver toxic treatments (54.7% vs 35.0%, p < 0.0001), in particular statins, antiepileptics and hydroxychloroquin (45.9% vs 18.7%, p < 0.0001) ( table 3) . patients with high transaminase levels were significantly more severe with a higher rate of admission to icu (40.0% vs 6.0%, p < 0.0001), ct-scan injury > 50% (71.6% vs 39.7%, p < 0.0001) and global mortality (26.7% vs 12.1%, p = 0.03). similar results were obtained for patients with cholestatic liver injury (table table 5 shows the association of factors with the composite severity endpoint (admission to icu, respiratory failure requiring mechanical ventilation, ct scan injury >50% and global mortality). elevated levels of ast or alt (6.20 1.84 ; 20.95, p = 0.003) and obesity were associated with severe disease (2.37 1.21 ; 4.64, p = 0.01). age, gender, diabetes, hypertension and hydroxychloroquin were not associated with disease severity. table 6 shows descriptive and univariate analyses comparing crp levels between the severe and non-severe group of patients. median crp was 41. characteristics of our study population are in line with prior studies, with a slightly male predominance (53.0%) and a median age of 60.0 years. main comorbidities were diabetes (29.5%) and hypertension (45.6%). notably, chinese cohorts displayed lower prevalence of diabetes (<10%) and obesity (with a median bmi of 22.6 kg/m 2 ) than this cohort. 1 6 . primary liver diseases were more frequent among chinese patients, with a reported prevalence of 2.0%; 1 6 this discrepancy may be explained by the higher rate of chronic hepatitis b in china. severe infection is known to be more frequent among those patients, but they had mostly imbalanced diabetes or hypertension, which was not the case in our study global mortality was also similar (16.0%), yet the number of admissions to icu (15.3%) was higher than previously reported 1 . thirty-six point three percent of patients showed liver abnormalities, in agreement with chinese data, reporting liver impairment in 14-53% of cases of covid-19 infections. 4 notably, abnormalities were mild and transient in this cohort; in particular, <10% of patients had levels greater than 5 times the uln at admission or during hospitalization. multivariate analysis showed that elevated levels of alt or ast, as well as obesity, were associated with more severe disease. obesity has already been identified as a predictive factor of severity 7 abnormal liver test could be an interesting tool for early detection of severe covid-19 infection. liver abnormalities (especially cytolytic profile) is more frequent among male patients, for reasons that remain unclear. we found no significant difference in prevalence of comorbidities such as underlying liver disease, diabetes or hypertension and for liver toxic drugs intake between men and women. further studies will be required to confirm and better understand the association between gender and cytolytic liver injury. liver injury in covid-19 infection can be due to several mechanisms. it may be directly caused by the virus: recent studies showed that the key receptor for sars-cov-2 cell j o u r n a l p r e -p r o o f entry, (ace2 receptor) was expressed in respiratory tract but also in other tissues, especially kidneys, enterocytes and cholangiocytes. 8 9 future work will be needed to determine the exact mechanism of liver damage. in this study, high level of ggt was the most frequent injury reported (25.3%), while alp was not always substantially elevated. it is known that ggt and alp are a reflect of the cholangiocyte function, even though it can be increased in injuries of other tissues. pathological studies of liver tissues from patients with sars-cov2 confirmed the presence of virus, although viral inclusions were not observed, suggesting that viral titers are relatively low, and that liver damage in sars-cov-2 infection is likely to be mediated by apoptosis. 10 an other possibility is that liver damage is induced by hepatotoxic drugs. in our study, 20.4% of patients had liver-toxic treatments on admission (mainly statins, antiepileptics and oral anticoagulants). twenty-seven point four percent of patients were treated by hydroxychloroquine during hospitalization. elevation of liver transaminases is a frequent secondary effect and has been a cause of early interruption. last, it is also possible that liver abnormalities are due to sepsis and tissue hypoxemia. levels of crp, a good marker of inflammation, were significantly higher in severe group (p = 0.011). histological examination showed apoptotic injuries such as vesicular steatosis and watery degeneration which may be due to hypoxemia. 6 infection is characterized by an important inflammatory response, resulting in cytokine storm and pneumonia-associated hypoxia. it might contribute to liver injury and even liver failure in patients who are critically ill. several limitations of this study should be noted. as a monocentric study, it may be difficult to generalize results to other regions of varying epidemiological characteristics. still, seine saint-denis, the suburb where montfermeil hospital is located, is one of the most affected region in france with a high level of hospitalization and death rate. because of the retrospective study design, only a few patients were known to have underlying liver disease. it is hard to know if lft abnormalities reflect their baseline liver function or a real effect of covid-19 infection. in this study, we mainly focused on the peakvalue. future work with regular and standardized follow-up at the beginning and during and hospitalization period would be interesting to precisely explore the kinetics of liver dysfunction. it would help to determine if elevated lft are a risk factor of severe disease or if severe disease induces liver dysfunction. comorbidities such as balanced diabetes and hypertension were not associated to the composite endpoint. combination therapy using hydroxychloroquin and azithromycin was also not associated with severe disease. a possible explanation is the lack of standardized criteria to begin a combination therapy: in montfermeil hospital, respiratory failure requiring oxygenotherapy greater than 4 liters per minutes associated with a respiratory rate greater than 28 per minute were one of those criteria, which differs from the definition of severe disease in our study. in conclusion: 36.3% of patients with covid-19 infection had abnormal liver test during the study period. liver tests abnormalities were associated with a poorer prognosis and could be a crucial biomarker for early detection of severe infection conflicts of interest stéphane nahon reports lecturer or advisory board fees from abbvie, msd, vifor pharma, pfizer, janssen and ferring. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f clinical characteristics of coronavirus disease 2019 in china epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms liver injury in covid-19: management and challenges radiological society of north america expert consensus statement on reporting chest ct findings related to covid-19. endorsed by the society of thoracic radiology, the american college of radiology, and rsna covid-19: abnormal liver function tests obesity and impaired metabolic health in patients with covid-19 specific ace2 expression in cholangiocytes may cause liver damage after multi-omics evaluation of gastrointestinal and other clinical characteristics of sars-cov-2 and covid-19 sars-associated viral hepatitis caused by a novel coronavirus: report of three cases key: cord-323736-zup9cp6s authors: ko, sheung‐fat; chen, yi‐ling; sung, pei‐hsun; chiang, john y.; chu, yi‐ching; huang, chung‐cheng; huang, chi‐ruei; yip, hon‐kan title: hepatic (31)p‐magnetic resonance spectroscopy identified the impact of melatonin‐pretreated mitochondria in acute liver ischaemia‐reperfusion injury date: 2020-07-21 journal: j cell mol med doi: 10.1111/jcmm.15617 sha: doc_id: 323736 cord_uid: zup9cp6s acute liver ischaemia‐reperfusion injury (iri), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. however, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. this study tested the hypothesis that (31)p‐magnetic resonance spectroscopy ((31)p‐mrs) findings could provide reliable living images to accurately identify the degree of acute liver iri and melatonin‐pretreated mitochondria was an innovative treatment for protecting the liver from iri in rat. adult male sd rats were categorized into group 1 (sham‐operated control), group 2 (iri only) and group 3 (iri + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). by the end of study period at 72 hours, (31)p‐mrs showed that, as compared with group 1, the hepatic levels of atp and nadh were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. the liver protein expressions of mitochondrial‐electron‐transport‐chain complexes and mitochondrial integrity exhibited an identical pattern to (31)p‐mrs finding. the protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial‐damaged biomarkers displayed an opposite finding of (31)p‐mrs, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. microscopic findings showed that the fibrotic area/liver injury score and inflammatory and dna‐damaged biomarkers exhibited an identical pattern of cellular stress signalling. melatonin‐pretreated mitochondria effectively protected liver against iri and (31)p‐mrs was a reliable tool for measuring the mitochondrial/atp consumption in living animals. acute liver ischaemia-reperfusion injury (iri), which has been reported to commonly occur during liver resection and transplantation surgery is a crucial factor for predictive of poor outcome of liver transplantation. [1] [2] [3] studies have further displayed that several key factors contribute to the hepatic injury at the initiation and during the progression of liver iri, including those of elevation of anaerobic metabolism, dysfunction of mitochondria, insult of oxidative stress, overload of intracellular calcium, activation of liver kupffer cells, infiltration of immune cells and release of inflammatory cytokines. [2] [3] [4] [5] [6] although the underlying mechanisms of hepatic iri have been extensively investigated, the clinical practice to prevent iri is still limited. additionally, despite the state-of-the-art advance of critical care for patients with acute hepatic failure, the in-hospital morbidity and mortality remain unacceptably high. [7] [8] [9] [10] [11] [12] therefore, it is of utmost importance to both clinician and medical researchers to explore a safe and efficacious therapeutic modality for patients with acute liver iri refractory to conventional treatment. [13] [14] [15] [16] however, prior to ascertain a novel, safe and efficacious therapeutic modality for acute liver iri, the core property that induces this disease entity must be first clarified. it is well recognized that mitochondria are especially rich in liver and heart. the function of mitochondria in liver or other organs not only serves as the primary energy source but also plays a pivotal role in extensive oxidative metabolism and normal function of the liver. 17, 18 however, severely impaired mitochondrial function and activity have clearly identified in setting of liver ischaemia-reperfusion 4, 19, 20 with subsequent alternation in energy metabolism, generation of reactive oxygen species (ros), inflammatory cell infiltration and cellular apoptosis [21] [22] [23] [24] and ultimately irreversible damages to mitochondria, 16, 25 exhausting atp in ischaemia-reperfusion liver. 4, 16, 21, [25] [26] [27] these findings 4, 16, [19] [20] [21] [22] [23] [24] [25] [26] [27] highlight that the quantity (ie amount) and quality (functional integrity) of mitochondria play extremely important roles on acute liver iri. accordingly, these aforementioned issues 4, 16, 21, [25] [26] [27] raise the hypothesis that restoration of mitochondrial function through exogenous mitochondrial transfusion may be a potential strategy for the treatment of acute hepatic iri. melatonin, acting as a scavenger of free radicals for cell membrane stabilization, has been identified to have powerful ancient antioxidant 28 for suppressing the generation of oxidative stress. 29 basic researches have exhibited that melatonin enters mitochondria through oligopeptide transporters and specifically targets at the mitochondria where it seems to function as an apex antioxidant. 30 in addition to cell uptake from circulation, melatonin is produced in the mitochondria as well. 30 it is well recognized that oxidative damage is a result of free radicals produced in cells, especially in mitochondria. 31 recent study has further demonstrated the protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial ca 2+ stress by melatonin's cascade metabolites. 32 thus, melatonin always plays a crucial role on protecting the mitochondrial number and functional integrity [30] [31] [32] through regulation of the oxidative stress and the preservation of atp/energy, resulting in protection of organs from iri. 16, 30, [33] [34] [35] [36] interestingly, previous studies 37, 38 16, 17, 21, 25, [28] [29] [30] [33] [34] [35] [36] this study tested the hypothesis that melatonin-pretreated exogenous mitochondria might offer an added benefit on protecting the liver against acute iri. additionally, this study further tested whether the 31 p-mrs examination could provide reliable living images to accurately identify the degree of atp consumption/depletion in hepatocytes, that is an indicator of acute liver ischaemia-reperfusion in rodent. whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. microscopic findings showed that the fibrotic area/ liver injury score and inflammatory and dna-damaged biomarkers exhibited an identical pattern of cellular stress signalling. melatonin-pretreated mitochondria effectively protected liver against iri and 31 p-mrs was a reliable tool for measuring the mitochondrial/atp consumption in living animals. the procedure and protocol have been described in our previous report 42 and were based on the other previous studies. 43, 44 in detail, the pathogen-free, adult male sprague dawley (sd) rats (n = 24) weighing 325-350 g were randomly divided into three groups: the procedure was described as follows: the left lobe of the liver was dissected free from the surrounding ligaments. hepatic ischaemia was induced by temporal obstruction of the vessels by placing a 4-0 silk loop around the hilar region of the left liver lobe in groups 2 and 3 animals, whereas sc animals received only laparotomy without undergoing hepatic ischaemia. reperfusion was started 60 minutes later when the hilar occlusion was released. all animals were killed by 72 hours after reperfusion. to elucidate the impact of acute liver ischaemia-reperfusion on hepatocyte damage and up-regulation of inflammatory reaction as well as the therapeutic effect of melatonin on the circulatory parameters, blood samples were collected before and after the experiments and plasma specimens were harvested for analyses of aspartate aminotransferase (ast) concentration, alanine aminotransferase (alt) and circulating levels of inflammatory cytokines. liver specimens were acquired and then stored at −80°c for individual study. tissues were also embedded in optimal cutting temperature compound or 4% buffered formaldehyde for cryo-sectioning and paraffin sectioning, respectively. for stabilization and attenuation of oxidative stress in isolated mitochondria, melatonin was given to donor rat (n = 3) at day 3 (50 mg), 2 (25 mg) and 1 (25 mg)/rat by intraperitoneal administration prior to acute liver ischaemia-reperfusion procedure. 16, 42 the procedure and protocol of liver mitochondrial isolation from donor sd rats have been previously described by our studies. 42, 45, 46 in detail, the rats (ie the donors) were fasted overnight prior to the mitochondrial isolation procedure, then killed and their gallbladders and livers carefully isolated and removed. immediately, the liver (3 g) was immersed in 50 ml of ice-cold ibc (10 mmol/l tris-mops, 5 mmol/l egta/tris and 200 mmol/l sucrose, ph 7.4) in a beaker, followed by rinsing the liver free of blood with ice-cold ibc. the liver was then minced with scissors in a beaker surrounded by ice. ibc was discarded during mincing and replaced with 18 ml of ice-cold fresh ibc. the liver was then homogenized with a teflon pestle. the homogenates were transferred to a 50-ml polypropylene falcon tube and centrifuged at 600 g for 10 minutes at 4°c. the supernatants were transferred to fresh tubes for centrifugation at 7000 g for 10 minutes at 4°c. the supernatants were discarded, and the pellets washed with 5 ml icecold ibc. again, the supernatants from the pellets were centrifuged at 7000 g for 10 minutes at 4°c. the supernatants were discarded and the pellets containing the mitochondria resuspended. the concentration of the mitochondrial suspensions was measured using the biuret method. each 10 mg of isolated mitochondria was labelled with 1 mol/l of mitotracker red cmxros (invitrogen, carlsbad, ca, usa) through incubation at 37°c for 30 minutes for tracing the mitochondria in the ischaemia-reperfusion liver. mitochondrial transfusion was performed for the study animals immediately after labelling (ie <3 hours after the isolation procedure). the liver mri examination was performed using a 9.4-t horizontalbore animal mr scanning system (biospec 94/20; bruker, ettingen, germany). this scanning system comprises a self-shielded magnet with a 20 cm clear bore and a bga-12s gradient insert (inner diameter: serial 31 p-mrs examinations were performed on each rat at baseline (preoperatively), before-surgery and day 1, 2 and 3 post-surgery. the 31 p-mrs was acquired using a simple pulse-acquisition sequence, with a pulse optimized to give a 90° excitation in the liver region with the added advantage of overtripping signal from the abdominal region. respiratory gating was not used as the spectra acquired were nonlocalized, and hence, the resulting spectrum was the average 31 p-mrs signal from the sensitive area of the coil, which was optimized to have the largest signal (90° pulse) in the liver. the 31 p-mrs data were collected with a tr of 3000 ms, 512 averages, and a spectral width of 8000 hz. data were analysed in the time cross via nmr data processing guide of paravision5.1 (bruker). the phosphomonoesters (pme), inorganic phosphate (pi), phosphodiester (pde), nadh and the three-nucleotide triphosphate (mainly adenosine triphosphate) peaks in the spectrum were fitted after application of 0.5-hz line broadening and manual phasing. after preparation of haematoxylin and eosin (he) stain, the degree of liver injury was determined according to our previous report with liver injury score defined as follows: 0 indicated no notable hepatocyte integrity impairment or sinusoidal distortion; 1 corresponded to mild hepatic injury with less than 25% of section involved; 2 indicated moderate hepatic injury with 25%-50% of section involved; 3 denoted severe hepatic injury with more than 50% involved. 42 for each animal, three liver sections were examined and three randomly selected high-power fields (hpfs) (ie the procedure and protocol of staining were based on our previous reports. 42, 45, 46 in detail, rehydrated paraffin sections ( the procedure and protocol for western blot analysis have been described in detail in our previous studies. 42, 45, 46 briefly, equal amounts (50 µg) of protein extracts were loaded and separated by sds-page using acrylamide gradients. after electrophoresis, the separated proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (amersham biosciences, amersham, uk). nonspecific sites were blocked by incubation of the membrane in blocking buffer (5% 2000; cell signalling) was used as a secondary antibody for 1-hour incubation at room temperature. the washing procedure was repeated eight times within 1 hour. immunoreactive bands were visualized by enhanced chemiluminescence (ecl; amersham biosciences) and exposed to biomax l film (kodak, rochester, ny, usa). for the purpose of quantification, ecl signals were digitized using labwork software (uvp™ biospectrum™ imaging system, upland, ca, usa). quantitative data were expressed as mean ± sd. statistical analysis was adequately performed by anova followed by bonferroni's multiple comparison post hoc test. sas statistical software for windows version 8.2 (sas institute, arlington, va, usa) was utilized. a probability value <0.05 was considered statistically significant. first, to elucidate how the circulating levels of inflammatory biomarkers and the liver enzymes were augmented after ischaemia-reperfusion procedure, the elisa method was utilized for examination of these parameters. the result demonstrated that by 72 hours after ischaemia-reperfusion procedure, circulating levels of tnf-α, il-6 and mpo, three indicators of inflammation, were significantly increased in iri than in sc and iri + mito and significantly increased in iri + mito than in sc. consistently, serum levels of ast and alt, two indices of hepatocyte integrity, showed an identical pattern compared with that of inflammation. these findings imply that melatonin-pretreated mitochondria therapy attenuated circulatory inflammatory markers and protected the liver from acute iri (figure 1 ). next, to clarify whether mri was a sensitive and reliable non-invasive tool for determining the degree of liver iri, the phosphorus were substantially reduced in iri than in sc and iri + mito, and significantly reduced in iri + mito than sc. however, γ-atp level was only relatively reduced in iri as compared with the other two groups but it did not reach statistical significance, whereas the parameters of pme and pde did not differ among the three groups. these findings suggested that exogenous mitochondrial administration enriched the hepatocyte mitochondria ( figure 2 ). it is well known that mitochondrial integrity and the stability of mitochondrial-electron-transport-chain complexes (metcc) are fundamentally important for an effective-energy generation of atp in the cells. we therefore measured the protein expressions related to these two cardinal factors. consistently, the protein expressions of cytosolic cytochrome c, drp1 and cyclophilin d, three indices of mitochondrial-damaged biomarkers, were significantly higher in iri than in sc and iri + mito, and significantly higher in iri + mito than in sc, suggesting iri depleted hepatocyte endogenous mitochondria. on the other hand, the protein expressions of complex i, ii, iii and v, four indictors of metcc, and mitochondrial cytochrome c, an index of mitochondrial integrity, were significantly higher in sc than in iri and iri + mito, and significantly higher in iri + mito than in iri, suggesting that exogenous mitochondrial transfusion enriched the mitochondria in the injured hepatocytes ( figure 3a-h) . we further identified that the protein expressions of nox-1 and nox-2, two indicators of oxidative stress, were significantly increased in iri than in sc and iri + mito and significantly increased in iri + mito than in sc. on the other hand, the protein expressions of ho-1, nqo1 and nfr2, three indicators of antioxidants, were significantly progressively increased from groups 1 to 3, suggesting an intrinsic response to ischaemia-reperfusion stimulation that was enhanced by exogenous mito therapy ( figure 3i-m) . molecular level of inflammatory reaction has been crystal clearly identified to be augmented in setting of acute ischaemia-reperfusion. 16, 33, 36 here, we also found that the protein expressions of il-1ß, tnf-α, p-nf-κb, mmp-2 and mmp-9, five indices of inflammation, were significantly increased in iri than in sc and iri + mito, and significantly increased in iri + mito than in f i g u r e 2 mri findings of hepatic phosphorylated metabolism by 72 h after liver ischaemia-reperfusion injury induction. a, illustrating the hepatic 31 p-magnetic resonance spectra (ie 31 p-mrs) examination for identifying the atp (an indicator of energy storage) in hepatocytes of sc, iri and iri + mito animals, respectively. b-g, the relative level of each metabolites was subtracted from the β-atp value. the data are expressed as mean ± sd (n = 8 per group). one-way anova followed by post hoc tukey-kramer test was used for statistical analysis. symbols (*, †, ‡) indicate significant differences (at 0.05 level). atp, adenosine triphosphate; iri, ischaemia-reperfusion injury; nadh, nicotinamide adenine dinucleotide; pde, phosphodiester; pi, inorganic phosphate; pme, phosphomonoesters; sc, sham control sc. additionally, the protein expressions of pi3k, p-akt and p-mtor, three biomarkers of cellular stress signalling, displayed an identical pattern of inflammation among the three groups, suggesting that melatonin-pretreated mitochondria effectively suppressed inflammatory reaction ( figure 3n -u). to assess whether the therapy of melatonin-pretreated mitochondria would offer benefit on ameliorating the liver injury score, microscopic finding of haematoxylin and eosin-stained liver sections was performed in each animal. the result showed that the liver injury score was significantly increased in iri than in sc and iri + mito and significantly increased in iri + mito than in sc. additionally, the masson's trichrome stain displayed that the fibrotic area exhibited an identical pattern of liver injury score among the three groups. these findings implied that melatonin-pretreated mitochondria preserved the liver parenchymal architecture (figure 4 ). to examine the cellular level of inflammatory cell infiltrations in liver parenchyma, the if microscope was performed. consistent finally, we found that the cellular expressions of γ-h2ax, an indicator of dna-damaged marker, and mmp-9, an indicator of acute innate inflammatory reaction, were significantly higher in iri than in sc and iri + mito, and significantly higher in iri + mito than in sc ( figure 6 ). as a non-invasive and accurate instrument, the advantage of utilizing hepatic 31 p-mrs for detecting the metabolism of different liver disease entities has been extensively discussed. [39] [40] [41] however, there is still lacking data to address the situation of atp metabolism and consumption in hepatocytes in setting of iri with the exogenous mitochondrial supply. additionally, a non-invasive tool with precise diagnostic feature is in urgent need for our daily clinical practice to identify the degree of atp consumption and depletion in setting of iri, especially in acute liver iri, has not yet been reported. these issues encourage us to carry out this experimental study. one novel finding in the present study was that the 31 p-mrs tool clearly identified that the level of atp in hepatocytes of living animals was remarkably reduced in iri animals, suggesting that the mitochondria/atp, indicator of energy, was significantly depleted in hepatocytes. however, this parameter was remarkably reversed in iri animals treated by melatonin-pretreated mito. accordingly, our findings, in addition to extending the findings of previous studies, 39-41 pinpoint the exciting potential of studying metabolic processes in the human liver in vivo. accurate diagnosis of disease is of course very important, and the effective treatment of the disease is no doubted essential. plentiful data have shown that exogenous mitochondrial transfusion not only effectively rescued the function and integrity of endogenous mitochondria but also refreshed the depleted mitochondria in organ, resulting in preserving the ischaemia-reperfusion-related organ dysfunction. 16, 46 the most important finding in the present study was that melatonin-pretreated exogenous mitochondria not only refreshed the mitochondria (ie increased atp) in hepatocytes but also attenuated the anatomical-histopathological changes of liver parenchyma (ie ameliorated the liver injury score and fibrosis) and circulatory level of ast and alt (ie two indices of liver function/ hepatocyte damage). our findings, in addition to strengthening the plentiful studies have previously established that iri always elicits inflammatory reaction, dna damage, apoptosis and generation of oxidative stress, resulting in organ damage, tissue necrosis and unfavourable outcomes. [45] [46] [47] [48] [49] additionally, increased oxidative stress in circulation and intracellular/mitochondrial compartment notably down-regulated mitochondrial function and depleted energy-storage capacity. 16, 46 an essential finding in the present study was that these above-mentioned parameters of molecular-cellular perturbations [45] [46] [47] [48] [49] were substantially increased in iri animals as compared to sc animals. in this way, our findings, in addition to being consistent with the findings from previous studies, [45] [46] [47] [48] [49] could, once again, explain why the liver enzyme, fibrosis and liver injury score were markedly increased in iri animals. of importance was that melatonin-pretreated mito therapy significantly suppressed inflammatory reaction, dna damage, oxidative stress, cellular apoptosis and fibrosis. finally, figure 7 schematically summarized the innovative findings of our study to facilitate readers' understanding. conclusively, we first developed the 31 p-mrs technique for precise diagnosis of atp energy metabolism in the liver. second, we created an animal model of acute liver iri and measured the alternations of molecular-cellular perturbations in liver parenchyma and circulation as well as identified the ultrastructural changes of liver parenchyma. finally, we clearly proved that melatonin-pretreated exogenous mitochondria effectively protected the liver against iri mainly through refreshment of the endogenous mitochondria which were at the gate of death in iri hepatocytes. it is well known that melatonin is a dietary supplement that is very safe and non-toxic to humankind. some recent data have even shown that melatonin therapy effectively protected the lung against covid-19 infection mainly through regulating the immune system, that is immunomodulation. [50] [51] [52] [53] these mentioned issues [50] [51] [52] [53] and the results of our study raise the need of consideration melatonin could be utilized for those of acute liver iri patients shortly, especially in those of patients who are refractory to the conventional treatment. this study has limitations. first, although the results were attractive and promising, study period was relatively short (ie the study period was only 72 hours). accordingly, the long-term impact of melatonin-pretreated mito therapy on protecting the liver against iri is still currently unclear. second, despite the impact of melatonin on suppressing the cellular infiltration was clearly clarified, the therapeutic impact of this regimen on regulating the macrophage polarization (ie m1/m2 ratio) was not investigated. third, although extensive works were accomplished in the present study, the exact underlying mechanisms of melatonin-pretreated mito therapy on protecting the liver against iri remain regrettably uncertain. accordingly, based on our results, the proposed schematic mechanism of melatonin-pretreated mito therapy on safeguarding the liver in setting of iri only was illustrated in figure 7 . in conclusion, 31 p-mrs provided a reliable method for elucidating the level, activity and metabolic rate of atp in hepatocytes of living animals. besides, the results of the present study demonstrated that melatonin-pretreated mito therapy effectively protected the liver against iri in rat. we thank the molecular imaging core of the center for translational research in biomedical sciences, kaohsiung chang gung memorial f i g u r e 7 schematically proposed mechanism of mitochondrial transfusion for reducing acute liver ischaemiareperfusion injury in rat. 31 p-mrs, 31 p-magnetic resonance spectroscopy; atp, adenosine triphosphate; il-6, interleukin 6; iri, ischaemia-reperfusion injury; mito, mitochondria; mpo, myeloperoxidase; ros, reactive oxygen species; sc, sham control; tnf-α, tumour necrosis factor alpha hospital, kaohsiung, taiwan, for technical and facility supports on 9.4t animal mri. the authors confirm that there are no conflicts of interest. the data that support the findings of this study are available from the corresponding author upon reasonable request. https://orcid.org/0000-0002-6305-5717 liver ischaemia and reperfusion injury mechanisms of hepatic ischemia-reperfusion injury and protective effects of nitric oxide ischemia/reperfusion injury in liver surgery and transplantation: pathophysiology impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury reperfusion injury and reactive oxygen species: the evolution of a concept oxidative stress and acute hepatic injury hypoxic hepatitis: underlying conditions and risk factors for mortality in critically ill patients hypoxic hepatitis epidemiology of acute liver failure acute liver failure update on acute liver failure update in intensive care medicine: acute liver failure. initial management, supportive treatment and who to transplant neuroinflammation in acute liver failure: mechanisms and novel therapeutic targets heme oxygenase-1 in liver transplant ischemia-reperfusion injury: from bench-to-bedside acute-on-chronic liver failure: consensus recommendations of the asian pacific association for the study of the liver (apasl): an update systemic combined melatonin-mitochondria treatment improves acute respiratory distress syndrome in the rat mitochondria in chronic liver disease energy metabolism and oxidative status of rat liver mitochondria in conditions of experimentally induced hyperthyroidism melatonin and mitochondrial function during ischemia/reperfusion injury evaluation of bioenergetic and mitochondrial function in liver transplantation the role of mitochondria in ischemia/reperfusion injury in organ transplantation hepatic ischemia reperfusion injury: a systematic review of literature and the role of current drugs and biomarkers hepatic ischemia/ reperfusion injury-a fresh look neutrophils: a cornerstone of liver ischemia and reperfusion injury advances in parametric mapping with cmr imaging pink1 activation and translocation to mitochondria-associated membranes mediates mitophagy and protects against hepatic ischemia/reperfusion injury renalase attenuates mouse fatty liver ischemia/reperfusion injury through mitigating oxidative stress and mitochondrial damage via activating sirt1 melatonin and tryptophan derivatives as free radical scavengers and antioxidants melatonin: a mitochondrial targeting molecule involving mitochondrial protection and dynamics melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas mitochondria: central organelles for melatonin's antioxidant and anti-aging actions protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial ca(2+) stress by melatonin's cascade metabolites c3-ohm and afmk in rba1 astrocytes protective effect of melatonin-supported adipose-derived mesenchymal stem cells against small bowel ischemia-reperfusion injury in rat additional benefit of combined therapy with melatonin and apoptotic adipose-derived mesenchymal stem cell against sepsis-induced kidney injury melatonin reduces dimethylnitrosamine-induced liver fibrosis in rats melatonin treatment improves adipose-derived mesenchymal stem cell therapy for acute lung ischemia-reperfusion injury non-invasive methods for studying brain energy metabolism: what they show and what it means quantitative imaging of brain energy metabolisms and neuroenergetics using in vivo x-nuclear (2)h, (17)o and (31)p mrs at ultra-high field in vivo hepatic 31p magnetic resonance spectroscopy in chronic alcohol abusers hepatic 31p magnetic resonance spectroscopy: a hepatologist's user guide in-vivo(31)p-mrs of skeletal muscle and liver: a way for non-invasive assessment of their metabolism melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia-reperfusion injury in rats through suppression of mitochondrial permeability transition spironolactone effect in hepatic ischemia/reperfusion injury in wistar rats effects of apocynin on liver ischemia-reperfusion injury in rats combined therapy with ss31 and mitochondria mitigates myocardial ischemia-reperfusion injury in rats shock wave therapy enhances mitochondrial delivery into target cells and protects against acute respiratory distress syndrome early administration of cold water and adipose derived mesenchymal stem cell derived exosome effectively protects the heart from ischemia-reperfusion injury preactivated and disaggregated shape-changed platelets protect kidney against from ischemia-reperfusion injury in rat through attenuating inflammation reaction short-interval exposure to ambient fine particulate matter (pm2.5) exacerbates the susceptibility of pulmonary damage in setting of lung ischemia-reperfusion injury in rodent: pharmacomodulation of melatonin melatonin: roles in influenza, covid-19, and other viral infections lungs as target of covid-19 infection: protective common molecular mechanisms of vitamin d and melatonin as a new potential synergistic treatment melatonin inhibits covid-19-induced cytokine storm by reversing aerobic glycolysis in immune cells: a mechanistic analysis covid-19: melatonin as a potential adjuvant treatment hepatic 31 p-magnetic resonance spectroscopy identified the impact of melatonin-pretreated mitochondria in acute liver ischaemia-reperfusion injury key: cord-303046-unksl7p4 authors: pawlotsky, jean-michel title: covid-19 and the liver-related deaths to come date: 2020-06-11 journal: nat rev gastroenterol hepatol doi: 10.1038/s41575-020-0328-2 sha: doc_id: 303046 cord_uid: unksl7p4 coronavirus disease 2019 (covid-19) itself and/or the use of hepatotoxic drugs might negatively affect the course and management of patients with pre-existing chronic liver diseases. however, the greatest effect of covid-19 on liver diseases will be indirect and delayed, resulting from the impending global economic crisis. coronavirus disease 2019 (covid-19) was first reported in december 2019 in wuhan, china and was considered harmless by many observers. as of may 30th 2020, according to the covid-19 dashboard by the center for systems science and engineering at johns hopkins university, usa, six million people worldwide have been diagnosed with covid-19 and more than 365,000 of them have died from its complications. meanwhile, the world economy is collapsing. covid-19, which is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was initially thought to present essentially as an upper and/or lower respiratory tract infection. we now know that sars-cov-2 induces a systemic disease attacking many organs, potentially causing major damage, including mortality, and long-term sequelae. surprisingly, the liver seems to be relatively spared by the virus. indeed, apart from patients with the most severe forms of the disease resulting from so-called cytokine storm, who die from multivisceral deficiencies including the liver, manifestations of the infection in the liver are generally mild and transient and have no effect on the course of covid-19 disease. studies based on single-cell rna sequencing in healthy liver tissues suggest that the sars-cov-2 cellular receptor, angiotensin-converting enzyme 2 (ace2), is present mainly on cholangiocytes, whereas expression is 20-fold lower in hepatocytes and ace2 is not expressed in kupffer cells or intrahepatic immune cells 1,2 . the low or lack of expression of ace2 in hepatocytes explains that sars-cov-2 does not actually cause viral hepatitis. covid-19 is gene rally associated with mild-to-moderate elevations of aspartate aminotransferase (ast) and, to a lower extent, alanine aminotransferase (alt) levels, which are more frequent in patients with severe disease than in those with a benign outcome 3 . total bilirubin levels can also be slightly elevated, whereas alkaline phosphatase and γ-glutamyltransferase elevations have been rarely reported 3 . nonspecific histological changes in the liver have been described in small autopsy series, ranging from moderate microvesicular steatosis with mild, mixed lobular and portal activity to focal necrosis, with detection of the virus in the liver in some cases 4 . changes in liver biochemistry might result from the systemic inflammatory response, pneumonia-induced hypoxaemia and/or drug-induced hepatic injury, especially in patients with severe forms of covid-19 requiring hospitalization. the frequency of abnormal liver tests was reported to increase during hospitalization, possibly owing to the administration of hepatotoxic drugs, including antibiotics, nonsteroidal anti-inflammatory drugs, herbal products and antiviral agents, such as ribavirin, interferons, or the fixed-dose combination of lopinavir and ritonavir 5 . elevations in aminotransferase levels have also been occasionally reported with other drugs used in clinical trials in patients with covid-19, including chloroquine and hydroxychloroquine, azithromycin, remdesivir, favipiravir, camostat or tocilizumab. however, liver test abnormalities disappear when the infection recovers and/or when hepatotoxic drugs are removed. patients with chronic liver disease do not seem to be at greater risk of acquiring the infection than other indivi duals in the general population, but the potential onset of covid-19 in these patients raises two questions: will patients with chronic liver disease develop a more severe form of covid-19; and will covid-19 aggravate the course of their liver disease and induce liver-related mortality? one study has suggested that 65 patients with non-alcoholic fatty liver disease (nafld) related to the metabolic syndrome were four times more likely to develop severe manifestations of covid-19 than 65 patients without nafld, regardless of the presence of diabetes 5 . however, it is unclear whether nafld itself plays a part, rather than the associated metabolic comorbidities that include obesity, diabetes and hypertension, which are well-known risk factors for developing severe covid-19. patients with cirrhosis seem to be at greater risk than other individuals for severe covid-19 and the occurrence of complications, including mortality, regardless of the aetiology covid-19 and the liver-related deaths to come www.nature.com/nrgastro of liver disease 6, 7 . the risk could also be increased in indivi duals with decomp ensated cirrhosis, but few cases have been reported. data from registries from a large, multicentre international cohort of patients with chronic liver disease suggest that patients with compensated cirrhosis are at increased risk of decompensation and death during the course of covid-19, even in the absence of respiratory symptoms 8 . the contribution of thromboembolic disorders to liver injury in patients with the most severe forms of covid-19 has been suggested 4 . such thromboembolic events might expose patients with pre-existing chronic liver disease to serious hepatic complications. as a result of lockdowns and suspension of usual clinical care activities for the benefit of patients with covid-19, the sars-cov-2 pandemic is having a major effect on the management of patients with chronic liver diseases, in particular those with cirrhosis, hepatocellular carcinoma and in liver transplantation programmes. the feared risk of acquiring covid-19 at the hospital also prevented many patients from being appropriately managed by hepatology teams. the effect of the pandemic on care of patients with cirrhosis was anti cipated to follow three waves: first, an intense period with prioritized high-acuity care with delayed elective procedures and routine care during physical distancing; then a challenging 'return to normal' following the end of physical distancing, with increased emergent decompensations, morbidity and systems of care overwhelmed by the backlog of deferred care; and finally, a protracted period of suboptimal outcomes characterized by missed diagnoses, progressive disease and loss to follow-up 9 . the covid-19 pandemic will also negatively affect the care and management of patients with hepatocellular carcinoma, generating delayed diagnosis, deferred treatment (including medical and surgical, such as access to liver transplantation), loss to follow-up and, ultimately, increased mortality. liver transplantation has been challenging during the covid-19 pandemic, as many centres had to virtually stop or drastically reduce their transplantation programmes owing to a dramatic reduction in the number of donors and the switch of many care facilities into covid-19 units. sars-cov-2 testing in donors and recipients has been implemented in many places, but the effect of covid-19 on the outcome of liver transplantation is unknown. the role of post-transplantation immune suppression on the course of covid-19 is also largely unknown owing to the scarcity of data. overall, information is still limited, but it is likely that covid-19 will result in a substantial, delayed increase in liver-related mortality that will become visible in the coming months and will substantially contribute to the overall pandemic-related mortality. the covid-19 pandemic will also negatively affect viral hepatitis elimination programmes. the world health organization has set the goal of eliminating viral hepatitis b and c as major public health threats by 2030. this aim includes reducing their incidence, prevalence, morbidity and mortality by means of prevention measures, including hepatitis b vaccination, extensive screening and improved access to care and antiviral treatments. during the covid-19 crisis, attention has been diverted from chronic viral hepa titis, despite the fact that global mortality attributed to viral hepatitis, which has been estimated by the who global hepatitis report 2017 to be approximately 1.5 million per year, currently remains higher than that from covid-19, while resources for public health interventions are already shrinking. lockdown, quarantine and social distancing, closing of harm reduction and treatment facilities, including primary care settings and general practitioners, will probably hamper the major efforts made to achieve the viral hepatitis elimination goals in many areas, further increasing indirect covid-19-linked mortality 10 . the greatest effect of covid-19 on liver-related morbidity and mortality remains to come, as a result of the global economic crisis that has already begun. bankruptcies, job losses, money and food shortage, social isolation and family issues will lead to increases in alcohol and drug use, whereas access to care will suffer from the collapse of health-care structures and organizations, and from government policies diverting resources elsewhere. this situation could translate into a substantial increase in blood-borne virus transmissions (in addition to the current opioid epidemic in north america, which is already rapidly increasing the incidence of new hepatitis c cases), as well as in alcoholic liver disorders and decompensations, resulting in many more patients with cirrhosis, hepatocellular carcinoma, liver transplantation and liver-related death. these unforeseen effects of the covid-19 pandemic will take years to become visible, but they are inevitable. in summary (box 1), sars-cov-2 infection appears to relatively spare the liver, with negligible liver-related mortality during the course of covid-19 (with the exceptions of liver failure in patients with very severe covid-19 infection leading to multivisceral deficiencies and of patients with decompensated cirrhosis). nevertheless, the effect of the covid-19 pandemic on liver-related mortality could be enormous, though hidden, delayed and not ascribed. similar conclusions apply to many other medical specialties and should be considered when drawing up a final assessment of the covid-19 pandemic, the policies put in place to combat it and the lessons to be learnt for the next pandemic. coronavirus disease 2019 (covid-19) has a modest effect on the liver, and liver-related mortality is unusual during the course of the disease, except in patients with severe pulmonary disease who die from multivisceral deficiencies including the liver or in patients with pre-existing advanced liver disease. most covid-19-induced liver-mortality will be delayed, resulting from deferred care for liver diseases, reduced funding for public health interventions and the global economic crisis, which will lead to increases in alcohol and drug use and in blood-borne virus transmissions, while access to care and funding are reduced. prevention of liver-related mortality following the covid-19 epidemic will require rapid resumption of care activities, in particular for patients with advanced cirrhosis or hepatocellular carcinoma and for those requiring liver transplantation, a massive investment in viral hepatitis elimination efforts and courageous economic and social measures to mitigate the health consequences of the coming economic crisis. single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection covid-19: abnormal liver function tests autopsy findings and venous thromboembolism in patients with covid-19 metabolic associated fatty liver disease increases covid-19 disease severity in non-diabetic patients clinical characteristics and outcomes of covid-19 among patients with pre-existing liver disease in united states: a multi-center research network study prognosis of covid-19 in patients with liver and kidney diseases: an early systematic review and meta-analysis high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry the covid-19 pandemic will have a long-lasting impact on the quality of cirrhosis care covid-19 and viral hepatitis elimination programs: are we stepping backward? liver int the author has served as an advisor for abbvie, gilead, glaxosmithkline, merck, regulus and siemens healthcare. covid-19 dashboard by the center for systems science and engineering at johns hopkins university, baltimore, maryland: https://systems.jhu.edu/ who global hepatitis report 2017: https://www.who.int/hepatitis/ publications/global-hepatitis-report2017/en/ key: cord-016880-q44623s8 authors: van hoek, b.; verkade, h.j.; porte, r.j. title: 22 levertransplantatie date: 2015-01-02 journal: leverziekten doi: 10.1007/978-90-313-7437-3_22 sha: doc_id: 16880 cord_uid: q44623s8 in 1963 verrichtte thomas starzl in denver de eerste levertransplantatie bij de mens. in 1966 werden in nederland de eerste twee (auxiliaire, zie par. 22.3.6) levertransplantaties verricht in leiden en arnhem, in 1968 startte cambridge. helaas resulteerden de eerste levertransplantaties niet in langetermijnoverleving als gevolg van niet-optimale operatietechniek, matige immuunsuppressie en onbekendheid met complicaties. eindstadium chronische leverziekte betreft met name levercirrose, maar ook bijvoorbeeld het budd-chiari-syndroom, in een eindstadium. dat wil zeggen dat er sprake is van leverfalen (zie hoofdstuk 7). de complicaties die hierbij optreden zijn vastgelegd in prognostische scores. de bekendste is de child-pugh-classificatie (tabel 22.1). bij een child-pugh klasse c, of b met progressie is er veelal een indicatie voor levertransplantatie. [1] tegenwoordig wordt de mayo end-stage liver disease-score (meld-score) veel gebruikt. deze correleert invers met de driemaandsoverleving bij cirrose, ook bij hogere scores -waar de child-pughscore geen onderscheidend vermogen meer heeft. [2] bij een meld-score tussen 15 en 17 is de kortetermijnoverleving bij cirrose zonder en met levertransplantatie gelijk (figuur 22.1). bij ontvangst van een lever met een hoge donor-risico-index [3] is er op korte termijn alleen nadeel en pas later voordeel van de levertransplantatie. [4] meestal vindt levertransplantatie dus pas plaats bij een meld-score van meer dan 15. [5] de meld-score is opgebouwd uit het totaal bilirubinegehalte, de inr en het creatininegehalte (te berekenen op www.mdcalc.com en www.eurotransplant.nl). sommige ziekten zijn wel een indicatie voor levertransplantatie, maar hebben een lage meld-score, zoals polycysteuze leverziekte. daarvoor zijn er in eurotransplant uitzonderingen geformuleerd zodat deze patiënten extra punten krijgen, waardoor levertransplantatie toch vaak op tijd mogelijk is. voor een deel van de patiënten met een meld-score onder 24 is mogelijk meld gecombineerd met het serumnatrium een betere voorspeller voor overleving, zoals in de uk-eld die in groot-brittannië wordt gebruikt. [ levertransplantatie voor levertumoren betreft met name het hepatocellulair carcinoom (hcc). aangezien hcc veelal in een cirrotische lever ontstaat, is resectie met voldoende behoud van functie vaak niet mogelijk. ook een centrale ligging van de tumor of portale hypertensie kunnen resectie te riskant maken. resectie is dus de eerste optie, maar indien dit niet mogelijk is wordt levertransplantatie overwogen. er moet dan geen extrahepatische tumor aanwezig zijn. ter beoordeling van de uitbreiding worden een ct-thorax, ct-of mri-abdomen (vierfasencontrast) en een skeletscan verricht. soms is een laparoscopie nodig. bij ascites wordt deze geanalyseerd op maligne cellen. een tumor mag niet te groot zijn in omvang of aantal en er mag geen macrovasculaire invasie zijn, daar anders de kans op terugkeer van hcc na levertransplantatie te groot is. veelal worden daarbij de 'milaan-criteria' aangehouden: een tumor van maximaal 5 cm of maximaal drie van ieder maximaal 3 cm. [8] de tumorvrije overleving na levertransplantatie is echter niet slechter met de zogenaamde ucsf-criteria, die iets ruimer zijn: een tumor van maximaal 6,5 cm of maximaal drie tumoren van maximaal 4,5 cm, met totale diameter van maximaal 8 cm. downstaging tot binnen de milaan-criteria maakt soms levertransplantatie mogelijk. bij een fibrolamellair hcc be-hoeven genoemde grenzen niet aangehouden te worden. uit analyse van patiënten die getransplanteerd zijn terwijl ze buiten de milaan-criteria vielen, blijkt dat met name micro-angio-invasie een slechte prognostische marker is en dat de overleving bij grotere of meer dan drie tumoren nog goed kan zijn als er geen micro-angio-invasie is. er wordt momenteel hard gezocht met onder andere micro-arrays naar andere merkers die het biologisch gedrag van de tumor weergeven, zodat de selectie voor levertransplantatie kan verbeteren. bij een deel van de patiënten keert hcc namelijk terug na levertransplantatie, met name in lever, longen of botten. om de wachttijd te overbruggen wordt zo mogelijk lokale ablatieve therapie van de tumor(en) gedaan met radiofrequente ablatie (rfa), percutane ethanolinjectie (pei), transkatheter arteriële chemoembolisatie (tace), lokale intrahepatische radiotherapie met yttrium (sirt) of combinaties daarvan. er zijn aanwijzingen dat door deze aanvullende multimodale therapie op de wachtlijst de tumorvrije overleving na levertransplantatie verbetert. [9] mogelijk verkleint immuunsuppressie met sirolimus ook de kans op recidief. waarschijnlijk is het ook goed om in ieder geval een halfjaar te wachten met levertransplantatie na het starten van genoemde aanvullende therapie: bij patiënten met een biologisch agressieve tumor wordt dan onterechte levertransplantatie vermeden. de multi-tyrosinekinase-en angiogeneseremmer sorafenib verlengt de overleving bij anders onbehandelbaar hcc; wellicht gaan deze of andere systemische behandelingen een rol spelen als (neo)adjuvante therapie bij hcc. het cholangiocarcinoom is in principe een contra-indicatie voor levertransplantatie, omdat deze tumor een zeer hoge recidiefkans na levertransplantatie heeft: er blijft vrijwel altijd tumorweefsel achter ten gevolge van microscopische lokale doorgroei. recentelijk is in onderzoekssetting levertransplantatie ingepast als eindstation na intensieve voorbehandeling met chemoradiotherapie en stagiëring, waarbij vele patiënten voor levertransplantatie afvielen. de patiënten die uiteindelijk levertransplantatie ondergingen, hadden een acceptabele tumorvrije overleving. [10] wellicht is er dus in een dergelijk strict protocol in de toekomst toch plaats voor levertransplantatie bij geselecteerde patiënten met cholangiocarcinoom. overige tumoren waarvoor bij geselecteerde patiënten levertransplantatie verricht wordt, zijn het hemangio-endothelioom, neuro-endocriene tumoren, hepatoblastoom (naast chemotherapie) en nog enkele zeldzame tumoren met een goede prognose na levertransplantatie. soms is levertransplantatie een optie bij grote goedaardige tumoren, al wordt dan meestal naar andere oplossingen gezocht. voor levertransplantatie bij metastasen van bijvoorbeeld een coloncarcinoom, is momenteel geen plaats wegens de hoge recidiefkans en de schaarste aan donorlevers. bij patiënten zonder voorafgaande leverziekte die hyperacuut, acuut of subacuut leverfalen ontwikkelen (zie hoofdstuk 7 voor kliniek, oorzaken en overige behandeling) is er vaak een indicatie voor hoogurgente levertransplantatie. [11] [12] [13] bij de indicatiestelling voor levertransplantatie worden meestal de criteria van het king's college hospital (kch-criteria) aangehouden (kader). bij virale etiologie (hepatitis b) werden in het verleden de clichycriteria gehanteerd, maar die zijn minder accuraat. de kch-criteria hebben een goede negatief voorspellende maar matige positief voorspellende waarde. dit laatste kan mogelijk verbeterd worden door het toevoegen van serumlactaat (> 3,5 mmol/l bij opname of > 3,0 mmol/l na resuscitatie met infuus 3-12 uur na opname). [ de positief en negatief voorspellende waarde van een meld-score onder of boven 33 is ongeveer gelijkwaardig aan de kch-criteria. ook de apache ii-score en de sofa-score zouden bruikbaar zijn. volgens een recente publicatie zou aanwezigheid van drie van de volgende zes criteria de noodzaak van een levertransplantatie beter voorspellen dan de kch-criteria en de meld-score: [16] -leeftijd 50 jaar of ouder; -interval tussen icterus en encefalopathie > 7 dagen; -encefalopathie graad 3-4; -hersenoedeem; -pt > 35 seconden; -creatinine > 132 mmol/l (1,5 mg/dl). totdat de overige scores goed gevalideerd zijn wordt aangeraden de kch-criteria, eventueel aangevuld met het serumlactaat te gebruiken. voor sommige indicaties, zoals de ziekte van wilson, is een aparte score ontworpen. [17] [18] vaak is het geïndiceerd om de preoperatief gestarte koeling, conti-nue venoveneuze hemofiltratie en antibiotica peroperatief en postoperatief enige tijd voort te zetten. met name moet niet alleen vóó r maar ook tijdens de levertransplantatie gewaakt worden voor toename van hersenoedeem en hypoglykemie. [19] j 22.1.4 enzymdeficiënties die gelokaliseerd zijn in de lever, die niet op andere wijze te behandelen zijn en die aanleiding geven tot irreversibele, levensbedreigende aandoeningen kunnen een indicatie zijn voor levertransplantatie. deze indicaties spelen vooral op de kinderleeftijd (zie par. 22.6). bij volwassenen gaat het bijvoorbeeld om familiaire amyloïdotische polyneuropathie. in zulke gevallen waarbij de lever op één enzym na normaal is, kan soms een dominolevertransplantatie plaatsvinden (zie par. 22.3.5). bij hemochromatose, de ziekte van wilson of alfa-1antitrypsinedeficiëntie is het leverfalen bij cirrose meestal de reden van levertransplantatie, al kan de ziekte van wilson zich als acuut leverfalen presenteren. [20] j 22.1.5 ongeveer 10 procent van de levertransplantaties is een retransplantatie. dit kan nodig zijn bij falen van een transplantatielever of bij recidiverende sepsis vanuit de donorlever, bijvoorbeeld bij galwegstricturen met cholangitis. in de eerste twee weken na de eerste levertransplantatie komt dit vooral door primaire non-functie of bij een arteria hepatica-trombose. later na transplantatie kan dit komen door bijvoorbeeld non-anastomotische stricturen in de galwegen of terugkeer van de oorspronkelijke ziekte. bij een vroeg falen van een transplantaat is er een hoge mortaliteit op korte termijn en mag een hoogurgente retransplantatie plaatsvinden; meer dan twee weken na een eerste levertransplantatie kan dit alleen in uitzonderingsgevallen. de eenjaarsoverleving van relevertransplantatie is lager (ongeveer 50%) dan van eerste levertransplantatie; dit komt vooral door de slechtere overleving bij vroege retransplantaties. levertransplantatie is tijdelijk of soms definitief niet mogelijk bij systemische infectie waarbij de infectiefocus niet in de lever ligt, bij extrahepatische maligniteit -tenzij follow-up genezing waarschijnlijk maakt -en bij andere aandoeningen die de prognose negatief beïnvloeden of kunnen leiden tot complicaties bij levertransplantatie (bijv. aandoeningen van hart of longen, zoals acute respiratory distress-syndroom, of extreme ondervoeding). ook indien een patiënt medicatie niet juist kan innemen, medische voorschriften niet begrijpt of niet in staat is de risico's van de behandeling en eisen die gesteld worden te begrijpen, is een levertransplantatie niet mogelijk. ook bij ernstige psychiatrie, actieve verslaving (uitgezonderd methadon), hersen-dood (met name bij comateuze patiënten, bijv. door inklemming van de hersenstam bij acuut leverfalen) en ernstige niet-reversibele portopulmonale hypertensie wordt geen levertransplantatie uitgevoerd. [20] obesitas kan levertransplantatie moeilijker maken, maar heeft in afwezigheid van comorbiditeit geen invloed op de overleving en is dus geen absolute contra-indicatie. [21] coronairlijden, nierinsufficiëntie, diabetes mellitus, obstructief longlijden (copd) en bindweefselziekten verkleinen de overlevingskans na levertransplantatie. [22] ook bij een meldscore van meer dan 30 daalt de overlevingskans na levertransplantatie vandaar dat levertransplantatie zo mogelijk voor dat moment moet plaatsvinden. seropositiviteit voor hiv is een relatieve contra-indicatie (met name bij hepatitis c). uitgebreide veneuze trombose tot in de vena mesenterica superior was vroeger een contra-indicatie, maar tegenwoordig zijn vaak oplossingen mogelijk in de vorm van renoportale anastomose of cavoportale transpositie. voordat iemand op de wachtlijst geplaatst wordt, is een uitvoerig vooronderzoek verricht en wordt alles in een multidisciplinair team besproken om de indicatie, de contra-indicaties en de timing goed af te wegen. de patiënt en zijn of haar naasten worden uitgebreid voorgelicht. ook tijdens de wachtlijstperiode wordt de patiënt geregeld onderzocht om veranderingen waar te nemen, medicatie bij te sturen en alles te optimaliseren voor de transplantatie. helaas overlijdt in nederland momenteel ongeveer 15 procent van de patiënten op de wachtlijst voordat er een donorlever beschikbaar is. de donorlever wordt volgens een standaardprocedure door een hiervoor gecertificeerde chirurg verwijderd bij de donor. dit is meestal een hersendode, heart-beating (hb) donor, soms een non-heart-beating (nhb) donor -ook deceased from cardiac death (dcd) donor genoemd. de tijd tussen het bij de donor spoelen van de lever met koude bewaarvloeistof en het van het ijs halen van de lever bij de ontvangeroperatie heet de koude ischemietijd, de tijd tussen dit moment en de reperfusie met bloed van de ontvanger is de warme ischemietijd. de totale ischemietijd moet onder de 24 uur -en liefst minder -blijven; onder de 10 uur is optimaal. bij nhb-donoren is er per definitie een hartstilstand, dus een extra eerste warme ischemietijd, die kan leiden tot extra ischemie en/of reperfusieschade aan de lever en galwegen. door strenge selectie van donoren en het kort houden van ischemietijden kunnen even goede resultaten als met hb-donoren gehaald worden. [23] [51] voor implantatie wordt een donorlever uitgebreid nagekeken en voorbereid op implantatie. ook vindt controle van de donor plaats op onder andere hiv en hepatitis b en c. de kwaliteit van donorlevers verschilt en kan bijvoorbeeld worden weergegeven in een donor risico-index. [3] eurotransplant alloceert levers op naam naar bloedgroep en gewicht en in volgorde van de meld-score, eerst binnen de regio (nederland en belgië zijn ieder een regio) en daarna erbuiten. bij hoge urgentie (acuut leverfalen of retransplantatie binnen twee weken) wordt direct volgens de eurotransplant-wachtlijst (dus ook over de grenzen heen) gealloceerd. het transplantatieteam beslist van tevoren of een door eurotransplant aangeboden lever geaccepteerd kan worden voor deze patiënt, op grond van gegevens van zowel donor als ontvanger. zo leidt een sterk vervette lever voor een oude ontvanger met hepatitis c tot een grote kans op complicaties. meestal wordt bij de ontvanger de zieke lever verwijderd en vervangen door een gehele donorlever op dezelfde plaats (orthotope levertransplantatie, olt). [24] de operatie bij de ontvanger bestaat uit drie fasen: (1) resectie van de eigen lever (met galblaas) via een bilaterale subcostale incisie, (2) de anhepatische fase (zonder lever, waarin bloedingen gestopt worden en de nieuwe lever ingehecht wordt) en (3) de fase na reperfusie van de donorlever. de eerste fase kan moeilijk zijn, met name bij ernstige portale hypertensie. soms is het nodig een tijdelijke portocavale shunt aan te leggen. vroeger werd de vena cava inferior, die immers door de lever loopt, met de lever verwijderd; er was dan een perfusiecircuit nodig om het bloed van de onderste helft van het lichaam naar het hart terug te brengen (figuur 22.3). tegenwoordig wordt de eigen lever van de vena cava 'afgepeld', waardoor deze cavo-cavale bypass niet meer nodig is. dit scheelt operatietijd en leidt tot minder bloedverlies. bij het inhechten wordt de onderzijde van de vena cava van de donor 'afgestapled' en de bovenzijde van de vena cava wordt side-to-side of end-to-side op de vena cava van de ontvanger gezet, de zogenaamde piggy-back-methode (figuur 22.4). na de cavo-cavale en de porto-portale anastomose volgt reperfusie. in deze fase kunnen grote hemodynamische schommelingen en verschuivingen in elektrolyten (bijv. kalium), het zuur-base-evenwicht en de stolling en kan fibrinolyse optreden. na de reperfusie wordt de arteriële anastomose gemaakt, meestal op de eigen leverarterie, soms via een conduit op de aorta. de ductus choledochus wordt vervolgens op de eigen ductus choledochus gezet, ofindien dat niet mogelijk is -op een roux-en-ydarmlis. bij de resectie is de galblaas verwijderd; de ontvanger krijgt geen galblaas terug, daar dit leidt tot infecties en steenvorming. tijdens de operatie worden vele laboratoriumwaarden frequent bewaakt. de stolling wordt integraal bewaakt met trombo-elastografie (teg) op de operatiekamer. zonodig wordt medicatie tegen hyperfibrinolyse gegeven. ook is een rapid infusion system aangesloten om bij veel bloedverlies in korte tijd veel bloed te kunnen transfunderen. zonodig worden met de cell-saver de cellen uit het verloren bloed gecentrifugeerd en met plasma van de bloedbank als autotransfusie teruggegeven. tegenwoordig is er meestal slechts weinig transfusie nodig. aan het eind van de vier tot tien uur durende operatie is te zien of de donorlever functioneert aan de stolling, het zuurstofverbruik en de functietesten. bij metabole ziekten en acuut leverfalen kan het bij hemodynamisch stabiele jongere patiënten aantrekkelijk zijn de eigen lever niet geheel te verwijderen en er een halve of hele donorlever naast te zetten. met een dergelijke auxiliaire levertransplantatie, worden bij geselecteerde patiënten goede resultaten bereikt. [25] [26] de oudere heterotope auxiliaire levertransplantatie (halt), waarbij een partieel transplantaat onder de eigen lever werd gezet, is grotendeels verlaten. tot nu toe wordt bij auxiliaire levertransplantatie in veel centra de auxiliair partieel orthotope levertransplantatie (apolt) gebruikt, met arteriali-satie van de vena portae, waarbij een deel van de eigen lever verwijderd wordt. recentelijk werd een verdere verbetering van de techniek ontwikkeld: de auxiliaire levertransplantatie met reno-portale anastomose (repalt) (figuur 22.5). [27] hierbij komt de portaalveneuze inflow van het transplantaat uit de niervene van de ontvanger, en is vrijwel geen resectie van de eigen lever nodig, wat een aantal voordelen heeft. het blijkt dat bij twee derde van de overlevenden van auxiliaire levertransplantatie regeneratie van de eigen lever optreedt. [26] de donorlever kan dan weer worden verwijderd -en soms zelfs hergebruikt -of de donorlever atrofieert door de afweeronderdrukkende medicatie uit te sluipen. [28] het voordeel van auxiliaire levertransplantatie voor de patiënt is dat deze zonder de riskante immuunsuppressie verder door het leven kan. [29] bij een hemodynamisch instabiele patiënt kan olt -of auxiliaire levertransplantatie met een grote resectie van de natieve lever [30] -beter zijn omdat resectie van de necrotische lever het toxic liver-syndroom -door cytokines -kan tegengaan. soms kan een lever van een donor die ouder is dan 40 jaar, meer dan 40 kg weegt en minder dan vijf dagen op de icu gelegen heeft, gesplitst worden voor een volwassene (rechterkwab, figuur 22.6) en een kind (de kleinere linkerkwab, figuur 22.7). [31] dit wordt een split levertransplantatie genoemd. ook kan in sommige gevallen een grote lever gesplitst worden voor twee volwassen ontvangers. [32] een nadeel van split levertransplantatie is dat de extra ischemietijd en het resectievlak leiden tot meer complicaties en een iets lagere overlevingskans dan bij olt. als dit overlijden op de wachtlijst te als iemand wegens een stofwisselingsdefect in de lever een levertransplantatie moet ondergaan, kan de verwijderde lever soms hergebruikt worden voor een andere -meestal urgente -ontvanger. dit kan bijvoorbeeld bij familiaire amyloïdotische polyneuropathie; het duurt dan ongeveer tien jaar voor de nieuwe ontvanger verschijnselen van polyneuropathie krijgt en geretransplanteerd moet worden. [33] j 22.3.6 donatie bij leven is in japan ontwikkeld, aanvankelijk als donatie van de linkerkwab van een ouder aan een kind. later werd ook de grotere rechterkwab gedoneerd van volwassene aan volwassene. uiteraard moeten potentiële donoren een uitgebreide beoordeling ondergaan. [34] dan nog gaat donatie van een rechterkwab gepaard met ongeveer 1 à 2 procent mortaliteit en 20 procent morbiditeit. bij donatie van segment 2 en 3, van bijvoorbeeld volwassene aan kind, ligt het donatierisico een factor 10 lager. het is bij volwassen ontvangers meestal nodig de rechterkwab te doneren omdat zowel de ontvanger als de donor voldoende levermassa over moeten houden (dat is als de lever > 0,8% van het lichaamsgewicht is). is dit niet het geval, dan treedt het smallfor-size-syndroom (sfs) op, met icterus en leverfalen. dit is voor een deel het gevolg van hyperperfusie wat leidt tot beschadiging van sinusoïdaal endotheel en een belemmerde afvloed. ook als de grote levervenen niet op de circulatie van de ontvangerworden aangesloten, kan een belemmerde afvloed en daardoor een functioneel sfs ontstaan. sinds kort worden ook twee linkerkwabben van twee donoren samen in een ontvanger getransplanteerd. [35] dit garandeert voldoende levervolume bij de ontvanger én de donoren. in azië, de verenigde staten en sommige centra in europa maakt levertransplantatie met een levende donor een substantieel percentage van het aantal levertransplantaties uit. in nederland is levertransplantatie met een levende donor begonnen, al is er vooralsnog terughoudendheid vanwege de risico's voor de donor. [36] de resultaten bij de ontvangers zijn zeker even goed als bij olt met een postmortale donor en mogelijk beter indien de overleving op de wachtlijst meegeteld wordt. [37] j 22.3.7 tijdens de operatie en daarna levenslang wordt immuunsuppressie gegeven om afstoting van de donorlever te voorkomen. [1] dit treedt nog maar bij ongeveer 20 procent van de patiënten op. de dosering kan na ongeveer drie maanden omlaag naar een onderhoudsdosis. meestal wordt begonnen met corticosteroïden, een calcineurineremmer (tacrolimus of cyclosporine) en eventueel mycofenolaatmofetil. ook wordt vaak in de eerste dagen na levertransplantatie een infuus met anti-cd25, bijvoorbeeld basiliximab, gegeven. verder is het gebruikelijk de corticosteroïden tussen drie en zes maanden verder af te bouwen en zo mogelijk te stoppen. rond de operatie geven de meeste centra intraveneus antibiotica, bijvoorbeeld gedurende 24 uur. sommige geven selectieve darmdecontaminatie met niet-resorbeerbare antibiotica -eventueel aangevuld met norfloxacine. in de eerste dagen wordt frequent laboratoriumonderzoek verricht en echografie van de buik verricht, onder andere om arteriahepaticatrombose uit te sluiten. ook wordt gelet op vochtcollecties (eventueel diagnostische punctie met granulocytgetal en microbiologisch onderzoek) en op galwegdilatatie (al is dat een laat optredend fenomeen bij afvloedbelemmering). meestal wordt in de eerste week een ct-abdomen verricht met dezelfde doelstellingen. de medicatie wordt ingesteld en aangepast op bloedspiegels; eventuele rejectie nabloeding of gallekkage kunnen redenen zijn voor relaparotomie. zo mogelijk wordt geprobeerd de patiënt eerst te stabiliseren. leverschade door ischemie en/of reperfusie leidt meestal tot een asat-en alat-piek in de eerste drie dagen als gevolg van necrose in zone 3; meestal dalen de aminotransferasen snel in de eerste dagen en herstelt deze schade. aminotransferasen boven 1500 in de eerste drie dagen zijn vaak een uiting van ernstige leverschade door ischemie en/of reperfusie en zo'n aminotransferasepiek kan gevolgd worden door een periode van intrahepatische cholestase. behalve de bekende complicaties van grote operaties zoals nierfalen, acuut respiratoir distress syndroom (ards) en hemodynamische instabiliteit, zijn er een aantal specifiek aan levertransplantatie gekoppelde problemen. primaire non-functie (pnf) komt bij 3 tot 5 procent van de levertransplantatie voor, primaire graft-disfunctie (pgd) of initiële slechte functie (ipf) bij 10 procent. manifestaties zijn: slechte stolling, bloeden uit buikdrains, hoge aminotransferasen, oplopend bilirubinegehalte, hypoglykemie en geen galuitvloed (al wordt dit niet vaak meer gezien nu er meestal geen externe galdrain gebruikt wordt). systemische uitingen zijn icterus, encefalopathie, hemodynamische instabiliteit, acidose en nierfalen. het wordt veroorzaakt door ischemie en/of reperfusieschade, soms bovenop al aanwezige schade in de donorlever door onder andere steatose, celoedeem, hersendood van de donor en shock. [38] er zijn enkele bekende risicofactoren voor pnf en pgd, zoals een hogere leeftijd van de donor, een sterk vervette donorlever, een hoog serumnatrium van de donor en de duur van ic-verblijf. bij de ontvanger zijn dit het creatinine-en bilirubinegehalte, de life support en de urgentiestatus op de wachtlijst. verder zijn er intraoperatieve factoren zoals de koude ischemietijd, de hoeveelheid bloedtransfusie en de warme ischemietijd. [39] aanvankelijke berichten dat infusie van prostaglandine e1 tegen pnf en pgd zou beschermen, konden niet worden bevestigd. bij nhb-donoren geven een leeftijd onder 45 jaar, warme ischemie bij de donor van minder dan 15 minuten en een koude ischemietijd van minder dan 10 uur bij een niet-urgente ontvanger een uitkomst die vergelijkbaar is met die bij hb-donoren. [23] j 22.4.2 trombose van de arteria hepatica (hat) komt bij ongeveer 1 procent van de levertransplantaties voor, met name bij aanwezigheid van arteriële varianten en vooral in de eerste dagen na transplantatie. hat uit zich meestal doordat asat en alat plotseling zeer fors stijgen. vervolgens ontstaan necrotische gebieden in de lever en/of galwegstricturen. een aneurysma ter hoogte van de arteriële anastomose kan het gevolge zijn van een lokale (mycotische) infectie. hat of schade aan de arteriële plexus kan ook leiden tot intrahepatische cholestase door downregulatie van galzuurtransporters in de hepatocytmembraan, alvorens stricturen van de grotere galwegen optreden. [40] bij een deel van de patiënten kan een hat die maanden tot jaren na levertransplantatie optreedt met opmerkelijk weinig symptomen gepaard gaan, maar in de acute fase kan alleen snelle interventie een retransplantatie voorkomen. soms is er een arteriële stenose die met een stent behandeld kan worden. [41] bij de donor wordt zonodig een arteriële reconstructie verricht. de noodzaak voor zo'n reconstructie en een hogere donorleeftijd zijn risicofactoren voor hat. zonodig wordt via een conduit een anastomose rechtstreeks op de aorta gemaakt. meestal wordt een end-to-endanastomose op de arteria hepatica gemaakt. direct na levertransplantatie en de volgende dag -en bij verdenking op hat -wordt de doorgankelijkheid van de arteria hepatica met echo-doppler gecontroleerd. als geen arterieel signaal gezien wordt, moet direct een ct-angiografie verricht worden. hat en trombose van de vena portae (pvt) kunnen gepaard gaan met infectie of uitingen van leverfalen zoals genoemd in paragraaf 22 terwijl vroeger een acute cellulaire rejectie bij 70 procent van de patiënten na levertransplantatie voorkwam, is dat nu -met de verbeterde immuunsuppressie -nog maar 20 procent. vroeger trad rejectie meestal in de eerste twee weken na levertransplantatie op, tegenwoordig ook wel later na het verlagen van de immuunsuppressie, na conversie naar een ander immuunsuppressivum of bij onjuiste inname van de medicatie. rejectie uit zich eerst door oplopende leverenzymen, daarna door vermoeidheid, drukgevoel in de rechterbovenbuik, en soms laat door koorts en icterus. [42] bij verdenking op rejectie moeten andere oorzaken uitgesloten worden en wordt een leverbiopsie verricht die deze diagnose ondersteunt: een portaal lymfocytair infiltraat, endotheliitis en even-tueel cholangitis. meestal worden daarvoor de banff-criteria aangehouden. behandeling met hoge doses corticosteroïden gedurende enkele dagen, in combinatie met het optimaliseren van de onderhoudsimmuunsuppressie, waarbij tijdelijk serumspiegels als vlak na levertransplantatie gehanteerd worden, is bijna altijd succesvol. onbehandelde rejectie leidt tot chronische rejectie met verlies van galgangen in de portadriehoekjes, ductopenie. waar rond 1990 nog bij 10 procent van de levertransplantaties chronische ductopene rejectie optrad, is dat nu nog bij 1 procent van alle levertransplantaties het geval, vooral bij niet-herkende late rejectie onder te lage immuunsuppressie of soms bij interferontherapie voor hepatitis c. chronische ductopene rejectie uit zich als cholestase en meestal is retransplantatie de enige optie. j 22.4.4 infectie bij een grote buikoperatie kunnen gemakkelijk infecties optreden, zeker bij een zieke patiënt die zware immuunsuppressie krijgt. infecties zijn belangrijke veroorzakers van morbiditeit en mortaliteit na levertransplantatie. [43] [44] bacteriële en soms mycotische infecties vlak na levertransplantatie veroorzaken vooral peritonitis, pneumonie, cholangitis en urineweginfecties. geïnfecteerde vochtcollecties of hematomen in de bovenbuik komen met name geregeld voor. de natuurlijke immuniteit van donor en ontvanger spelen een rol bij de kans op infecties na levertransplantatie. [45] bij koorts of andere tekenen van infectie is snelle diagnostiek (soms inclusief ascitespunctie of bronchoscopische lavage) en behandeling nodig. [46] door selectieve darmdecontaminiatie is waarschijnlijk een verschuiving van gramnegatieve naar meestal minder heftig verlopende grampositieve infecties te bewerkstelligen en door gerichte profylaxe is het aantal gistinfecties te verminderen. [47] ook virale infecties kunnen optreden, bijvoorbeeld primo-infectie met of reactivatie van het cytomegalovirus (cmv). in het eerste jaar vindt geregelde controle van cmv-dna in het bloed plaats, met pre-emptieve therapie met (val)gancyclovir zodra deze test positief wordt. bij combinatie van een seropositieve donor en een seronegatieve ontvanger met betrekking tot igg-anti-cmv wordt (val)gancyclovir profylactisch gegeven gedurende drie maanden. [48] het epstein-barr-virus (ebv) kan (bij 1% van de levertransplantaties) leiden tot posttransplantatielymfomen (ptld); die zijn tegenwoordig vaak curatief te behandelen en voor een deel mogelijk te voorkomen door ebv-dna-metingen in het eerste jaar, met zonodig aanpassen van de immuunsuppressie. [49] als een patiënt zich na levertransplantatie presenteert met koorts moet onmiddellijk diagnostiek op de spoedeisende hulp plaatsvinden en wordt direct gestart met intraveneuze antibiotica (bijv. combinatie van cefuroxim en gentamycine). als na 48 uur de kweken negatief blijven, kan daarmee vaak gestopt worden en anders worden de antibio-tica daarna gericht op geleide van de kweek. als een cholangitis de oorzaak kan zijn, kan een ercp nodig zijn ter uitsluiting en behandeling van galwegstricturen. j 22.4.5 galwegcomplicaties uiten zich vaak door cholestase en cholangitis. bij galwegstricturen zijn er bij echografie vaak geen uitgezette galwegen zichtbaar, mogelijk doordat de patiënt zich onder immuunsuppressie snel met cholangitis presenteert. diagnostiek vindt plaats met behulp van mrcp of ercp. kort na levertransplantatie kan er een gallek op de anastomose zijn, meestal met een galcollectie ernaast, een biloom. een klein gallek is vaak via ercp met papillotomie en het plaatsen van een endoprothese te behandelen (figuur 22.8). soms, met name bij een biliodigestieve anastomose, is percutane transhepatische cholangiografische (ptc) galwegdrainage nodig; deze kan tegenwoordig gedeeltelijk door ercp met de dubbelballonendoscoop vervangen worden. bij een biloom wordt dan meestal tevens een percutane drain in het biloom geplaatst, met kweek van de inhoud en bij infectie tijdelijk behandeling met antibiotica. een groot gallek vereist soms chirurgie (reanastomose of conversie naar biliodigestieve anastomose met een roux-en-y-lis). de oorzaak van een gallek is vaak de matige doorbloeding van het onderste deel van de ductus choledochus van de donor. dit is ook de oorzaak van anastomotische stenosen. die treden vooral op in het eerste halfjaar na levertransplantatie en zijn meestal goed via ercp of ptc met dilatatie en endoprothese of percutane drainage te behandelen. ook hiervoor is soms reoperatie nodig. [50] niet-anastomotische galwegstricturen kunnen alleen de donorcholedochus betreffen (type a), de bifurcatie (type b), ook het gebied daarboven (type c) of de gehele lever (type d). ze worden ook wel ischemic-type biliary lesions (itbl) genoemd, omdat de meeste van deze niet-anastomotische stricturen in het eerst halfjaar ontstaan en vooral aan ischemie en/of reperfusieschade gerelateerd lijken te zijn. latere stricturen lijken meer aan cmv en bijvoorbeeld terugkerende scleroserende cholangitis gerelateerd te zijn. [51] stricturen van type a en type b zijn vaak wel via ercp of ptc te behandelen, bij type c en type d kan retransplantatie nodig zijn, al kunnen sommige patiënten met onderhoudsantibiotica langdurig vrij van cholangitis blijven. bij olt met een lever van een hartdode donor treden meer galwegstricturen op, al kan de patient-en transplantaatoverleving bij goede selectie van donoren hetzelfde zijn als bij olt met een lever van een hersendode donor. [52] j 22.4.6 tumoren vaker dan posttransplantatielymfomen komen huidtumoren voor na levertransplantatie. als de immuunsuppressie een mtor-remmer (sirolimus of everolimus) bevat, komen minder huidtumoren voor. patiënten die levertransplantatie hebben ondergaan wegens door alcoholgebruik veroorzaakte cirrose, hebben een verhoogde kans op orofarynxen slokdarmtumoren. na levertransplantatie wegens primaire scleroserende cholangitis en bij inflammatoire darmziekte is de kans op colonkanker sterk verhoogd en wordt jaarlijkse colonoscopie met biopsieën ter uitsluiting van dysplasie aangeraden. ook van andere tumoren is de incidentie enigszins hoger na levertransplantatie dan in de gewone populatie. j 22.4.7 het hepatitis b-virus (hbv) moet bij een hbsagpositieve patiënt onderdrukt worden tegen terugkeer van actieve hbv-infectie in de nieuwe lever. hiervoor wordt een combinatie van anti-hbs-antistoffen en replicatieremmers gebruikt, waarmee dit meestal goed lukt. het is wel nodig de hbv-dnaload te vervolgen en zonodig de hbv-medicatie aan te passen. als hepatitis c nog aanwezig is ten tijde van levertransplantatie, kan dit in 60 procent van de gevallen leiden tot enige mate van chronische hepati-tis na levertransplantatie; ongeveer 10 procent van de patiënten krijgt actieve hepatitis c en ontwikkelt versneld cirrose in het transplantaat, leidend tot een verminderde overleving en retransplantatie. ook auto-immuunhepatitis, primaire biliaire cirrose en psc kunnen in het transplantaat terugkeren. mogelijk kan de activiteit daarvan met veranderde immuunsuppressie en ursodeoxycholzuur verminderd worden. ook een aantal andere aandoeningen, waaronder hepatocellulair carcinoom, kan terugkeren in de getransplanteerde lever. de calcineurineremmers cyclosporine en tacrolimus kunnen leiden tot chronisch nierfalen en hypertensie. om deze reden staan schema's waarbij in het eerste jaar van een calcineurineremmer naar een mtor-remmer gegaan wordt momenteel in de belangstelling. bij cyclosporine kan haargroei in het gelaat optreden, bij tacrolimus -vooral in combinatie met prednison -komt vaker diabetes mellitus voor. corticosteroïden kunnen osteopenie en osteoporose verergeren of doen ontstaan en leiden tot cosmetische bezwaren, water-en zoutretentie, hypertensie en soms psychische klachten. azathioprine en mycofenolaat-mofetil kunnen leiden tot maagklachten en de laatste ook tot diarree en beide kunnen cytopenie geven. mtor-remmers (sirolimus, everolimus) kunnen leiden tot huidklachten of -bij hoge doses -mondulcera. dit zijn slechts enkele voorbeelden. vaak zijn bijwerkingen de reden om de immuunsuppressie te wijzigen, soms moet medicatie bijgegeven worden wegens bijwerkingen (antihypertensiva, orale antidiabetica, insuline). verhoogde gevoeligheid voor infectie blijft een van de belangrijkste bijwerkingen van immuunsuppressie. immuunsuppressie verhoogt de kans op maligniteiten, meestal van de huid, in ongeveer 1 à 2 procent van de olt's treden lymfomen op. dit zijn vaak aan het ebv-virus gerelateerde lymfomen waarbij met verlagen van immuunsuppressie, anti-cd20 en eventueel chemotherapie vaak volledige remissie te bereiken is. keel-en slokdarmkanker bij ex-alcoholisten en colonkanker bij olt voor psc zijn reeds genoemd. wellicht zijn in de toekomst schema's die immuuntolerantie opwekken mogelijk, waardoor minder immuunsuppressie benodigd zal zijn. j 22.5 in figuur 22.9 is de verbetering van de patiëntoverleving na levertransplantatie in de loop er jaren weergegeven. momenteel is er een goede patiëntoverleving in gespecialiseerde centra: een jaar na levertransplantatie is dat 90 procent en na vijf, tien en vijftien jaar respectievelijk 80, 75 en 70 procent. [53] de overleving van het transplantaat (graft survival) ligt meestal ongeveer 10 procent lager. de grootste risico's op overlijden liggen in de eerste drie maanden na levertransplantatie, en dit houdt verband met de genoemde complicaties. veel patiënten voelen zich vanaf drie maanden na de levertransplantatie veel beter dan gedurende lange tijd daarvoor, al is de kwaliteit van leven niet altijd optimaal. zo komt vermoeidheid vaak voor na levertransplantatie waarvoor diverse oorzaken kunnen zijn, zoals bijwerkingen van medicatie en stress. velen kunnen sporten, de helft van de patiënten gaat na levertransplantatie werken. als mensen zich beter voelen is het belangrijk het nut van goede inname van medicatie, onthouding van alcohol, geregelde controles en het direct ondernemen van actie bij koorts nog eens te onderstrepen. levertransplantatie bij kinderen verschilt van die bij volwassenen met betrekking tot indicaties, pretransplantatiezorg ten aanzien van optimaliseren van groei en ontwikkeling, operatietechnische aspecten en epidemiologie van postoperatieve complicaties, zoals posttransplantatie lymfoproliferatieve ziekte (ptld). j 22.6.1 cholestatische leverziekte is bij 50 tot 75 procent van de kinderen de indicatie voor levertransplantatie. andere belangrijke indicaties zijn metabole ziekten en acuut leverfalen (elk 10-15%). eindstadium cholestatische leverziekte ten gevolge van biliaire atresie is wereldwijd verantwoordelijk voor ongeveer de helft van alle levertransplantaties bij kinderen. biliaire atresie (galgangatresie) heeft een incidentie van 1 op 18.000-20.000, hetgeen voor nederland betekent dat de diagnose jaarlijks bij tien nieuwe patiënten wordt gesteld. andere cholestatische leverziekten die bij kinderen een indicatie voor levertransplantatie kunnen betekenen, zijn onder andere de ziekte van alagille (een autosomaal dominante aandoening met gelaatsafwijkingen (dysmorfie), congenitale hartafwijkingen, cholestatische geelzucht en botafwijkingen), congenitale leverfibrose, vormen van progressieve familiaire intrahepatische cholestase (pfic) en neonatale hepatitissyndromen. j 22.6.1.1 galgangatresie als indicatie voor levertransplantatie galgangatresie is een ernstige, potentieel fatale leverziekte. de aandoening is gekenmerkt door een progressieve, inflammatoire destructie van de extrahepatische en soms ook van de intrahepatische galwegen. meestal wordt de ziekte op de leeftijd van 4 tot 6 weken ontdekt bij overigens vaak (nog) goed gedijende zuigelingen die worden onderzocht wegens geelzucht, ontkleurde ontlasting en donkere urine. een belangrijke valkuil is het klinisch sluipende beloop waarbij er in de eerste fase geen ontkleurde ontlasting en/of donkere urine behoeven te zijn. onbehandeld leidt galgangatresie tot terminaal leverfalen op de leeftijd van 6 maanden tot 2 jaar. de oorzaak is onopgehelderd. bij 10 tot 15 procent van de kinderen gaat de aandoening gepaard met een belangrijk verschil tussen levertransplantaties bij kinderen en bij volwassenen is het feit dat de meeste postmortale orgaandonoren volwassenen zijn. hierdoor kan bij kinderen slechts zelden gebruikgemaakt worden een volledige (kinder)donorlever. als alternatief werd daarom eind jaren tachtig van de vorige eeuw in parijs de leverreductietechniek (reduced size-levertransplantatie) geïntroduceerd, door bismuth en houssin. [ [56] voor het splitsen van donorlevers wordt gebruikgemaakt van chirurgische technieken die ook worden gebruikt bij leverresecties en die ervaring met leverchirurgie en goede kennis van de leveranatomie vereisen. behalve transplantatie van gereduceerde en gesplitste levers bestaat er sinds een aantal jaren ook de mogelijkheid van donatie door een levende donor. [57] vaak is de donor een van de ouders van het zieke kind. bij de levende donor wordt een resectie verricht van de linkslaterale segmenten van de lever, die vervolgens gebruikt worden voor transplantatie bij het kind. j 22.6.4 de postoperatieve situatie bij kinderen verschilt op een aantal punten van die bij volwassenen. in de eerste plaats is er bij jonge kinderen die een cholestatische leverziekte hadden vaak sprake van een ernstige groeiachterstand. in de jaren na transplantatie kan meestal een forse inhaalgroei bereikt worden. desondanks blijkt uit eigen historische gegevens dat ongeveer de helft van de getransplanteerde kinderen uiteindelijk een lengte bereikt die lager is dan -1,3 sd van hun 'targetlengte'. [58] het is aannemelijk dat een deel van deze achterblijvende groei kan worden toegeschreven aan het gebruik van corticosteroïden als immuunsuppressie. de laatste jaren is het corticosteroïdengebruik in de verschillende immunosuppressieve schema's sterk verlaagd. een tweede specifieke situatie na kinderlevertransplantatie is de verhoogde kans op een primoinfectie met het epstein-barr-virus (ebv) en het hieraan gekoppelde verhoogde risico op posttransplantatie-immunoproliferatieve ziekte (ptld). de meeste kinderen worden op jonge leeftijd getransplanteerd, op een moment dat zij immunonaïef zijn tegen ebv. zij lopen dan ook een hoge kans op het ontwikkelen van een primo-infectie met ebv onder immuungesupprimeerde omstandigheden. [59] de ebv-bron kan het getransplanteerde orgaan zijn, het virus kan afkomstig van ebv-positieve oudere kinderen of volwassenen of er kan sprake zijn van een normale nosocomiale besmetting. ptld wordt in eerste instantie behandeld met het verlagen of volledig staken van de immuunsuppressie, met de hierbij horende risico's op rejectie van de lever. bij onvoldoende effect kunnen andere middelen noodzakelijk zijn, inclusief monoklonale antilichamen (anti-cd20) en chemotherapie. een derde aandachtspunt na levertransplantatie bij kinderen is de ontwikkeling. enerzijds kan de ontwikkeling vertraagd zijn geraakt door een langdurige ziekteperiode voor de transplantatie. anderzijds blijkt postoperatief de periode van puberteit en adolescentie tot psychologische druk te leiden in het gezin. in deze fase kan extra begeleiding nodig zijn om de autonomie van zorg en medicijngebruik succesvol van de ouders aan het kind over te dragen. j 22.6.5 de langetermijnoverleving na levertransplantatie bij kinderen is goed. het overlevingspercentage een jaar na transplantatie bedraagt 85 tot 90 procent en dit percentage blijft in de daaropvolgende jaren redelijk stabiel. de tienjaarsoverleving bij kinderen na levertransplantatie bedraagt 75 tot 80 procent. uit groningse ervaringen is gebleken dat circa 7 procent van de kinderen binnen tien jaar na de (eerste) levertransplantatie een retransplantatie nodig heeft vanwege fibrose en uiteindelijk cirrose van het transplantaat. de pathogenese van dit transplantaatverlies is niet geheel duidelijk en onderwerp van nader onderzoek. tot slot kan het niet genoeg benadrukt worden dat levertransplantatie bij kinderen vaak niet alleen maar levensreddend is; de kwaliteit van overleven maakt het meestal mogelijk om 'gezonde' levensdoelen te realiseren in termen van relaties, gezinsvorming, opleiding en werk. hepatocellular carcinoma: latest developments successful liver transplantation for hilar cholangiocarcinoma intensive care management of acute liver failure acute liver failure: summary of a workshop brain edema in acute liver failure prognostic impact of lactate in acute liver failure blood lactate but not serum phosphate levels can predict patient outcome in fulminant hepatic failure early indicators of prognosis in fulminant hepatic failure: an assessment of the model for end-stage liver disease (meld) and king's college hospital criteria wilson's disease in children: 37-year experience and revised king's score for liver transplantation revised king's college score for liver transplantation in adult patients with wilson's disease mild hypothermia for the treatment of acute liver failure -what are we waiting for? protocol indicatiestelling en selectie voor levertransplantatie bij volwassenen in nederland charlton m. the impact of obesity on long-term outcomes in liver transplant recipients-results of the niddk liver transplant database modified charlson comorbidity index for predicting survival after liver transplantation factors affecting graft survival after liver transplantation from donation after cardiac death donors techniques of orthotopic liver transplantation hepatocyte transplantation followed by auxiliary liver transplantation -a novel treatment for ornithine transcarbamylase deficiency auxiliary versus orthotopic liver transplantation for acute liver failure a novel technique for auxiliary partial liver transplantation with reno-portal anastomosis and avoidance of the hepatoduodenal ligament reuse of auxiliary liver grafts in second recipients with chronic liver disease auxiliary liver transplantation for acute liver failure emergency subtotal hepatectomy: a new concept for acetaminophen-induced acute liver failure: temporary hepatic support by auxiliary orthotopic liver transplantation enables long-term success predictors of survival after in vivo split liver transplantation: analysis of 110 consecutive patients split liver transplantation domino liver transplantation donor evaluation and hepatectomy for living-donor liver transplantation living donor liver transplantation using dual grafts from two donors: a feasible option to overcome small-for-size graft problems? postsurgical complications in livingrelated liver donors a2all study group. improvement in survival associated with adult-to-adult living donor liver transplantation use of severely steatotic grafts in liver transplantation: a matched case-control study initial poor function in the age of old donors: prognostic factors dearterialization of the liver causes intrahepatic cholestasis due to reduced bile transporter expression endovascular stent placement in patients with hepatic artery stenoses or thromboses after liver transplant mechanisms of disease: the evolving understanding of liver allograft rejection incidence and timing of infections after liver transplant in italy gram-positive bloodstream infections in liver transplant recipients: incidence, risk factors, and impact on survival mannose binding lectin gene polymorphisms confer a major risk for severe infections after liver transplantation infectious complications limit the outcome of liver transplantation in medical urgency code 2 patients risk stratification and targeted antifungal prophylaxis for prevention of aspergillosis and other invasive mold infections after liver transplantation impact of targeted oral ganciclovir prophylaxis for transplant recipients of livers from cytomegalovirus-seropositive donors posttransplantation lymphoproliferative disorder -the great mimic in liver transplantation: appraisal of the clinicopathologic spectrum and the role of epstein-barr virus a prospective study of standardized nonsurgical therapy in the management of biliary anastomotic strictures complicating liver transplantation nonanastomotic biliary strictures after liver transplantation, part 2: management, outcome, and risk factors for disease progression similar graft and patient survival in liver transplantation with brain death and controlled cardiac death donors with restrictive acceptance criteria long-term results after liver transplantation reduced size orthotopic liver graft in hepatic transplantation in children orgaan-en weefseldonatie en transplantatie. nederlandse transplantatie vereniging transplantation einer spenderleber auch bei zwei empfänger (splittingtranspantation): eine neue methode in der weiterentwicklung der lebersegementtransplantation levertransplantatie met een levende donor growth and final height after liver transplantation during childhood the value of prospective monitoring of ebv dna in blood samples of pediatric liver transplant recipients key: cord-283120-hyzk59qv authors: sharma, ashish; jaiswal, pragya; kerakhan, yasameen; saravanan, lakshmi; murtaza, zeba; zergham, azka; honganur, nagaraj-sanchitha; akbar, aelia; deol, aran; francis, benedict; patel, shakumar; mehta, deep; jaiswal, richa; singh, jagmeet; patel, urvish; malik, preeti title: liver disease and outcomes among covid-19 hospitalized patientsa systematic review and meta-analysis date: 2020-10-16 journal: ann hepatol doi: 10.1016/j.aohep.2020.10.001 sha: doc_id: 283120 cord_uid: hyzk59qv introduction and objectives: the coronavirus disease 2019 (covid-19) pandemic has been a challenge globally. in severe acute respiratory syndrome (sars) epidemic 60% of patients had hepatic injury, due to phylogenetic similarities of the viruses it is assumed that covid-19 is associated with acute liver injury. in this meta-analysis, we aim to study the occurrence and association of liver injury, comorbid liver disease and elevated liver enzymes in covid-19 confirmed hospitalizations with outcomes. materials and methods: data from observational studies describing comorbid chronic liver disease, acute liver injury, elevated aspartate aminotransferase (ast), alanine aminotransferase (alt) levels and outcomes of covid-19 hospitalized patients from december 1, 2019, to june 30, 2020 was extracted following prisma guidelines. adverse outcomes were defined as admission to intensive care unit (icu), oxygen saturation <90%, invasive mechanical ventilation (imv), severe disease and in-hospital mortality. odds ratio (or) and 95% confidence interval (95%ci) were obtained. results: 24 studies with 12882 confirmed covid-19 patients were included. overall prevalence of cm-cld was 2.6%, covid-19-ali was 26.5%, elevated ast was 41.1% and elevated alt was 29.1%. cm-cld had no significant association with poor outcomes (pooledor:0.96;95%ci:0.71–1.29; p = 0.78). covid-19-ali (1.68;1.04–2.70; p = 0.03), elevated ast (2.98;2.35–3.77; p < 0.00001) and elevated alt (1.85;1.49–2.29; p < 0.00001) were significantly associated with higher odds of poor outcomes. conclusion: our meta-analysis suggests that acute liver injury and elevated liver enzymes were significantly associated with covid-19 severity. future studies should evaluate changing levels of biomarkers amongst liver disease patients to predict poor outcomes of covid-19 and causes of liver injury during covid-19 infection. the who declared coronavirus disease 2019 (covid-19) as a global pandemic on march 11, 2020 (1) . with approximately 5 million cases and over 160,220 deaths, the usa remains the worst affected country as of august 8, 2020 (2) . following this, india (4.3m), brazil (4.1m), and russia (1m), are among the other countries that are greatly affected. total cases worldwide are around 27 million, and thus it remains an emergency of international concern (3). severe acute respiratory syndrome coronavirus 2 (sars-cov2) mainly affects the respiratory system but as we are gaining more insight about this novel disease, many published studies have also provided evidence of its organotropism and multisystem organ inflammation nature (4, 5) . multiple studies have observed association of elevated liver enzymes in patients with covid-19 infection assuming that it can cause liver damage either via direct hepatotoxic injury with viral infection, or drug toxicity, or immune mediated response. in the past, it has been reported that 60% of patients developed liver damage due to the sars epidemic (6) . since sars-cov-2 belongs to the same coronavirus family, we assume it may cause liver injury. the biliary epithelium has expression of angiotensinconverting enzyme (ace-2) receptor, which is also the binding site of sars-cov-2 (7) . the ace2 receptor expression in hepatocytes has been shown to be upregulated in animals' models of liver injury but hepatocytes have lower expression in humans (8, 9) . (10) . hence, we aim to systematically study the occurrence of liver injury, comorbid liver disease and elevated liver enzymes in covid-19 confirmed hospitalizations and also identify their association with outcomes. j o u r n a l p r e -p r o o f the aim of the study is to evaluate the role of the comorbid chronic liver disease (cm-cld), elevated liver enzymes and covid-19 associated acute liver injury (covid-19 ali) in predicting the outcomes in confirmed covid-19 hospitalized patients. covid-19 confirmation was evaluated by combined findings of rt-pcr, serology, symptoms, and mri chest in majority of those studies. poor outcomes were defined by intensive care unit (icu) admission, oxygen saturation<90%, invasive mechanical ventilation (imv) utilization, severe disease, and in-hospital mortality. study-specific poor outcomes and definitions of cm-cld, covid-19 ali and cut-off levels of liver enzymes in each individual study are mentioned in table 1 . a systematic search was conducted on published studies using prisma guidelines (11) abstracts were reviewed, and articles were retrieved and reviewed for availability of data on comorbid liver disease, elevated liver enzymes, acute liver injury and outcomes of covid-19 patients. studies which gave details on outcomes were selected for quantitative analysis. pm and dm independently screened all identified studies and assessed full-texts to decide eligibility. any disagreement was resolved through discussion with another reviewer up. from the included studies, we extracted the following variables including comorbid liver disease, elevated ast and alt levels, acute liver injury and outcomes. details on binary outcomes like icu vs. non-icu admission, severe vs non-severe disease, imv vs no-imv use, oxygen saturation <90% vs >90%, in-hospital mortality vs discharged alive and survivors were collected using prespecified data collection forms by two authors (pm and dm) with a common consensus of author (up) upon disagreement. we have presented the study characteristics like the first author's last name, publication month and year, country of origin, sample size, mean or median age, males, outcomes and definitions of comorbid liver disease and acute liver injury and cut offs for elevated ast and alt levels assessed in that individual study in table 1 (references for the studies are in supplemental file 1.1). data analysis was performed using review manager version 5.3 (the nordic cochrane centre, the cochrane collaboration, copenhagen, denmark). if the study has more than one outcome comparison j o u r n a l p r e -p r o o f then we have used data from the most severe outcome in the analysis to minimize the overall selection bias of our study. the maentel-haenszel formula was used to calculate dichotomous variables to obtain odds ratios (ors) along with its 95% confidence intervals to describe the association of comorbid liver disease, elevated liver enzymes, acute liver injury and outcomes of covid-19 patients in each study. random-effect models were used regardless of heterogeneity to estimate the combined effect and its precision, to give a more conservative estimate of the ors and 95%ci. the i² statistic was used to assess statistical heterogeneity and i² >50% was considered significant heterogeneity. the p<0.05 was considered significant. publication bias was assessed visually using funnel plots and the newcastle-ottawa scale (nos). newcastle ottawa scale (nos) (13) was used to assess the quality and bias in the included studies, which rates selection, comparability and outcome (supplemental file1.2) . all studies were assessed to be of high quality. the pooled or and 95% ci are represented in the form of forest plots. each square on the chart area represents individual study and the area of each square is equivalent to the weight of the study, which is the inverse of the study variance. the diamond represents the pooled or and the width corresponds to the 95% ci. j o u r n a l p r e -p r o o f discussion in our meta-analysis, we found that acute liver injury and elevated alt and ast levels were associated with poor outcomes. however, our study could not provide significant evidence of the effect of preexisting chronic liver disease on covid-19 patient outcomes. in support of our findings, a recent metaanalysis found that the frequency of underlying chronic liver disease was not statistically different between severe and non-severe diseases (14) . however, another study by singh et al. reported that patients with preexisting liver disease, particularly cirrhosis, are at higher risk for hospitalizations and mortality (15) . few studies have reported that the frequency of patients with liver injury was higher in severe cases compared to mild cases, consistent with our findings of liver injury associated with poor outcomes (16) (17) (18) . there are several theories proposed for liver damage in covid-19 such as direct effect of the virus on hepatocytes or biliary epithelium via angiotensin-converting enzyme 2 (ace 2) receptors expression, liver injury related to increased immune response (cytokine storm) and immune mediated damage, drug toxicity (because of drugs like acetaminophen, antivirals and hydroxychloroquine), and liver failure occurring in patients having multiorgan dysfunction (19) (20) (21) . a study by chai et al., has reported that expression of ace 2 receptors was 57.9 % in bile duct cells (cholangiocytes) and 2.6% in hepatocytes (7). cholangiocytes play a vital role in liver regeneration and immune response (22) . hence one possible theory for liver injury in covid-19 patients is destruction of cholangiocytes by sars-cov-2 virus via ace 2 receptors. additionally, it was also observed that expression of ace 2 in hepatocytes increases in cases of liver injury (9) . recently, post mortem liver biopsies of covid-19 patients have shown moderate microvesicular steatosis and mild lobular and portal activity, indicating the injury could have been caused by either sars-cov-2 infection or druginduced liver injury (23) . whether this liver injury is caused by the virus itself or is due to a severe j o u r n a l p r e -p r o o f inflammatory response with liver damage or sepsis or multisystem organ failure or drug toxicity is not well understood (24) . therefore, temporal relationship of covid-19 induced liver damage cannot be established. furthermore, increased liver enzymes (ast and alt) occur in the setting of hepatocyte damage (abnormal liver function). according to initial studies, more than a third of patients had elevated ast and alt (transaminitis) which was associated with longer hospital stay(17, 25, 26) . in a study done by cai et al., 76 .3% of covid-19 patients had abnormal liver tests while 21.5% developed liver injury during hospitalization, which was defined by alt, ast, total bilirubin and gamma-glutamyl transferase levels elevated to more than 3x the upper limit of normal (27). the study also found that patients with abnormal liver tests had significantly higher odds of developing severe pneumonia (27). these findings are also consistent with our study that shows a significant elevation in ast and alt among covid-19 patients may be helpful in predicting poor outcomes among these patients. sars-cov-2 is phylogenetically similar to sars-cov and mers and, studies have given evidence of association of sars-cov and mers with liver injury. and elevated transaminases associated with severe disease (20, (28) (29) (30) (31) (32) .additionally, autopsies of sars patients found not only virus particles in the hepatocytes and hepatic vascular endothelial cells but also significant increase in mitotic cells, with eosinophilic bodies and ballooning hepatocytes, suggesting that sars-cov may induce liver cells apoptosis and thus cause liver injury (33, 34) . hence, liver injury due to sars-cov, mers and sars the data used in this study is deidentified and collected from the studies published online thus informed consent or irb approval was not needed for this study. the data is collected from the studies published online, publicly available, and specific details related to data and/or analysis will be made available upon request. j o u r n a l p r e -p r o o f who director-general's opening remarks at the media briefing on covid-19 -11 covid-19 coronavirus pandemic. worldometer multiorgan and renal tropism of sars-cov-2 age-adjusted risk factors associated with mortality and mechanical ventilation utilization amongst systematic review and meta-analysis. sn comprehensive clinical medicine covid-19 and liver specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection. biorxiv chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2 upregulation of hepatic angiotensin-converting enzyme 2 (ace2) and angiotensin-(1-7) levels in experimental biliary fibrosis comorbidity and its impact on 1590 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for sars coronavirus. a first step in understanding sars pathogenesis clinical characteristics and mechanism of liver injury in patients with severe acute respiratory syndrome risk factors related to hepatic injury in patients with corona virus disease 2019. medrxiv cholangiocyte pathobiology associated with acute respiratory distress syndrome. the lancet respiratory medicine covid-19 and liver dysfunction: current insights and emergent therapeutic strategies clinical features of covid-19-related liver functional abnormality clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19: abnormal liver function tests organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways clinical significance of hepatic derangement in severe acute respiratory syndrome clinical characteristics and mechanism of liver damage in patients with severe acute respiratory syndrome clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-associated viral hepatitis caused by a novel coronavirus: report of three cases , and pneumonia can be seen in imaging. severe cases: meeting any of the followingrespiratory distress, respiratory rate ≥ 30 breaths/min; spo2 ≤ 93% at rest; and pao2/fio2 ≤ 300. patients with >50% lesion progression within 24 to 48 hours. critical/extremely severe cases: if they have one of the following: respiratory failure requiring mechanical ventilation, shock, and other organ failure requiring icu treatment. ***patients were included in the mild disease group if they did not need high-flow oxygen support and in the severe disease group if they were provided with high-flow oxygen support. #not mentioned; ##general covid-19 included following criteria:(i) obvious alleviation of respiratory symptoms (e.g. cough, chest distress and breath shortness) after treatment; (ii) maintenance of normal body temperature for ≥3 days without the use of corticosteroid or antipyretics; (iii) improvement in radiological abnormalities on chest ct or x-ray after treatment; (iv) a hospital stays of ≤10 days.otherwise, it was classified as refractory covid-19; $ all the studies mentioned chronic liver disease as a comorbidity. ^liver enzyme abnormalities, ^^acute liver injury defined as an increase in alanine aminotransferase (alt) over two times the upper limit of the normal range (uln) or an increase in conjugated bilirubin or a combined increase in aspartate aminotransferase (ast), alkaline phosphatase and total bilirubin provided that one of them was above two times uln. ^^^ acute liver injury was defined as jaundice with a total bilirubin level of 3mg/dl or higher and an acute increase in alt of at least 5 times the upper limit of the normal range and/or an increase alkaline phosphatase of at least twice the upper limit of the normal range. ^*liver injury was judged alt and ast levels @ studies considered ast (aspartate aminotransferase) and alt (alanine aminotransferase) levels >40iu/l as elevated levels.references of the included studies in meta-analysis is in esupplemental file 1 key: cord-003921-8r8z0otz authors: nakamura, kojiro; kageyama, shoichi; kupiec-weglinski, jerzy w. title: the evolving role of neutrophils in liver transplant ischemia-reperfusion injury date: 2019-01-29 journal: curr transplant rep doi: 10.1007/s40472-019-0230-4 sha: doc_id: 3921 cord_uid: 8r8z0otz purpose of review: hepatic ischemia-reperfusion injury (iri), an inevitable event during liver transplantation, represents a major risk factor for the primary graft dysfunction as well as the development of acute and chronic rejection. neutrophils, along macrophages, are pivotal in the innate immune-driven liver iri, whereas the effective neutrophil-targeting therapies remain to be established. in this review, we summarize progress in our appreciation of the neutrophil biology and discuss neutrophil-based therapeutic perspectives. recent findings: new technological advances enable to accurately track neutrophil movements and help to understand molecular mechanisms in neutrophil function, such as selective recruitment to ir-stressed tissue, formation of neutrophil extracellular traps, or reverse migration into circulation. in addition to pro-inflammatory and tissue-destructive functions, immune regulatory and tissue-repairing phenotype associated with distinct neutrophil subsets have been identified. summary: newly recognized and therapeutically attractive neutrophil characteristics warrant comprehensive preclinical and clinical attention to target iri in transplant recipients. liver transplantation (lt) has become the standard of care for patients with end-stage liver disease and those with liver malignancies [1] . hepatic ischemia-reperfusion injury (iri), an inevitable event during lt, represents a major risk factor for the primary graft dysfunction as well as the development of acute and chronic rejection [2, 3] . hence, minimizing iri is this article is part of the topical collection on immunology * jerzy w. kupiec-weglinski jkupiec@mednet.ucla.edu important not only for improving clinical outcomes but also for successful use of marginal liver grafts and expansion of donor organ pool available for transplantation. despite its obvious clinical importance, however, the mechanisms accounting for liver iri are only partially understood and effective preventive or therapeutic strategies remain to be established. in the initial stage of liver iri, ischemic insult renders hepatic cells sensitive to glycogen consumption, oxygen deprivation, ph changes, and adenosine triphosphate (atp) depletion [4] . these are followed by enhanced production of reactive oxygen species (ros), higher intracellular calcium concentration, and organelle damage, leading to the initial parenchymal cell death [5] . the reperfusion itself disturbs liver metabolism and evokes inflammatory cascades leading to aggravated hepatocellular damage. innate immune activation plays a central role in this inflammatory response via cytotoxic mechanisms and dynamic cross-talk with adaptive immune cell repertoires, ultimately converting immunologically quiescent hepatic milieu into an inflammatory organ [6, 7] . in unstressed human liver, kupffer cells (liver-resident macrophages) account for about 15% of total hepatocellular population and 80-90% of whole body macrophages [8] . in iri-lt pathophysiology, both kupffer cells (donor-origin) and liver-infiltrating bone marrow-derived macrophages (recipient-origin) play dominant roles in priming innate immune responses [9] [10] [11] , with the majority of studies focusing on macrophage regulation [12, 13] . on the other hand, neutrophils are dominant immune cells in the steady-state blood circulation (50-70% in human, 10-25% in mouse [14, 15] ), constantly patrolling and serving as the first line of defense against invading pathogens [16] . likewise, in ir-stressed blood-perfused liver, neutrophils are recruited to the injury site, contributing to sterile inflammation and enhancing the hepatocellular damage. indeed, despite being long considered as "non-specialized" innate effector cells, neutrophil infiltration into hepatic sinusoidal lumen is now considered as one of reliable biomarkers of liver iri [17, 18] . in this review, we first summarize progress in our appreciation of the neutrophil biology, and then discuss therapeutic prospects of their targeting for the treatment of inflammatory states, such as iri in lt recipients. neutrophils, the largest circulating fraction of leukocytes, are continuously generated from myeloid precursors in the bone marrow in a process of "granulopoiesis" (daily production reaches up to 2 × 10 11 cells) [16, 19] . the crucial signals for neutrophil activation are provided by danger-associated molecular patterns (damps), i.e., endogenous molecules constitutively expressed in nuclear, cytoplasm, and extracellular matrix under basal conditions. although indispensable for homeostasis maintenance, once released in response to tissue injury, damps are detected by and become critical triggers of the inflammatory cell activation. a growing number of damps have been identified to date, such as atp, histone, high mobility group box 1 (hmgb1), with complementary pattern recognition receptors (prrs) crucial in cell recognition and signaling pathways (representative are listed in table 1 ). the initial parenchymal cell damage results in the release of damps from damaged/dead/moribund cells that stimulate local sentinel kupffer cells and liver sinusoidal endothelial cells (lsecs). kupffer cells sense neighboring cell death by receiving damps signals and produce il1β, which up-regulates intercellular adhesion molecule-1 (icam-1) on lsecs. neutrophils are then recruited via integrin αmβ2 (mac1)-dependent adhesion to endothelial icam-1. this neutrophil adhesion mechanism requires atp-induced activation of p2x7 receptor and nod-like receptor pyrin domaincontaining-3 (nlrp3) inflammasome [20] . indeed, mice depleted of kupffer cells by clodronate showed reduced caspase, il1, and neutrophil recruitment, whereas impaired p2x7r signaling was accompanied by equivalently impaired neutrophil accumulation in a liver thermal injury model [21] . activated kupffer cells, along with parenchymal cells, also release chemokines detected by g protein coupled receptors (e.g., cxcr2) on neutrophils and thereby recruit them to the site of inflammatory damage. in addition, lsecs themselves can sense damps via tlr9 to release il1β and il18 [22] (fig. 1a) . because of the unusual hepatic microvasculature, circulating neutrophils can move directly to integrinmediated adhesion devoid of selectin-mediated rolling. in line with this scenario, mac-1 or icam-1 neutralizing antibodies effectively alleviated liver iri [23, 24] . histone and hmgb1 are among the most tested damps in the model of liver iri. the levels of circulating histones are significantly increased in liver iri, and anti-histone neutralizing antibodies suppress tlr9/myd88 signaling while decreasing inflammatory hepatic damage [25] . treatment with hmgb1 neutralizing antibody alleviated iri in wt but not tlr4deficient mouse recipients [26] . likewise, recombinant thrombomodulin (hmgb1 inactivator) mitigated liver iri in wt but not tlr4-knockout mouse [27•] . since broad spectrum of immune cells express tlrs, the hmgb1-tlr4 inflammatory axis is unlikely to be exclusive for neutrophil regulation. indeed, hepatocyte-specific hmgb1 deficient mice showed decreased hepatic necrosis and neutrophil accumulation, whereas the number of their macrophages remained unchanged in acetaminophen-induced liver injury model [28] . in contrast, hepatocyte-specific hmgb1-deficient mice exhibited exaggerated liver ir-damage, implying hmgb1 is essential for hepatocyte resistance against hypoxia [29] . clearly, extracellular damps are promising therapeutic targets to manage liver iri by alleviating innate, including neutrophil, cell responses. following adhesion to the endothelium, neutrophils are guided by kupffer cell-derived chemokines (cxcl1, cxcl2), bind to cxcr2 on the luminal surface of lsecs, and form the chemotactic gradient at the injury site [20] (fig. 1b) . consistent with such a mechanism, reparixin (an allosteric cxcr2 antagonist) decreased neutrophil infiltration and mitigated liver iri [30] . in addition, cxcl1 blocking antibody alleviated hepatic infiltration in necrotic cellinduced neutrophil mobilization model [31] , whereas in a carbon tetrachloride (ccl4)-induced acute liver injury, defective cxcl2 expression in tlr2-knockout or s100a9-knockout mice was accompanied by suppressed hepatic neutrophil recruitment [32] . in the following dynamic stage, neutrophils moving on the lsecs luminal side migrate underneath the sinusoidal wall to the damaged site. mitochondrial n-formyl peptides (fmit), cleavage products of mitochondrial proteins, are critically involved in this process serving as chemoattractant damps. damaged cells release fmit and create a gradient that attracts neutrophils through neutrophilspecific formyl peptide receptor 1 (fpr1) [33] . honda neutrophils move on the lsecs luminal side toward the damaged site, guided by kupffer cell-derived chemokine (cxcl1/cxcl2) gradient. c neutrophils detect mitochondrial n-formyl peptides (fmit) via formyl peptide receptor 1 (fpr1) and migrate underneath lsecs toward the injury site. d neutrophils express and secret matrix metalloproteinase 9 (mmp9) to degrade extracellular matrix (ecm) during migration reported that cyclosporine h (fpr1 antagonist) decreased the number and crawling velocity of neutrophils while alleviating liver iri and inhibiting the accumulation of neutrophils into laser irradiation-induced necrotic areas [34•] . interestingly, at this time point, neutrophils are attracted by two opposing chemoattractant gradients, cxcl2 mostly anchored on the luminal lsecs side and fmit emanated from parenchymal damaged cells. the final signaling via fmit-fpr1 overrides the cxcl1/cxcl2-cxcr2 axis, making neutrophils to preferentially migrate toward the fmit while ignoring the cxcl2 attraction (fig. 1c) . such a chemotactic hierarchy has been consistently observed in pathogen-triggered infection where bacterial formylated peptides are dominant to endogenous chemoattractant [35] , as well as in sterile inflammation where neutrophils preferentially migrated toward injured cells rather than soluble chemokines [21] . indeed, marques et al. demonstrated that cxcr2 and fpr1 antagonists alleviated neutrophil migration in acetaminophen-induced liver damage, while simultaneous cxcr2 and fpr1 antagonism achieved maximum protection [36] . hence, both cxcl2 and fmit are critically involved in the hepatic neutrophil migration in a dynamic and sequential manner. the extracellular matrix (ecm), a network of proteins and sugars in all solid organs, is the essential scaffold and structural cell support as well as tissue regulator of cell adhesion, migration, differentiation, proliferation, and survival. once neutrophils migrate across lsec, they move toward damaged site while simultaneously degrading ecm. this process is dominated by matrix metalloproteinases (mmps), zincdependent endopeptidases involved in the breakdown of ecm, and tissue inhibitor of metalloproteinases (timps), which proteolytically regulate mmps activity. mmp2 and mmp9 are the most prominent in the pathophysiology of liver iri [37] . in liver iri, mmp9 is predominantly expressed by ly6g-positive neutrophils, while neutrophils are the only cells in the body which can release mmp9 free of its endogenous inhibitor timps, and therefore are uniquely capable of delivering highly active mmp9 to digest its fibronectin and collagen type iv substrates [38] . fibronectin, a major ecm component of the subendothelial space of disse in normal liver, interacts with integrin receptor α4β1 on activated neutrophils to induce mmp9 [39] (fig. 1d) . hamada et al. demonstrated that mmp9 deficient neutrophils were impaired in their ability to migrate across fibronectin in a transwell culture system, whereas genetic disruption or exogenous blocking with mmp9 antibody decreased neutrophil infiltration and cytoprotection in a mouse hepatic iri model [40] . meantime, ir-insult markedly increases timp1 expression, an endogenous mmp9 inhibitor. indeed, timp1 deficient mice had markedly increased mmp9 activity and massive neutrophil infiltration in their ir-stressed livers, which has led to 60% death. in contrast, virus-mediated gene transfer of timp1 increased timp1 expression primarily on parenchymal cells, suppressed gelatinolytic activity of neutrophil mmp9, attenuated neutrophil infiltration, and alleviated the hepatocellular damage [41, 42] . furthermore, lt patients experiencing acute rejection had elevated serum mmp9 activity at 1 week, implying its role as a potential biomarker in clinical lt [43] . neutrophils recruited or activated in liver sinusoids do not cause hepatic damage per se, as they can exert cytotoxicity only after migrating across the endothelium and in close proximity to the parenchymal cells [17] . tissue damage is mediated primarily by ros and proteases, with kupffer cells being principal ros producers early after reperfusion, and neutrophil oxidative burst becoming the main source of ros in the later iri phase. indeed, by 6 h of reperfusion (the peak of hepatocellular damage in a murine iri-lt model), infiltrating ly6g-positive neutrophils elaborated large amounts of ros metabolite (4-hydroxynonenal) [11] . neutrophils generate ros through nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which consists of a rho gtpase, two membrane-bound phox (phagocytic oxidase) proteins (p22 phox and gp91 phox ), and three cytosolic phox proteins (p40 phox , p47 phox , and p67 phox ). patients encoded with hypofunctional mutations in gp91 phox or p47 phox were protected from transiently induced upper limb iri [44] , while apocynin (gp91 phox inhibitor) suppressed ros metabolite (8hydroxy-2-deoxyguanosine) and alleviated liver iri in methionine/choline-deficient diet conditioned rats [45] . meanwhile, neutrophils contain large quantities of serine proteases in the azurophilic granules, such as neutrophil elastase, proteinase 3, and cathepsin g. after arriving at the damaged site, neutrophils degranulate and release them to exert, in combination with ros, their effector functions. neutrophil elastase, a 29 kda chymotrypsin-like serine protease which degrades ecms causes organ damage, whereas sivelestat (neutrophil elastase inhibitor) is widely used in treating acute lung injury in humans. we and others have reported that sivelestat inhibited adhesion and migration of neutrophils to vascular endothelium and protected mouse livers against iri [46, 47] . furthermore, a randomized clinical study in patients that underwent hepatic resection revealed sivelestat treatment reduced post-operative serum hmgb1 and il6 levels [48] . myeloperoxidase (mpo), a heme protein synthesized during myeloid differentiation, is often used as a neutrophil activity marker. in the presence of physiological chloride concentrations, mpo reacts with hydrogen peroxide to catalyze formation of hypochlorous acid/hypochlorite and other oxidizing species [49] , and thus, it is also considered as a cytotoxic molecule in liver pathophysiology. however, its cytotoxicity in liver iri remains to be rigorously re-evaluated as a recent study demonstrated mpo was required for hepatoprotection in endotoxin-induced inflammation [50•] . with recent data showing efficacy and tolerability of azd3241 (an orally absorbed irreversible human mpo inhibitor) in parkinson patients [51] , the thorough evaluation of mpo in liver lt settings seems warranted. activated neutrophils can enhance inflammatory tissue damage by releasing neutrophil extracellular traps (nets), i.e., extracellular scaffolds of dna fibers decorated with histone, granule-derived antimicrobial peptides, and enzymes, such as neutrophil erastase, cathepsin g, and mpo [52] . generation of ros by nadph oxidase and activation of protein-arginine deiminase 4 (pad4), an enzyme that converts arginine to citrulline on histones, are essential steps for chromatin decondensation in the net formation. ne and mpo translocated into the nucleus further unfold chromatin, leading to the nuclear membrane break, chromatin release into cytosol where it is further decorated with granular and cytosolic proteins, and the emission of these traps into extracellular space [53] . nets function was originally described as the efficient means to immobilize, catch, and eliminate pathogens, whereas recent studies have recognized its critical involvement in noninfectious inflammatory states, including ir-stress. indeed, biopsies obtained from human kidney transplant recipients with post-transplant acute tubular necrosis exhibited increased nets formation [54•], while bronchoalveolar lavage fluid collected from human lung transplant recipients with primary graft dysfunction contained extensive nets [55] . furthermore, several net-targeting agents successfully attenuated iri in murine models, including pad4 inhibitors (yw3-56, yw4-03), which reduced nets formation and decreased severity of liver iri [56•]; pad inhibitor (clamidine), which suppressed nets and alleviated renal iri [54•]; or dnase i combined with recombinant tissue-type plasminogen activator, which inhibited nets and alleviated myocardial iri [57] . additional functional aspects of nets are detailed below. in addition to pro-inflammatory and tissue-destructive functions, recent studies have identified previously unappreciated novel role of neutrophils to control inflammation and resolve tissue damage. first, neutrophils retain the potential to inhibit t cell cytotoxicity. arginase-1 (arg1, a tertiary granule content) is a manganese-containing enzyme expressed in mouse myeloid cells, such as macrophages. however, in humans, it has been detected selectively in neutrophils [58, 59] , whereas activated neutrophils release arg1 during degranulation process, which inhibit t cell proliferation and expansion [60, 61] . neutrophil elastase (an azurophilic granule content) cleaves cd2, cd4, and cd8 on peripheral blood t lymphocytes, leading to reduced il2 production and suppressed t cell cytotoxicity [62] . neutrophil-derived ros may also suppress t cells via inactivation of coffilin, an actin-remodeling protein, which impairs formation of the immune synapse and cell activation [63] . moreover, as regulatory t cells (treg) are less sensitive to inhibition by ros [64] , production of ros by neutrophils effectively creates a treg dominant environment [65] . several lines of evidence indicate that neutrophil can limit inflammation response in other innate immune cells. selective neutrophil ablation or genetic mpo deletion in endotoxinchallenged mice unexpectedly enhanced inflammation responses and increased mortality, indicating that neutrophils and neutrophil-derived mpo can contribute to host resistance by limiting endotoxin-driven innate immune pathology [50•] . cathepsin g (an azurophilic granule content) cleaves nkp46 and impairs nkp46-mediated responses of nk cells, including ifnγ production and cell degranulation [66] . neutrophilderived microvesicles cause macrophages to generate lipid pro-resolving mediators and tgfβ, leading to counter inflammatory response [67] . dead neutrophils secret cytosolic protein annexin a1 (anxa1), which limits neutrophil migration by interacting with formyl peptide receptor 2 (fpr2) [68, 69] and prompts neutrophil apoptosis [70] . clearance of apoptotic neutrophils by macrophage efferocytosis may then switch macrophages to anti-inflammatory il10/tgfβ dominant phenotype, leading to tissue-repairing environment as well as treg dominated immune tolerance. analogous to the concept of pro-inflammatory (m1) and anti-inflammatory (m2) macrophage phenotypes [71] , tissuespecific microenvironment may promote neutrophil polarization into two distinct phenotypes [72] . thus, n1 neutrophils are anti-tumoral/pro-inflammatory, having increased tnfα expression and reduced arginase/cxcr4/mmp-9/vegf levels, whereas n2 neutrophils are pro-tumoral/anti-inflammatory and characterized by decreased tnfα and increased arginase/cxcr4/mmp-9/vegf expression [73, 74] . several cytokines may contribute to n1/n2 polarization, with tgfβ promoting neutrophils to acquire n2 phenotype by simultaneously inhibiting n1 neutrophil generation [72] . endogenous ifnβ imprints neutrophils to n1 phenotype, which restrict tumor angiogenesis and enhance inflammatory cytotoxicity [75] . a recent study demonstrated that analogous to m1/m2 macrophage polarization, lps and ifnγ drove peripheral blood neutrophils toward n1 phenotype while il4 polarized toward n2 phenotype [76] . in addition, murine infarctedmyocardium was infiltrated by day 1 with n1 dominant neutrophils expressing pro-inflammatory markers, whereas n2 neutrophils increased by day 7 concomitantly with enhanced levels of anti-inflammatory markers, supporting putative role of n2 neutrophils in the resolution of inflammation and promoting tissue repair [76] . noteworthy, rosiglitazone (peroxisome proliferator-activated receptor-γ agonist) treatment in a mouse brain stroke model shifted neutrophil population toward n2 phenotype (31% vs. 77%), suppressed inflammation, enhanced neutrophil clearance, and alleviated brain damage [77] . although macrophage m1/m2 plasticity is broadly accepted [78] , neutrophils generally have short lifespan (6-8 h in blood stream), their plasticity remains unknown. future studies need to elucidate whether n1/n2 polarization is dominated by re-programming of existing neutrophils or environmental influence on de novo neutrophil populations. by virtue of technological advances and efforts to identify neutrophil heterogeneity, additional antiinflammatory neutrophil subsets have been recognized. studies by tirouvanziam et al. identified a cd63+/mhc-ii+/cd80+/cd294+ human neutrophil subset in mature cystic fibrosis airway, which retained anabolic/pro-survival phenotype and suppressed t cell function [60, 79] . pillay et al. found a subset of mature human cd16 bright /cd62l dim neutrophils in endotoxin-challenged blood, which suppressed t cell proliferation via neutrophil mac-1 dependent manner [80] . neutrophils may also contribute to tissue-repairing processes following infectious or sterile inflammatory organ damage [81] . clinical observations support this concept, as patients with leukocyte adhesion deficiency type 1 (an autosomal recessive disorder characterized by defects in neutrophil adhesion ability) experience delayed wound healing [82] . in the beginning of tissue repair process, neutrophils acting as professional phagocytes remove tissue debris. indeed, in a thermal hepatic injury model, neutrophil depletion resulted in far more debris remaining at the injury site [83• ]. next, neutrophil apoptosis shifts the phagocytosing macrophages to the resolution phase (m2), which governs tissue-repairing environment. m2 macrophages secrete il1ra, il10, tgfβ, and vegf, leading to fibroblast differentiation into myofibroblasts, synthesis of interstitial fibrillar collagens by myofibroblasts, and expression of mmps/timps that control ecm turnover [84] . since neutrophils can release mmp9 free of its endogenous inhibitors (timps), they are capable of delivering highly active mmp9, a key proangiogenic factor degrading ecm, to release ecm-bounded vegf and other growth factors [85] . a series of recent studies have deepened our understanding of that process. for instance, christoffersson et al. identified a cd11b+/gr1+/cxcr4 high neutrophil subset in a syngeneic mouse pancreatic islet transplant model, which was recruited via vegf-a depending pathway. it contained higher amount of mmp9 than those recruited to an inflammatory stimulus, and contributed to revascularization via mmp9 dependent mechanism [86] . they also found an analogous cd49d+/vegfr1 high /cxcr4 high neutrophil population in mouse and human, which was recruited by hypoxia-induced vegf-a [87] . in a liver thermal injury model, limited period of neutrophil depletion (~48 h) resulted in delayed revascularization at 7 days and the presence of non-healing injury area at 4 weeks, suggesting the requirement for neutrophil in liver homeostasis [83•] . likewise, in a transplantation model of bioengineered u-graft, which contained an unassembled suspension of vascular cells embedded in a hydrogel, host-derived alternatively polarized neutrophils (n2) contributed to graft revascularization [88• ]. in a successful response to an acute injury, it is crucial to prevent tissue damage by promoting the local resolution of inflammation through the removal of neutrophils from the stressed site [89] . apoptosis, a highly organized programmed cell death program, is considered a favorable neutrophil death mechanism because the orderly cell elimination limits uncontrolled release of toxic neutrophil contents and damps, which potentially may cause further tissue damage [90] . morphologically, apoptotic neutrophils show the condensation of chromatin and its migration to the nuclear periphery, fragmentation of nuclear dna, and the blebbing of cell membranes leading to apoptotic body formation. as discussed, engulfment of apoptotic neutrophils by macrophages occurs in the resolution phase of inflammation and repair from tissue injury by turning off production of pro-inflammatory cytokines and launching an anti-inflammatory transcriptional program characterized by the release of il10 and tgfβ [84] . in contrast to the organized apoptotic cell death, neutrophil necrosis is a traumatic and highly detrimental event resulting in disintegration of the nuclear envelope and cytoplasmic membranes. neutrophils unavoidably undergo necrosis if the insult is too severe or the step-by-step apoptosis procedure fails to be achieved in a timely fashion, a process called a "secondary necrosis." necrotic neutrophil passively releases toxic effector molecules as well as damps leading to further damage and inflammation in neighboring tissues [90] . netosis is the process by which neutrophils produce and release nets. during netosis, intracellular proteins become exposed to the extracellular space, which results in the potential presentation of autoantigens to the host immune system and the release of damps that amplify ongoing immune reactions [53] . in this inflammatory aspect, the netosis seems closer to necrosis than apoptosis. on the other hand, the function of netosis to promote coagulation and thrombosis have been now recognized [91, 92] , whereas a recent study from mcdonald et al. has revealed net-platelet-thrombin axis that promotes intravascular coagulation and microvascular dysfunction in endotoxin-induced liver injury model [93•] . considering neutrophils preferentially undergo necrosis/ netosis rather than apoptosis if the stress/infection is severe [94] , the function of netosis to block blood flow to/from the severely damaged site seems to be favorable by averting the spread of damps/pathogens to remote organs and preventing distant organ injury. in this context, nakazawa et al. revealed nets inhibitor alleviated acute kidney injury while concomitantly reducing serum nets levels and mitigating remote organ injury in the lung, liver, heart, and brain [54• ]. in contrast, homozygous pad4 knockout in mrsa challenged mice was accompanied by decreased nets and reduced lung injury despite higher bacterial counts, increased levels of inflammatory cytokines, and equivalent overall survival as compared to wt counterparts. the heterozygous pad4 knockout mice unexpectedly showed improved survival as compared with wt counterparts or homozygous knockout mice [95•] ; in a mouse model of peritonitis, degradation of nets by dnase attenuated organ damage only when combined with antibiotics [96] ; in an experimental model of a gout (acute sterile inflammatory reaction to monosodium urate crystals), netosis-deficient mice developed exacerbated and chronic disease that could be reduced by adoptive transfer of aggregated nets [97] . therefore, the influence of nets inhibition depends on disease etiology and severity of the inflammation response. because of increased incidence of acute respiratory distress syndrome (ards) and acute kidney injury in lt patients [98, 99] , it is important to evaluate potential clinical value of nets reducing therapeutic strategies. it has long been accepted that apoptosis is the most common and preferred cell death program to terminate neutrophil activity. however, recent evidence suggests that neutrophils do not necessarily die in an inflamed site, and instead can leave the site of tissue damage in a process termed as a "reverse migration." this phenomenon was first visualized in zebrafish larvae in which most of the recruited neutrophils leave a damaged site and traffic to a distal site [100, 101] . a recent study combining intravital imaging and photoactivation techniques in a mouse thermal hepatic injury model demonstrated that neutrophils recruited to the stressed tissue neither underwent apoptosis, necrosis, nor phagocytosis by monocytes, but instead reversely migrated to the circulation, became arrested within the lung without causing local tissue injury, and then homed to the bone marrow where they ultimately underwent apoptosis [83•] . in contrast, in hepatic iri or acute pancreatitis model, activated neutrophils were redistributed to produce remote organ injury [102] [103] [104] . it is noteworthy that neutrophils undergoing transendothelial migration in vitro expressed specific marker, icam1 high / cxcr1 low , which was resistant to apoptosis and produced more ros [102] , whereas patients with acute pancreatitis who developed acute lung injury had more icam1 high / cxcr1 low expressing neutrophils in their circulation [104] . this implies the potential for neutrophil reprograming or existence of active/inactive neutrophil subsets after reverse migration. however, there is no rigorous evidence as to why and how reversely migrating neutrophils can be toxic in some cases but inactive in other pathology states. neutrophils have long been recognized as important players in liver iri, while great majority of studies focused on their proinflammatory and tissue-destructive functions. indeed, neutrophil-targeting strategies mostly narrowed at reducing their target organ infiltration. table 2 lists representative preclinical studies on neutrophil targeting in liver iri [23, 24, 30, 34•, 40, 45, 47, 56•, 105-107] . it is important to keep in mind that lt recipients are immunocompromised while neutrophils constitute the forefront of pathogen host defense. hence, therapies limiting neutrophil sequestration and/or effector functions may in turn increase the risk of infections in transplant recipients. despite preclinical studies showing therapeutic efficacy, global neutrophil depletion is unlikely to be clinically feasible. more specific maneuvers to inhibit neutrophil adhesion, migration, and effector functions are required, though it remains to be determined whether and how such therapies can achieve optimal balance between effective host immune surveillance while controlling cytodestruction. albeit non-specifically targeting neutrophil functions, the reduction of extracellular damps such as hmgb1 or histone seems favorable, because damps are the immune-activating signals released from host cells but not from pathogens. of note, one of the hmgb1 inactivators (art-123, recombinant thrombomodulin), originally shown to mitigate liver iri in tlr4 depending manner [27•] , is now used clinically for disseminated intravascular coagulation [108, 109] . in agreement with recent data [54•, 56•] , inhibition of nets formation by specifically targeting highly destructive neutrophil function seems biologically beneficial. however, futures studies need to evaluate potential risks, as this maneuver may spread abundant damps or pathogens, which otherwise should be isolated inside the nets areas. since dnase has been successfully applied for the treatment of cystic fibrosis patients and the nets digestion is considered to be a part of its beneficial effects [110, 111] , these investigations are clearly warranted. although a number of recent studies revealed neutrophil immunoregulatory and tissue-repairing capabilities, little is known about these functions in liver iri. this might be due to the overwhelming focus on the "toxic" side of the neutrophil biology. although t cells, particularly of cd4+ phenotype, are indispensable for the activation/regulation of sterile inflammation in liver iri [112, 113] , it seems that the presence of cd4+ t cells per se rather than ag-specific de novo t cell activation is needed for immune regulation [114] . hence, the ability of neutrophils to reduce t cell activation may be insufficient to significantly impact iri outcomes. in addition, the severe hepatocellular damage produced in currently used murine iri models may lead to a failure in detecting discrete immunomodulatory and/or tissue-repairing neutrophil functions. future studies need to focus on neutrophil involvement in the resolution of inflammation and recreation of tissue homeostasis. experimental evidence indicates the antiinflammatory (n2) neutrophil polarization by tgfβ, il4 or rosiglitazone [75] [76] [77] . although the neutrophil plasticity has not been rigorously proven, the n1 to n2 phenotype shifting should be of a potential therapeutic interest. neutrophil apoptosis is a favorable way of its functional termination, leading to an immunomodulatory shift of phagocytosing kupffer cells and prevention of the damps burst, while excessive induction of neutrophil apoptosis may cause unnecessary immune suppression. indeed, ectoine, a natural cell protectant, improved inflammatory resolution in a sterile inflammation lung model by preferentially prompting apoptosis in activated but not inactivated neutrophils, reducing neutrophil numbers and improving resolution of the lung pathology [115] . furthermore, a recent randomized clinical trial in elderly patients highlight the efficacy of inhaled ectoine in neutrophilic lung inflammation [116] . studies by christoffersson et al. have identified vegf-a-induced cxcr4 high unique neutrophil subset, which retained the ability of revascularization in a pancreatic islet transplant model [86, 87] , while a study by wang et al. [83•] implied cxcr4 expressing noninflammatory neutrophil subset capable of homing back to bone marrow in the "reverse migration" mechanism [117] . thus, one may envision a great potential for future regimens shifting neutrophils into tissue-resolving cxcr4-positive dominant population. due to technological advances, a remarkable progress has been recently made in our appreciation of the neutrophil biology, especially its immune regulatory and tissue-repairing functions. live-cell and deep-tissue imaging combined with single cell flow cytometry analyses [118] allow to accurately track neutrophil functions and interactions in real-time. unlike lysm reporter mice, unable to distinguish between neutrophil and monocyte/macrophage lineages, a newly developed murine strain with a more specific ly6g promoter [119] has enabled to investigate in vivo neutrophil functions more selectively and unequivocally. since efficient strategies against hepatic iri have not been yet developed, newly discovered and therapeutically attractive neutrophil functions, as discussed in this review, warrant future comprehensive preclinical and clinical attention in transplant recipients. conflict of interest the authors declare that they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. particular interest, published recently, have been highlighted as: • of importance challenges to liver transplantation and strategies to improve outcomes the utility of marginal donors in liver transplantation ischaemia-reperfusion injury in liver transplantation-from bench to bedside increased ischemic injury in old mouse liver: an atp-dependent mechanism mechanisms of hepatic injury and protective effects of nitric oxide liver ischemia and 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peripheral blood leukocyte counts in mice using cardiac, tail, foot, and saphenous vein puncture methods neutrophil recruitment and function in health and inflammation neutrophils-a key component of ischemia-reperfusion injury recombinant relaxin protects liver transplants from ischemia damage via hepatocyte glucocorticoid receptor: from bench-to-bedside neutrophils, from marrow to microbes sterile inflammation in the liver intravascular danger signals guide neutrophils to sites of sterile inflammation acetaminopheninduced hepatotoxicity in mice is dependent on tlr9 and the nalp3 inflammasome functional inactivation of neutrophils with a mac-1 (cd11b/cd18) monoclonal antibody protects against ischemiareperfusion injury in rat liver efficacy of intraportal injection of anti-icam-1 monoclonal antibody against liver cell injury following warm ischemia in the rat endogenous histones function as alarmins in sterile inflammatory liver injury through toll-like receptor 9 in mice the nuclear factor hmgb1 mediates hepatic injury after murine liver ischemia-reperfusion thrombomodulin attenuates inflammatory damage due to liver ischemia and reperfusion injury in mice in toll-like receptor 4-dependent manner the hmgb1/rage axis triggers neutrophil-mediated injury amplification following necrosis hepatocyte-specific high-mobility group box 1 deletion worsens the injury in liver ischemia/reperfusion: a role for intracellular high-mobility group box 1 in cellular protection intravital microscopic evaluation of the effects of a cxcr2 antagonist in a model of liver ischemia reperfusion injury in mice kupffer cellderived tnf-alpha promotes hepatocytes to produce cxcl1 and mobilize neutrophils in response to necrotic cells a tlr2/s100a9/cxcl-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse circulating mitochondrial damps cause inflammatory responses to injury intravital imaging of neutrophil recruitment reveals the efficacy of fpr1 blockade in hepatic ischemia-reperfusion injury this study revealed the efficacy of fpr1 antagonist to inhibit neutrophil migration and alleviate liver iri an intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure matrix metalloproteinase-2 (mmp-2) gene deletion enhances mmp-9 activity, impairs parp-1 degradation, and exacerbates hepatic ischemia and reperfusion injury in mice angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix fibronectin-alpha4beta1 integrin interactions regulate metalloproteinase-9 expression in steatotic liver ischemia and reperfusion injury metalloproteinase-9 deficiency protects against hepatic ischemia/reperfusion injury timp-1 deficiency leads to lethal partial hepatic ischemia and reperfusion injury adeno-associated virus-mediated gene transfer of tissue inhibitor of metalloproteinases-1 impairs neutrophil extracellular trap formation and ameliorates hepatic ischemia and reperfusion injury assessment of serum matrix metalloproteinases mmp-2 and mmp-9 after human liver transplantation: increased serum mmp-9 level in acute rejection role of nadph oxidase in endothelial ischemia/reperfusion injury in humans ischemia-reperfusion injury in fatty liver is mediated by activated nadph oxidase 2 in rats sivelestat sodium hydrate inhibits neutrophil migration to the vessel wall and suppresses hepatic ischemia-reperfusion injury the inhibition of neutrophil elastase ameliorates mouse liver damage due to ischemia and reperfusion the effect of the neutrophil elastase inhibitor sivelestat on early injury after liver resection myeloperoxidase: friend and foe this study demonstrates the unexpected requirement for neutrophils and neutrophil-derived mpo in hepatoprotection against endotoxin-induced liver injury and highlights a novel immunoregulatory neutrophil function effect of the myeloperoxidase inhibitor azd3241 on microglia: a pet study in parkinson's disease neutrophil extracellular traps: double-edged swords of innate immunity an emerging role for neutrophil extracellular traps in noninfectious disease histones and neutrophil extracellular traps enhance tubular necrosis and remote organ injury in ischemic aki neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of dnase-based reperfusion strategy human eosinophil granulocytes do not express the enzyme arginase arginase i is constitutively expressed in human granulocytes and participates in fungicidal activity mature cystic fibrosis airway neutrophils suppress t cell function: evidence for a role of arginase 1 but not programmed death-ligand 1 l-arginine depletion blunts antitumor t-cell responses by inducing myeloid-derived suppressor cells cleavage of lymphocyte surface antigens cd2, cd4, and cd8 by polymorphonuclear leukocyte elastase and cathepsin g in patients with cystic fibrosis oxidation of cofilin mediates t cell hyporesponsiveness under oxidative stress conditions naturally occurring regulatory t cells show reduced sensitivity toward oxidative stress-induced cell death neutrophils in homeostasis, immunity, and cancer neutrophils can disarm nk cell response through cleavage of nkp46 specific lipid mediator signatures of human phagocytes: microparticles stimulate macrophage efferocytosis and pro-resolving mediators endogenous lipid-and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin a4 receptor annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages monocyte and macrophage heterogeneity polarization of tumor-associated neutrophil phenotype by tgfbeta: "n1" versus "n2" tan on the dual roles and polarized phenotypes of neutrophils in tumor development and progression an overview of the role of neutrophils in innate immunity, inflammation and hostbiomaterial integration neutrophils responsive to endogenous ifn-beta regulate tumor angiogenesis and growth in a mouse tumor model temporal neutrophil polarization following myocardial infarction n2 neutrophils, novel players in brain inflammation after stroke: modulation by the ppargamma agonist rosiglitazone macrophage plasticity, polarization, and function in health and disease profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways a subset of neutrophils in human systemic inflammation inhibits t cell responses through mac-1 neutrophils in tissue injury and repair immunodeficiency diseases caused by defects in phagocytes visualizing the function and fate of neutrophils in sterile injury and repair resolution of inflammation: an integrated view human neutrophils uniquely release timp-free mmp-9 to provide a potent catalytic stimulator of angiogenesis vegf-a recruits a proangiogenic mmp-9-delivering neutrophil subset that induces angiogenesis in transplanted hypoxic tissue identification and characterization of vegf-aresponsive neutrophils expressing cd49d, vegfr1, and cxcr4 in mice and humans host non-inflammatory neutrophils mediate the engraftment of bioengineered vascular networks phagocyte partnership during the onset and resolution of inflammation neutrophil cell death in response to infection and its relation to coagulation expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction tissue factor expression in neutrophil extracellular traps and neutrophil derived microparticles in antineutrophil cytoplasmic antibody associated vasculitis may promote thromboinflammation and the thrombophilic state associated with the disease platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice infection-induced netosis is a dynamic process involving neutrophil multitasking in vivo interesting study highlighting the ability of excessive nets inhibition to cause pathogen spread and increase inflammation neutrophil extracellular traps induce organ damage during experimental and clinical sepsis aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines acute respiratory distress syndrome after orthotopic liver transplantation early acute kidney injury after liver transplantation: predisposing factors and clinical implications resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish a zebrafish compound screen reveals modulation of neutrophil reverse migration as an anti-inflammatory mechanism the junctional adhesion molecule jam-c regulates polarized transendothelial migration of neutrophils in vivo leukotriene b4-neutrophil elastase axis drives neutrophil reverse transendothelial cell migration in vivo reversemigrated neutrophils regulated by jam-c are involved in acute pancreatitis-associated lung injury interferon regulatory factor 3 deficiency leads to interleukin-17-mediated liver ischemia-reperfusion injury the fibrinderived peptide bbeta15-42 attenuates liver damage in a rat model of liver ischemia/reperfusion injury bruton tyrosine kinase inhibition attenuates liver damage in a mouse warm ischemia and reperfusion model the n-terminal domain of thrombomodulin sequesters high-mobility group-b1 protein, a novel antiinflammatory mechanism soluble thrombomodulin attenuates sinusoidal obstruction syndrome in rat through suppression of high mobility group box 1 neutrophil elastase enhances sputum solubilization in cystic fibrosis patients receiving dnase therapy molecular mechanisms of net formation and degradation revealed by intravital imaging in the liver vasculature the emerging role of t cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse t-cell immunoglobulin mucin-3 determines severity of liver ischemia/reperfusion injury in mice in a tlr4-dependent manner cd4 t cells promote tissue inflammation via cd40 signaling without de novo activation in a murine model of liver ischemia/reperfusion injury recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation reduction of neutrophilic lung inflammation by inhalation of the compatible solute ectoine: a randomized trial with elderly individuals neutrophil mobilization and clearance in the bone marrow turning on the lights inside neutrophils catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes key: cord-277535-u283k70i authors: vaja, rakesh; rana, meenal title: drugs and the liver date: 2020-09-22 journal: nan doi: 10.1016/j.mpaic.2020.07.001 sha: doc_id: 277535 cord_uid: u283k70i the liver is a major organ with multiple functions. a number of drugs are metabolized by the liver during phase 1 and 2 reactions which include complex processes involving cytochrome p450 enzymes. genetic and acquired variability in cytochrome p450 activity may have profound effects on pharmacokinetics. additionally, drugs can also modify how the liver functions and cause dysfunction or even failure of the organ both by a direct effect on the liver or by alteration in liver blood flow. it is important to recognize the signs and symptoms of liver failure in patients and identify possible causes including drug interactions. furthermore, once a patient has been recognized to be suffering with liver dysfunction or failure drug choice and dosing regime will need to be rationalized. paracetamol overdose can have severe and life threatening consequences for patients due to its effect on liver function. it is the leading cause of acute liver failure in the uk, 1 correct and early management is crucial and will be discussed within this article. the liver receives approximately 30% of cardiac output. uniquely it receives both arterial blood from the hepatic artery and venous blood from the portal veins. the portal vein supplies 70e75% of hepatic blood flow but only 50% of oxygen supply, the remaining blood flow and oxygen supply being from the hepatic artery. anatomically the liver is divided into two lobes and further into functional lobules based around a central vein, which contains blood from the hepatic arterial and portal venous circulations. blood arriving to the liver flows into the sinusoids which are spaces lined by hepactocytes. blood then drains towards the centre of the lobule and the central vein then hepatic vein to return blood back to the heart via the inferior vena cava. it is the portal veins taking blood directly from the gut to the liver which allows for first pass metabolism, making the liver susceptible to ingested drugs as they are absorbed from the gastrointestinal tract and transported to the liver. the liver has a broad range of functions categorized in table 1 . the liver metabolises a wide range of drugs the end result being to produce water soluble compounds which can be excreted in the bile. this results from phase 1 reactions mediated by cytochrome p450 including oxidation, reduction and hydrolysis reactions. this is followed by phase 2 reactions which are conjugative. the cytochrome p450 family are a group of enzymes found mainly in the liver, which perform oxidation and reduction reactions (phase 1) using iron to enhance the water solubility of drugs to aid excretion. cyp450 enzymes are so named as they are bound to membranes within the cell and contain a haem pigment that absorbs light at a wavelength of 450nm when exposed to carbon monoxide. after reading this article you should: c understand the mechanisms of drug metabolism by the liver c have an appreciation of alterations to drug choice and dosing regimens in patients with liver disease due to their altered pharmacokinetics c know the management of a patient with paracetamol overdose there are many different isoforms of cyp450, classified according to their amino acid sequencing into families, subfamilies and individual genes. their importance can be seen in certain subgroups that lack particular genes. an example pertinent to anaesthesia is deficiency in cyp2d6 which metabolises codeine to morphine, these patients therefore find codeine ineffective. conversely there is a small subgroup of people of saudi arabian and ethiopian decent with very high expression of 2d6 who metabolize codeine into vast amounts of morphine. refer to table 2 for more details. an individual more detailed breakdown of cyp450 genes is beyond the scope of this article. some drugs can induce or inhibit cyp450 enzymes which have the sequential effect on the metabolism of other drugs, either increasing or reducing it respectively. possibly the most important example is cyp3a4 which metabolises many substrates and is induced by rifampicin, carbamazepine, phenytonin and dexamethasone. of interest to anaesthesia this will increase metabolism of opioids, benzodiazepines and local anaesthetics. another well cited example is the increased metabolism of the oral contraceptive pill and its reduction in efficacy. for a more exhaustive list of substrates, inducers and inhibitors see table 3 (see table 4 ). a number of non-cytochrome p450 dependent reactions occur in the liver eg oxidation of dopamine and alcohol and hydrolysis of amides and esters (eg lignocaine and pethidine respectively). a predominant rise in aspartate aminotransferase (ast) and alanine aminotransferase (alt) signals hepatocellular injury or death. this can be caused by drug reactions or toxicity (e.g. paracetamol), viral hepatitis, autoimmune hepatitis, alcoholic hepatitis, ischaemic hepatitis secondary to profound hypotension, and rare causes such as wilson's disease. an obstructive pattern has a rise predominantly in alkaline phosphatase (alp) and gamma-glutamyltransferase (ggt), these are canalicular enzymes and suggest cholestasis. this is caused by obstruction, either calculi or tumour (primary biliary, pancreatic or metastases), and liver disease such as primary biliary cirrhosis. pharmacological causes include antibiotics, anabolic steroids and oral contraceptives. a mixed pattern can be seen in sepsis, some drug reactions, cholangitis, congestive cardiac failure and alcoholic liver disease. halothane hepatitis can cause raised liver enzyme assays, raised bilirubin and jaundice. an isolated rise in unconjugated bilirubin may be attributed to gilbert's syndrome or haemolysis. the sars-cov-2 virus uses angiotensin-converting enzyme 2(ace-2) receptors to gain entry into cells. 3 liver particularly in the ductal region has abundance of these receptors, hence may be susceptible to sars-cov-2 3 elevated lft have been reported widely in hospitalized patients with covid-19, the range of elevation is highly variable from 14 to 58%. 4 surprisingly, the pattern of elevation mimics hepatocyte damage (ast/alt higher than bilirubin or alp) rather than cholangiocytic damage, as would have been expected given the density of ace-2 receptors is higher in the ductal system. 4 additionally low albumin has been seen and is a marker of severe disease. 5 one study reported longer hospital stay in patients with elevated lft. 6 an international registry has suggested that has many as 25% of patients with covid-19 may present with hepatic decompensation in absence of respiratory symptoms. 7 the above findings hold important implications for anaesthetists, especially in preop assessments. most drugs given in anaesthesia and intensive care are given intravenously, thus having a bioavailability of 1. however some maybe given orally or nasogastrically and absorbed enterally. the absorption will be affected by delayed gastric emptying or reduced by diarrhoea and increased gastric transit time seen in liver failure. additionally if vasopressors are used there maybe splanchnic vasoconstriction with associated reduced absorption. volume of distribution is a theoretical calculated volume within which a dose of a drug is dissolved. hepatic dysfunction can cause fluid retention and will increase the volume within which drugs are present, particularly those which usually remain in the plasma, thus increasing their volume of distribution and reducing their plasma concentration. 2 in liver disease, protein synthesis may be reduced. these proteins are important as binding sites for drugs and as such alter the amount of free drug available, volume of distribution, half life and duration of action. an important example is albumin. hypo-albuminaemia will increase the proportion of free drug which is active, therefore doses of highly protein bound drugs may need to be reduced, for example phenytoin and benzodiazepines, aspirin and warfarin. 8 another protein produced by the liver, a 1 acid glycoprotein binds basic drugs such as carbamazepine, propanolol, alprenolol and imipramine as well as steroids. bilirubin can also compete for protein binding sites, so raised levels can increase amount of free drugs, the effect however is less in vivo than in vitro. 1 problems with absorption of enterally delivered drugs have been described. once absorbed these drugs undergo the "first pass effect" by the liver before reaching the systemic circulation. in liver failure the degree of metabolism will be reduced, therefore the extraction ratio will also be reduced and more drug will reach the systemic circulation, thus increasing bioavailability. prevalence of ultrarapid metabolisers metabolism of drugs in liver disease depends on liver blood flow. this can be reduced in a cirrhotic liver as portovenous shunting in the form of varices which are created and blood is diverted directly into the systemic circulation by-passing the liver. thus first pass metabolism is reduced. drug metabolism by the liver may also be reduced by the use of vasopressors on intensive care which reduce liver blood flow due to varying degrees of splanchnic vasoconstriction. the phase 1 and 2 reactions performed by the liver are affected and metabolism and thus extraction ratios are reduced. drugs can be divided into those with high extraction ratios >0.7, for example fentanyl and morphine and low extraction ratios <0.3 such as lorazepam, diazepam and methadone. most drugs have low extraction ratios <0.3 that is they have poor permeability and are metabolized by the liver but poorly extracted, therefore clearance is limited by reduced metabolism not by blood flow. those with high extraction ratios >0.7 are highly permeable and clearance is dependent on blood flow. 9 hepatic dysfunction is not uncommon within the intensive care setting affecting 11e54% of critically ill patients depending on definitions used. 2 there is currently no tool akin to renal clearance to indicate degree of liver dysfunction. 9 therefore clinicians use liver function blood tests, international normalized ratio (inr), serum albumin and clinical scores such as the child pugh score act as a surrogate for function. more recently the model for end stage liver disease (meld score) and the meld-na have been used to more accurately predict the severity of liver dysfunction. 10, 11 although their correlation with pharmacokinetic function not well understood. due to the alterations discussed in pharmacokinetics in liver dysfunction drug choices, dosages and frequency may need to be rationalized and altered accordingly. for example the induction dose and maintenance dose, for either anaesthesia or sedation, needs to be reduced. historically inhalational agents, particularly halothane have been implicated in causing hepatitis. the risk is related to the generation of trifluroacteyl chloride (tfa) by metabolism of agents, which is implicated in toxicity 12 around 20% of administered dose of halothane is metabolized by the liver, more specifically by cytochrome p450. this is a relatively high percentage when compared to more modern inhalational agents, for example, 0.2% isoflurane, 0.02% desflurane and 3% sevoflurane. even though sevoflurane undergoes 3% metabolism it does not generate tfa and hence is not linked to immune mediated injury 13 sevoflurane metabolism produces fluoride which is not linked to hepatotoxicity. 14 inhalational agents themselves cause a dose dependent reduction in hepatic blood flow (hbf). isoflurane and sevoflurane result in relatively lower reduction in hbf at 1 mac as compared to desflurane. 15 as long as hypotension is avoided and above effects are kept in mind desflurane is probably the safest choice of inhalational agent due to its' low rate of metabolism and rapid and predictable emergence from anaesthesia. 13 there are two types of halothane hepatitis. type 1 which is mild, transient and has a relatively high incidence (25e30%). type 2 caused by oxidative metabolism of halothane in the liver leading to fever, jaundice, and dramatically elevated serum transaminases. the compounds synthesized by oxidation then bind to trifluoroacetylate proteins in the hepatic endoplasmic reticulum causing cellular dysfunction, it is thought to occur in genetically predisposed individuals. the committee on safety of medicines in 1986 recommended the avoidance of halothane in patients with a history of previous adverse reactions, those who had received halothane within 3 months unless clinically necessary, and those with a history of unexplained jaundice or pyrexia following previous halothane anaesthesia. iv anaesthetics: the induction agents have a marked effect on haemodynamics and may cause sudden precipitious fall in blood pressure. in clinical practice a standard induction dose need not be altered. however they should be titrated slowly to effect. there are no current recommendations on the use of tiva (total intravenous anaesthesia) in patients with liver disease. research is sparse and conflicting. some earlier reports suggested that inhalational anaesthesia results in smaller elevation of liver enzymes than tiva with propofol-fentanyl. 16 a more recent study however suggested slightly lower rate of elevation in lft after using tiva. 17 opiates morphine is metabolized by the liver to active metabolite morphine-6-glucoronide which has potent analgesic properties, and morphine-3-glucuronide, which has no analgesic properties but has adverse neurotoxic side effects such as confusion and respiratory depression. as both metabolites are excreted renally, they accumulate in renal failure. in liver failure morphine itself may accumulate as extraction ratio is reduced thereby enhancing further the effect of morphine. 13 therefore dose of morphine should be reduced. the same is true of fentanyl and alfentanil dose, as although there is no active metabolite they also rely on hepatic metabolism. remifentanil may be good choice as its metabolism is by plasma esterases and it has no active metabolites. a review of pain management in patients with liver disease by the american association for study of liver diseases (aalsd) in 2018 states in general most opioids have prolonged half life. their recommendations include increase the dosing intervals (6e12hr) and using immediate release preparations over extended release 18 in july 2013 the medicine and healthcare products regulatory agency (mhra) produced a drug safety update that restricted the use of codeine in children. 19 this was prompted by case reports of four children suffered serious harm following the administration of codeine in the immediate post-operative period. codeine should only be used to relieve acute moderate pain in children older than 12 years and only if it cannot be relieved by other painkillers such as paracetamol or ibuprofen alone. a significant risk of serious and life-threatening adverse reactions has been identified in children with obstructive sleep apnoea who received codeine after tonsillectomy or adenoidectomy (or both). codeine is now contraindicated in all children younger than 18 years who undergo these procedures for obstructive sleep apnoea. codeine is converted to morphine in the liver by the cyp2d6 enzyme. the extent of conversion of codeine to morphine depends on genetic variations of cyp2d6. people can be classified as: poor; intermediate; extensive; or ultra-rapid metabolisers. poor metabolisers convert very little codeine into morphine and therefore have little or no pain relief, ultra-rapid metabolisers or extensive metabolisers have an excessive amount of morphine in their blood following ingestion of codeine. ethnic origin is an important factor in genetic variability. up to 10% of caucasians are poor metabolisers whereas up to 29% of patients of african origin may be ultra-rapid metabolisers. (table 2) . this genetic variability leads to different plasma morphine concentrations in patients leading to different analgesic effects as well as side effects including respiratory depression. codeine is contraindicated in all patients of any age known to be cyp2d6 ultra-rapid metabolisers and should not be used by breastfeeding mothers because it can pass to the baby through breast milk and potentially cause harm. nsaids are contraindicated for systemic use in most liver disease patients, because of increased bioavalibilty, the high risk of precipitating gastrointestinal bleeding and renal failure. 20 pregabalin and gabapentin are not metabolized in the liver and can be considered for use. these drugs are renally excreted therefore patients with hepatorenal syndrome warrant cautious adminstration. 18, 20 gabapentin is considered as first line non opiod drug for analgesia. among the tricyclic antidepressants nortryptilline appear safer than amitriptyline and imipramine. 20 an increased dose of non-depolarising neuromuscular blockers (nmb) may be required in liver disease possibly due to altered pharmacology anaesthesia and intensive care medicine xxx:xxx protein binding and increased volume of distribution. however, those which are metabolized by the liver have a prolonged duration of action, atracurium as metabolized in the plasma has a more predictable duration of action. however, it is worth noting that in prolonged usage concentrations laudanosine from hoffmann degradation may accumulate with the potential to provoke epileptiform activity on electro encephalography (eeg). 13 this is of greater concern if the patient has concomitant renal failure or impaired blood brain barrier. the metabolism of succinylcholine may be prolonged due to reductions in pseudocholinesterase concentrations, but clinically this is of little significance. suggamadex is a unique reversal agent for amniosteriodal nmb which acts by chelating the nmb. it is not metabolized and is excreted almost exclusively unchanged by the kidneys within 24 h. data regarding use of sugammadex, in patients with liver dysfunction is limited. however as sugammadex is almost entirely excreted renally, no dose reduction is required in patients with mild to moderate liver dysfunction. in patients undergoing liver transplant sugammadex is able to reverse neuromuscular block maintained by rocuronium continuous infusion. 21 however it is important to note that the sugammadex recovery time in this population was found to be considerably longer than in other surgical settings, and should be considered in clinical practice. dexmedetomidine is a highly selective alpha-2 receptor agonist, with analgesic, anxiolytic and sedative. it is primarily metabolized in the liver and may have a prolonged half-life in patients with liver disease. dexmedetomidine has potential protective effects on the liver and intestine during hepatectomy and intraoperative use of dexmedetomidine during liver surgery is subject to ongoing research. 22 additionally patients with elevated bilirubin and bile salts secondary to jaundice may show bradycardias limiting its use. 23 paracetamol overdose paracetamol is the commonest drug taken in overdose in the uk to date, it can result in liver failure and in some cases fatal. 24 hepatocelluar necrosis can occur if as little as 7.5g of paracetamol is ingested. normal pathways of metabolism are saturated and hepatic glutathione stores are exhausted. patients are often initially asymptomatic for the first 24 h before reporting nausea, vomiting, right upper quadrant pain with progressive derangement of liver function tests (lfts) after 18 h. the mhra produced new guidelines for treatment of paracetamol overdose in 2012; the changes included an updated nomogram and a simplified treatment schedule. 25 the administration of acetylcysteine has previously been based on the rumack-matthew nomogram, which divided treatment groups into high and low risk. the dose was then calculated on a weight-based table, which increased the risk of drug error. the updated nomogram has a single treatment line (figure 1) 25 ; thereby eliminating the need for assessing whether the patient falls into the high-risk category. it also advises that in cases of staggered overdoses there should be no delay in the administration of acetylcysteine. another cause for concern was the adverse events that had been reported following the bolus dose of acetylcysteine, this has been addressed by increasing the duration over which it is infused, from 15 min to 60 min. acetylcysteine should be administered when the plasma paracetamol level is on or above a single treatment line joining points of 100mg/l at 4 h and 15mg/l at 15 h after ingestion. a baseline full blood count (fbc), urea and electrolytes (u&es), lfts and coagulation screen along with an arterial blood gas sample should also be done at the earliest opportunity. intravenous preparations of paracetamol were licensed in the uk in 2004 and it is routinely used in anaesthetic practice. however, there have been some concerns regarding the dosage and administration; especially in adults and children under 50kg, patients with pre-existing hepatic dysfunction and the elderly. children and infants weighing less than 10kg should receive the reduced dose of 7.5mg/kg not exceeding the daily dose of 30mg/ kg, whilst those >10kg can be prescribed up to 15mg/kg not exceeding the daily dose of 60mg/kg. both the mhra and the npsa have issued alerts, regarding the correct dose prescription as there have been reported cases of accidental overdose due to ml to mg conversion and the administration of 1g in adults weighing less than 50kg. the key issue is that with intravenous paracetamol plasma levels will peak immediately after administration. the traditional nomograms used to predict plasma levels after overdose refer to oral ingestion where the levels peak some hours after. indicators of severe paracetamol poisoning which is likely to require referral to a specialist liver centre include: inr of >2.0 at 24 h, >4 at 48 h or >6 at 72 hours; renal impairment (creatinine >200 micromol/l); hypoglycaemia; metabolic acidosis despite rehydration; hypotension despite resuscitation or encephalopathy. 26 the only other treatment in fulminant liver failure is transplantation. paracetamol as analgesic in chronic liver disease given the potential of paracetamol to cause liver injury, there is a common misconception that these patients should never take paracetamol. however, various studies have shown that if taken in appropriate doses, paracetamol is one of the safest analgesics for patients with cirrhosis. limiting the total daily dosage to 2e3 g/day with thrice daily dosing is generally recommended. 27 patients should be educated about over-the-counter and prescription medications that may also contain acetaminophen to avoid overdose. remdesvir: elevated liver enzymes are commonly observed in clinical trials of patients with remedevir. 28 the elevated values rarely warrant treatment discontinuation. current recommendations suggest that if the enzymes are elevated to 5 times or more above baseline, to discontinue the drug. tocilizumab: alt elevations are frequent but fulminant hepatitis is rare. the risk of re activation of hbv should be kept in mind if patient had chronic liver disease secondary to viral etiology. 28 steriods: low dose dexamethasone is probably safe in patients with chronic stable liver diease. however use of methylprednisolone in high doses may reactivate hbv and increase risk of spontaneous bacterial peritonitis (sbp) in severe cases. 28 hydoxychloroquine: data is limited but generally has not been associated with elevations in alt levels and is an extremely rare cause of drug induced liver injury. 28e31 changing patterns of causation and the use of transplantation in the united kingdom drug dosing considerations for the critically ill patient with liver disease sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor liver injury in covid-19: the current evidence hypoalbuminemia predicts the outcome of covid-19 independent of age and co-morbidity liver injury during highly pathogenic human coronavirus infections high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry differentiated effects of liver cirrohosis on albumin binding sites for diazepam, salicylic acid and warfarin anaesthesia for the patient with liver disease a model to predict poor survival in patients undergoing transjugular intrahepatic portosystemic shunts meld-na (the new meld) and peri-operative outcomes in emergency surgery buggy donal j. pharmacology of anaesthetic agents ii: inhalation anaesthetic agents anaesthesia for patients with liver disease the effects of prolonged lowflow sevoflurane anesthesia on renal and hepatic function blood supply to the liver in the human after 1 mac desflurane in comparison with isoflurane and halothane the effect of isoflurane or propofol anaesthesia on liver injury after partial hepatectomy in cirrhotic patients comparison of the postoperative liver function between total intravenous anesthesia and inhalation anesthesia in patients with preoperatively elevated liver transaminase levels: a retrospective cohort study pain management in patients with cirrhosis analgesics in patients with hepatic impairment: pharmacology and clinical implications sugammadex versus neostigmine after rocuronium continuous infusion in patients undergoing liver transplantation dexmedetomidine reduces intestinal and hepatic injury after hepatectomy with inflow occlusion under general anaesthesia: a randomized controlled trial bile acids induce arrhythmias: old metabolite office for national statistics) paracetamol overdose: an evidence based flowchart to guide management the therapeutic use of analgesics in patients with liver cirrhosis: a literature review and evidence-based recommendations care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper liver injury in covid-19: management and challenges covid-19: abnormal liver function tests pharmacokinetics in disease states modifying hepatic and metabolic function key: cord-324529-xbrdtxnz authors: wang, ming; yan, weiming; qi, weipeng; wu, di; zhu, lin; li, weina; wang, xiaojing; ma, ke; ni, ming; xu, dong; wang, hongwu; chen, guang; yu, haijing; ding, hongfang; xing, mingyou; han, meifang; luo, xiaoping; chen, tao; guo, wei; xi, dong; ning, qin title: clinical characteristics and risk factors of liver injury in covid-19: a retrospective cohort study from wuhan, china date: 2020-10-07 journal: hepatol int doi: 10.1007/s12072-020-10075-5 sha: doc_id: 324529 cord_uid: xbrdtxnz background: coronavirus disease 2019 (covid-19) has rapidly become a major international public health concern. this study was designed to evaluate the clinical characteristics and risk factors of covid-19-associated liver injury. methods: a fraction of 657 covid-19 patients were retrospectively analyzed. clinical and laboratory data were derived from electronic medical records and compared between patients with or without liver injury. multivariate logistic regression method was used to analyze the risk factors for liver injury. results: among 657 patients, 303 (46.1%) patients had liver injury with higher rate in severe/critically ill patients [148/257 (57.6%)] than those in moderate cases [155/400 (38.8%)]. the incidence of liver injury was much higher in male [192/303 (63.4%)] than female [111/303 (36.6%)], and in severe/critical patients [148/303 (48.8%)] with percutaneous oxygen saturation ≤ 93% [89/279 (31.9%)] or peak body temperature ≥ 38.5 °c [185/301 (61.5%)] on admission. liver injury-related inflammations included increased white blood cells, neutrophils and decreased lymphocytes. more patients with liver injury than without had increased serum il-2r, tnfα, ferritin, hscrp, pct, esr, γ-gt, and ldh. multivariate regression analysis revealed that increasing odds of liver injury were related to male, higher serum hscrp (≥ 10 mg/l), and neutrophil-to-lymphocyte ratio (nlr) (≥ 5). moreover, more deceased patients (14/82 (17%)) had significantly elevated serum tbil than discharged patients [25/532 (4.7%)]. conclusion: liver injury is a common complication in covid-19 patients. the potential risk factors of liver injury include male, hscrp and nlr score. a close monitor of liver function should be warned in covid-19 patients, especially in severe/critical individuals. electronic supplementary material: the online version of this article (10.1007/s12072-020-10075-5) contains supplementary material, which is available to authorized users. the ongoing outbreak of coronavirus disease 2019 (covid -19) has been recently becoming a pandemic [1] . at present, it is believed that sars-cov-2 mainly invades the respiratory system. most patients have fever, cough, chest distress and dyspnea. furthermore, systemic hyperinflammation driving hypercoagulability is common in these patients [2] . however, accumulating evidences have suggested that some covid-19 patients have different degrees of liver dysfunction [3] [4] [5] [6] . of 99 patients with covid-19 enrolled in wuhan jinyintan hospital, among whom 43 patients with different degrees of abnormal liver function were admitted to intensive care unit (icu). one patient presented with obvious liver injury (alanine aminotransferase (alt) 7590 u/l, aspartate aminotransferase (ast) 1445 u/l) [3] . huang et al. showed that more patients admitted to icu had elevated ast levels [4] . wang et al. showed that patients admitted to icu had significantly higher alt levels [5] .moreover, guan et al. extracted the currently largest cohort regarding 1099 mainly moderate sars-cov-2 infected patients and showed 39.4% with severe disease had elevated ast and 28.1% had elevated alt, and the proportion was 18.2% and 19.8% in patients with non-severe disease [6] .given that the number of patients in these studies is relatively small, information about the clinical characteristics of liver injury in these patients is scarce. so far, to our knowledge, there are few reports that analyzed risk factors of liver injury in covid-19 patients. this current study aims to analyze the clinical characteristics of covid-19 patients with liver injury, and evaluate the potential risk factors for the development of liver injury. all 657 patients included in the study were admitted from january 13, 2020 to february 25, 2020 in tongji hospital. the clinical characteristics and outcomes (death or discharge) were monitored up to march 28, 2020. tongji hospital was urgently reconstructed and has been assigned by chinese government as a designated hospital for severe or critically ill covid-19 patients. according to the guidance provided by the chinese national health commission,all confirmed covid-19 patients were diagnosed by sars-cov-2 nucleic acid detection of respiratory tract specimens. the clinical data and laboratory data of patients were obtained from electronic medical records. the clinical data included sex, age, percutaneous oxygen saturation, heart rate, respiration rate, peak temperature during the course of the disease, duration of fever,comorbidities and treatment in the patients. the laboratory data included blood routine examination, liver function, renal function, coagulation function, creatine kinase (ck), creatine kinase-mb (ck-mb), hypersensitive cardiac troponin i (ctni), n terminal pro-brain type natriuretic peptide (nt-probnp), hypersensitive c-reactive protein (hscrp), erythrocyte sedimentation rate (esr), procalcitonin (pct), ferritin, cytokines (interleukin 1β (il-1β), interleukin 2 receptor (il-2r), interleukin 6 (il-6), interleukin 8 (il-8), interleukin 10 (il-10), tumor necrosis factor α (tnfα)),hepatitis b surface antigen and hepatitis c virus antibody examinations. all patients were classified as being moderate, severe, or critically ill according to the guidance for covid-19 (6th edition) released by the national health commission of china [7] (supp . table s1 ). liver injury was defined as serum level of alt or total bilirubin (tbil) greater than upper limit of normal value (uln). the uln of alt or tbil was 40u/l and 26 μmol/l, respectively, regardless of whether the patients had a history of chronic liver diseases. continuous variables were described as median and interquartile range (iqr), and tested using non-parametric mann-whitney u test. categorical variables were described as number and percentage, and tested using χ 2 test, fisher's exact test, as appropriate. multivariate logistic regression models were used to explore the risk factors for liver injury. wilcoxon signed-rank test was used for paired continuous variables. spss (version 22.0) was used for all analyses. p value < 0.05 was considered statistically significant. from january 13, 2020 to february 25, 2020, a fraction of 991 patients were enrolled from tongji hospital for this study. among them, 657 patients with confirmed sars-cov-2 infection were subsequently included for further analyses. as of march 28, 2020, 532 patients recovered and were discharged, 82 patients died and 43 patients remained hospitalized. as shown in table 1 , among 657 patients, 303 (46.1%) had liver injury. as shown in table 2 as shown in table 1 , liver injury appeared in 277 (42.2%) of 657 patients with alt elevation and in 32 patients (4.9%) with tbil elevation, 160 (24.4%) of 657 patients had γ-gt elevation. there were significant differences in laboratory findings between covid-19 patients with or without liver injury, including higher white blood cell, neutrophil and lower lymphocyte count in liver injury patients than those without. more [54/301 (17.9%)] patients with liver injury developed leukocytosis (white blood cell count ≥ 10 × 10 12 /l) than those [27/354 (7.6%)] without. there were no differences in nt-probnp and ctni levels between patients with or without liver injury ( table 2) . elevated hscrp (> 10 mg/l) appeared more frequent in patients with liver injury [230/274 (83.9%)] than those without [220/334 (65.9%)]. moreover, serum esr level was significantly higher in patients with liver injury compared with those without. in terms of cytokines, more patients with liver injury had elevated serum il-2r, il-6 and tnfα than those without. to further investigate the correlation of inflammation with liver injury, a series of inflammation-based scores including mgps, plr, nlr, and car, lmr, pni scores were calculated and compared (table 3 ). more patients with liver injury have mgps score over 2 points than those without. the plr, nlr, and car scores were significantly higher in patients with liver injury than those without. the lmr and pni scores were significantly lower in patients with liver injury than those without. the correlation between some surrogate markers of systemic inflammation response syndrome (sirs), such as il-2r, il-6, tnfα, hscrp, ferritin and pct, and alt, tbil and γ-gt levels, was investigated. and the results showed serum alt, tbil and γ-gt all correlated with hscrp and ferritin; meanwhile, tbil and γ-gt correlated with il-2r, tnfα and pct (supp . table s2 ). we also observed the effect of medications on liver injury. during hospitalization, some patients received one or more medications including antiviral drugs (oseltamivir, lopinavir/ritonavir, or arbidol), interferon inhalation, antibiotics, systemic glucocorticoid and non-steroidal anti-inflammatory drugs (nsaids the proportion of cases with γ-gt level above 1-2, 2-5 or greater than 5 times the uln was higher in severe/ critical patients than those in moderate patients. after considering the p value of univariate analysis, clinical significance and data integrity, 593 patients were enrolled and sex, metabolic disorder, viral hepatitis, hscrp, nlr and peak temperature were selected as variables of logistic regression models. the independent risk factors for liver injury were as follows: male (or 2.038, 95% ci 1.443-2.879, p < 0.001), hscrp ≥ 10 mg/l (or 1.733, 95% ci 1.118-2.687, p = 0.014), and nlr ≥ 5 (or 2.154, 95% ci 1.486-3.124, p < 0.001) ( table 4 ). the dynamic changes of hscrp and nlr with or without liver injury were further observed every 5 days after admission. both hscrp and nlr levels declined over the course of 15 days with higher values in patients with liver injury compared with those without (supp. figure s1 ). data were expressed as median (interquartile range, iqr) or n (%). p values were calculated by mann-whitney u test, χ 2 test, as appropriate. patients with at least one of the following three conditions are classified as metabolic disorders: 1. diabetes mellitus, 2. hypertension, 3. dyslipidemia to understand correlation of alt and tbil levels with disease progression, the dynamics of alt and tbil levels in 207 patients (21 deceased patients and 186 discharged patients) with complete data were analyzed from admission to the 15th day with an interval of every 5 days (fig. 1a, b) . during hospitalization, the alt levels of the discharged patients slightly increased from admission to day 5, then gradually decreased thereafter until discharge. whereas the alt levels of those deceased patients rapidly increased from the 5th day after admission until day 15. tbil levels of discharged patients stayed within normal range during the whole course, whereas it increased significantly in the deceased patients after admission, until day 15. the increase of alt from day 5 to day 15 was significantly higher in deceased patients than those in discharged patients (fig. 1c) . although the increases of tbil levels in deceased patients from day 5 to day 15 were notable, there was no statistical significance compared with discharged patients (fig. 1d) . furthermore, tbil levels of 1-2 or above 2 times the uln [7/82 (8.5%) and 7/82 (8.5%)] in deceased patients were markedly higher than those in discharged patients [24/532 (4.5%) and 1/532 (0.2%)]. the alt levels showed no significant differences between the two groups (table 5 ). the present study showed that liver injury was more prevalent in male, severe or critically ill patients with percutaneous oxygen saturation ≤ 93% or peak temperature ≥ 38.5 °c on admission, and comprehensively delineated the risk factors for covid-19-associated liver injury. liver injury-related inflammations included increased white blood cells, neutrophils and decreased lymphocytes. more patients with liver injury had increased serum il-2r, tnfα, ferritin, hscrp, pct, esr, γ-gt, and ldh. in addition, male, elevated hscrp (≥ 10 mg/l) and nlr (≥ 5) were risk factors for covid-19-associated liver injury. and patients with abnormal liver function tests (lft) had higher risks of progressing to severe disease. covid-19-associated liver injury is defined as any liver damage occurring during disease progression and treatment of covid-19 in patients with or without pre-existing liver diseases. in our cohort, there were 26 patients with elevated tbil alone. according to data analysis of these patients, the results showed that the bilirubin elevation was due to direct bilirubin elevation as main body (15 cases) or both direct bilirubin and indirect bilirubin elevation (8 cases) or indirect bilirubin elevation as main body accompanied by the rise of ast (3 cases). although all the 26 patients showed a degree of liver injury, hemolytic jaundice or gilbert's disease cannot be entirely excluded and requires further monitor in follow-up study. liver injury is not uncommon in previous studies in patients infected with severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [8] . likewise, abnormal lfts were frequently seen in patients with covid-19, with incidence rates exceeding 50% in some studies. liver injury was presented in 30 out of 113 deceased patients from our previous reports [9] . however, patients with covid-19 rarely develop acute or acute-on-chronic liver failure [4, 6, 9] . taken together, these studies demonstrated that the majority of cases of covid-19-associated liver injury occurred in severe or critically ill patients. consistent with the above findings, current study in a relatively larger cohort of severe/critical patients further demonstrated liver injury occurred more frequently in these patients [148/257 (57.6%)] than in moderate ones [155/400 (38.8%)]. serum alt, tbil and γ-gt levels were markedly higher in severe and critically ill patients than in moderate patients, with a peak alt of 728 u/l and peak tbil of 174.1 μmol/l, respectively. the increase of alt from day 5 to day 15 after admission was significantly greater in deceased patients than in discharged patients, and the proportion of cases in deceased patients with serum tbil levels of 1-2 or above 2 times the uln was markedly higher than that in discharged patients. these results provided the concrete evidence that impaired liver function was more frequent in severe and critically ill patients. moreover, multivariate regression analysis showed that hscrp ≥ 10 mg/l, nlr ≥ 5 and male were the risk factors for liver injury in our study, which was consistent with lei's results [10] . as a traditional inflammatory marker, crp is an acute-phase protein used for the diagnosis and follow-up of infection or tissue damage. li et al. [11] showed crp was independently associated with hepatic injury in patients with covid-19. nlr, as a reliable marker of systemic inflammation and infection, has been considered as a predictor of bacterial infections, including pneumonia. liu et al. [12] reported that nlr might predict severe illness in the early stage of covid-19. zhang et al. [13] identified nlr as an independent risk factor for severe covid-19. current studies indicated that there might be a sex predisposition to covid-19, with men more prone to being affected likely due to much higher smoking rate in men [14] . underlying mechanisms involved in liver injury in patients with covid-19 are complex and interactive, including immune reconstitution in the presence of sars-cov-2, direct drug toxicity, systemic inflammation with liver involvement induced by cytokine storm or pneumoniaassociated hypoxia [15] . previous studies indicated that t lymphocytes particularly cd4 + t and cd8 + t cells decreased increase in tbil from day 5 to day 15 after admission. p values were calculated by mann-whitney u test, or wilcoxon signed-rank test, as appropriate in severe covid-19 patients [16] , and immune function dysregulation in some cases may be associated with the development of acute respiratory distress syndrome (ards), collateral damage to multiple organs, such as liver, kidney and heart as well as death [17] . the exaggerated immunemediated inflammation is known as "cytokine storm". similar to our study, accumulating evidence suggested that patients with severe covid-19 might have a cytokine storm syndrome. changes of inflammatory cytokines following sars-cov-2 infection may cause or contribute to liver damage. the underlying mechanism involved in cytokine accumulation in covid-19 and subsequent liver injury warrants further investigation. recent study [18] demonstrated no significant increase in alp and γ-gt in patients with covid-19. in contrast, among 657 cases in the present study, more severely/critically ill patients [83/257 (32.3%)] than moderate patients [77/400 (19.3%)] had elevated γ-gt level. previous pathological findings of covid-19 in the liver showed moderate microvesicular steatosis and active inflammation in the hepatic portal area but no viral inclusions identified in the liver tissue [19] . the latest research showed that angiotensin-converting enzyme 2 (ace2) was expressed in specific cholangiocytes in healthy liver tissues [20] and suggested liver injury in covid-19 patients may not be only due to hepatocyte damage but cholangiocyte dysfunction. in our study, elevated γ-gt and bilirubin in patients with covid-19 suggested that the virus may cause liver impairment by targeting cholangiocytes. the pathogenesis of covid-19-associated liver injury warrants further investigation. the impact of preexisting chronic liver diseases on the liver function of covid-19 patients remains not fully elucidated. ji et al. recently reported patients with nonalcoholic fatty liver disease (nafld) were at higher risk of disease progression, and developing abnormal liver function during hospitalization in covid-19 patients [21] . singh et al. [22] found covid-19 patients with preexisting liver disease, notably cirrhosis, were at higher risk for hospitalizations and mortality. guan et al. reported that patients with preexisting chronic hepatitis b did not have more severe disease compared to the overall population [6] . our study showed that 52 (7.9%) patients with underlying viral hepatitis (hepatitis b and hepatitis c)developed liver dysfunction, and more patients with underlying viral hepatitis developed liver injury than those without [31/303 (10.2%) vs 21/354 (5.9%)], which may be partly due to inflammatory responses and immune disorder of viral hepatitis. further multivariate regression analysis showed that underlying viral hepatitis and metabolic disorder were not independent risk factors for liver injury, which is consistent with recent report [23] , suggesting that liver injury cannot be entirely attributed to the preexisting chronic liver diseases in covid-19 patients. considering the administration of antivirals, antibiotics, nsaids and glucocorticoids in the treatment of patients, potential drug-induced liver injury (dili) deserves special concern. given data provided is limited, based on recent results, we cannot draw a definitive conclusion about whether lopinavir/ritonavir increase the risk of developing liver damage [18, 24, 25] . our study revealed a higher proportion of patients with liver injury had received systemic glucocorticoid. nevertheless, we found a slightly reduced risk of hepatic injury in patients treated with arbidol. this is likely due to the possibility that arbidol may effectively suppress sars-cov-2 replication, which may be a beneficial pathway to lower the risk of the subsequent viral-induced liver injury. it has recently been reported that arbidol combined with lopinavir/ritonavir achieved significantly higher rates of viral clearance and favorable clinical outcome compared with lopinavir/ritonavir used alone [26] . these findings further supported the hypothesis that effective antiviral therapy may prove useful in preventing or slowing the progression of the covid-19 disease. our study has some limitations. first, nearly one third of critically ill patients developed disorders of consciousness on admission, which might result in a loss of some information (particularly a detailed history and subjective symptoms). additionally, some physical examination and laboratory tests were not done in all the patients, and missing data or important tests might lead to bias of clinical characteristics. second, the impact of covid-19 in patients with preexisting chronic liver diseases, such as viral hepatitis, nafld, and alcohol-related liver disease, remains to be evaluated. third, in the early period of epidemic, owing to some environmental constraints, such as the explosive growth of the infected population and the shortage of medical supplies, the results may be difficult to interpret accurately. more intensive surveillance or individually tailored therapeutic approach is needed for severe cases, especially among patients with liver or other important organ dysfunction. in conclusion, immune-mediated inflammation may lead to covid-19-associated liver injury. the potential risk factors for liver injury include male, hscrp and nlr score. moreover, patients with abnormal lfts had higher risks of progressing to severe disease. attention should be paid to monitor liver function during the course of covid-19, especially in severely/critically ill patients. the data provided may be useful in helping us extend our understanding of the disease so that early effective intervention could be carried out promptly. appropriate intervention and reasonable supportive care are expected to reduce the inflammatory response and then further reduce liver injury of patients with covid-19. a novel coronavirus from patients with pneumonia in china coagulation abnormalities and thrombosis in patients with covid-19 epidemiological and clinical 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smoking predisposition in patients with covid-19 liver injury in covid-19: management and challenges clinical and immunological features of severe and moderate coronavirus disease 2019 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical features of covid-19-related liver damage pathological findings of covid-19 associated with acute respiratory distress syndrome specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection nonalcoholic fatty liver diseases in patients with covid-19: a retrospective study clinical characteristics and outcomes of covid-19 among patients with pre-existing liver disease in united states: a multi-center research network study coronavirus disease 2019 (covid-19) and prevalence of chronic liver disease: a metaanalysis characteristics of liver tests in covid-19 patients a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study we thank all the patients and their families involved in this study, as well as numerous doctors, nurses and civilians working together to fight against the sars-cov-2.author contributions mw and wmy share first authorship, and the order in which they are listed was determined by workload. qn, dx (dong xi), and wg designed the study and had full access to all data in the study and take responsibility for the integrity of data and the accuracy of the data analysis. mw and dx contributed to patient recruitment, data collection, data analysis, data interpretation, literature search, and writing of the manuscript. wmy, wpq, and dw contributed to patient recruitment, data collection, data analysis, and writing of the manuscript. lz, wnl, xjw, and km had roles in patient recruitment, data collection, and data interpretation. mn, dx (dong xu), hww, gc, hjy, hfd, myx, mfh, and tc had roles in the patient management, data collection, and clinical management. xpl contributed to data analysis, data interpretation, and writing of the manuscript. all authors had access to the study data and reviewed and approved the final manuscript.funding this work was funded by grants from the tongji hospital for pilot scheme project and partly supported by the chinese national thirteenth five years project in science and technology (2017zx10202201), national commission of health, and by chinese national major science and technology (2018zx09733001-002-006). open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-279667-ikfduu2k authors: ronnje, louise; länsberg, john-kalle; vikhareva, olga; hansson, stefan r.; herbst, andreas; zaigham, mehreen title: complicated covid-19 in pregnancy: a case report with severe liver and coagulation dysfunction promptly improved by delivery date: 2020-09-04 journal: bmc pregnancy childbirth doi: 10.1186/s12884-020-03172-8 sha: doc_id: 279667 cord_uid: ikfduu2k background: it has been proposed that pregnant women and their fetuses may be particularly at risk for poor outcomes due to the coronavirus (covid-19) pandemic. from the few case series that are available in the literature, women with high risk pregnancies have been associated with higher morbidity. it has been suggested that pregnancy induced immune responses and cardio-vascular changes can exaggerate the course of the covid-19 infection. case presentation: a 26-year old somalian woman (g2p1) presented with a nine-day history of shortness of breath, dry cough, myalgia, nausea, abdominal pain and fever. a nasopharyngeal swab returned positive for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. her condition rapidly worsened leading to severe liver and coagulation impairment. an emergency caesarean section was performed at gestational week 32 + 6 after which the patient made a rapid recovery. severe covid-19 promptly improved by the termination of the pregnancy or atypical hellp (hemolysis, elevated liver enzymes and low platelet count) exacerbated by concomitant covid-19 infection could not be ruled out. there was no evidence of vertical transmission. conclusions: this case adds to the growing body of evidence which raises concerns about the possible negative maternal outcomes of covid-19 infection during pregnancy and advocates for pregnant women to be recognized as a vulnerable group during the current pandemic. the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has exposed vulnerable populations to an unprecedented global health crisis. from the knowledge gained from previous human coronavirus outbreaks, it has been proposed that pregnant women and their fetuses are particularly at risk for poor outcomes [1] . the maternal and neonatal outcomes of pregnant women with covid-19 is limited to a handful of case reports which present diverse results. obstetricians are still learning about covid-19 presentation and progression in pregnancy and even though the majority of pregnancies infected by covid-19 have good outcomes, a recent systematic analysis [1] showed that up to 3% of pregnancies were associated with severe maternal morbidity. it was indicated that mothers with a complicated medical history could be at increased risk for severe outcomes. furthermore, experts are of the opinion that the clinical recommendations for managing covid-19 in pregnancy should be based on lessons learned from the current epidemic [2] which emphasizes the importance of presenting covid-19 cases associated with complex clinical management. we therefore present a case report of a young woman, pregnant in the third trimester, diagnosed with covid-19, showing severe liver and coagulation impairment. a 26-year-old somalian woman (gravida 2, para 1) who had been living in sweden for a year presented at the emergency department of skåne university hospital in malmö on april 17th, 2020 pregnant at 32 + 1 weeks of gestation. she was transferred to the infectious diseases department with suspicion of covid-19. a diagnostic test, based on quantitative real time polymerase chain reaction (qrt-pcr), from a nasopharyngeal swab, was positive for sars-cov-2. she had recently moved from stockholm to malmö. in 2015 she had a normal vaginal delivery in somalia. the patient had an appendectomy and a cholecystectomy in somalia. her medical history also included hypothyroidism, currently treated with 150 ug levothyroxine daily. the body mass index (bmi) on admission to prenatal care was 47 kg/ m 2 with length 163 centimetres (cm), weight 126 kg (kg). apart from the obesity, her pregnancy had been without complications. she had received an intramuscular injection of anti-d immunoglobulin at 28 + 5 weeks of gestation since she was rhesus d (rhd) negative and the fetus was rhd positive. on admission, the patient described a nine-day history of shortness of breath, dry cough, myalgia, nausea, abdominal pain and fever (fig. 1) . she had significant abdominal pain on admission but the surgeon did not find any signs of an acute abdominal event. the patient had also noticed reduced fetal movements for the last two days. obstetric examination including cardio-tocography (ctg) and an abdominal ultrasound showed no abnormalities. the patient's respiratory rate was 22 breaths/minute, oxygen saturation 95%, blood pressure 116/71, pulse 113 beats/minute and temperature 37.2 degrees celsius ( o c). the laboratory tests are shown in table 1 . the patient was given morphine, paracetamol and oxycodone for pain relief and also received thromboprophylaxis, dalteparin 7500 units /day subcutaneously. no additional oxygen was needed. on day 2 (18/4/2020), the patient was relatively stable apart from two short episodes of fever up to 38.9 o c. due to risk for preterm labour, the patient received 12 milligrams (mg) of betamethasone intramuscularly to aid fetal lung maturity. daily fetal monitoring using ctg showed no signs of fetal distress. the patient was discharged on day 3 (19/4/2020) with a planned obstetric follow-up including fetal growth assessment after recovery. she was prescribed dalteparin for four weeks. the patient returned to the emergency department the next day (20/4/2020) with a sore throat and severe difficulties in swallowing. apart from tachypnoea (25-35 breaths/minute) and tachycardia (118 beats/minute), other vital signs were normal. after examination, she was discharged with a prescription of betamethasone tablets for three days (6, 4 and 3 mg) for swallowing difficulties and potassium supplements for the hypokalaemia noted in the blood tests ( table 1) . the patient was readmitted to the infectious diseases department the next day (21/4/2020) (fig. 1) . her covid-19 symptoms (cough, myalgia, abdominal pain and fever) had worsened and she now presented with dyspnoea. at readmission, the patient's respiratory rate 42 breaths/minute, blood pressure 114/61, pulse was 120 beats/minute and temperature 38.9 o c. during episodes of coughing, her oxygen saturation fell to 86%, but with 5 l of oxygen on mask the saturation rose to 99%. laboratory tests are shown in table 1 . her condition deteriorated on day 2 (22/4/2020) of the readmission. in addition to the generalized pain and tenderness, the pain in her right upper abdomen had worsened. blood tests showed elevation of aspartate aminotransferase (asat), interleukin-6 (il-6) and ferritin concentrations. there was impaired coagulation as shown by a prolonged activated partial thromboplastin time (aptt), high d-dimer, falling platelet count and decreased level of anti-thrombin iii (table 1 ). hemolysis was indicated by a fall in the hemoglobin concentration and rising lactate dehydrogenase levels although haptoglobin concentrations only were slightly elevated (table 1) . despite her worsening condition, the patient felt active fetal movements and normal intermittent ctg controls were registered. intravenous antibiotic treatment with cefotaxime (2 g, 3 times daily) was initiated due to suspicion of concomitant bacterial infection (table 1 ). blood and urine cultures were taken but since the general condition of the patient had worsened, a decision was made to deliver by caesarean section (32 + 6 gestational weeks), on maternal indication. the operation was performed in spinal analgesia in an operating theatre with negative air ventilation. the local hospital guidelines were followed to prevent the spread of covid-19 [3] . an uncomplicated operation was completed within 40 min and the total blood loss was 200 millilitres (ml). after two hours in the post-operative unit, the patient returned to the ward and received thromboprophylaxis, dalteparin at a total dose of 10.000 units divided in two doses. a computed tomography (ct) lung scan, performed later the same day, showed bilateral diffuse, ground-glass opacities with both peripheral and perihilar distribution, but no signs of pulmonary embolism (fig. 2) . due to deranged liver values, the patient was unable to receive paracetamol and due to the covid-19 infection not able to receive ibuprofen [4] . the pain relief was managed by administering 2.5 ml of intravenous morphine as needed. however, the patient's condition worsened during the night and on examination, the patient was somnolent and lethargic but answered adequately when woken up (reaction level scale 2). the patient's pain was mostly localized to the upper right quadrant of the abdomen and epigastrium. her uterus was well contracted and there were no signs of postoperative complications such as bleedings and local infection. on examination, the patient had miotic pupils that reacted poorly to light stimulation. even though the patient only was given 7.5 mg of morphine over the course of 8 h, a morphine over-dose was suspected, and intravenous morphine was replaced by a combination of orally administered naloxone and oxycodone. post-operative mobilization was initiated one day after surgery where after the patient made a steady recovery. table 1 illustrates the drastic improvement in the patient's blood tests on day 3 (23/4/2020) of the readmission. the patient was discharged in good health on the 30th of april, 2020 with thromboprophylaxis planned for 6 weeks postpartum and a follow-up visit to the obstetrics clinic. a male baby was delivered with birth weight 2085 g (37th percentile), birth length 48 cm (99th percentile) and head circumference 33.5 cm (99th percentile). the cord was clamped immediately after birth and the baby was shown briefly to the mother before being taken to a neonatal resuscitation station. at 1-minute after delivery, the baby had a normal heart rate but was gasping and had absent tone and no spontaneous movements. positive pressure ventilation by a t-piece (neopuff) was given with peak inspiratory pressure (pip) 20 cmh 2 o and positive end expiratory pressure (peep) between 5 and 7 cmh 2 o intermittently during the first seven minutes of life. at ten minutes, the baby was spontaneously breathing, albeit grunting, through t-piece continuous positive airway pressure (cpap) with preductal oxygen saturation between 90-95% at fio 2 (fraction of inspired oxygen) 0.4. fine crackles could be heard on lung auscultation. intermittent intercostal retractions were also seen. skin colour, tone and reflex irritability improved gradually during the stabilisation process. apgar score; 1 min: 4 (appearance 1, pulse 2, reflex irritability 0, activity 1, respiration 0), 5 min: 6 (appearance 1, pulse 2, reflex irritability 1, activity 1, respiration 1), 10 min: 8 (appearance 2, pulse 2, reflex irritability 1, activity 1, respiration 2). vitamin k was given intramuscularly and a nasogastric tube was inserted. nasal cpap with peep 6 cmh 2 o was started. arterial umbilical cord blood gas showed mild combined respiratory and metabolic acidosis with ph 7.28, partial pressure of oxygen (po 2 ) 3.45 (kilopascal) kpa, partial pressure of carbon dioxide (pco 2 ) 6.97 kpa and base excess − 6.8. the baby was put in an incubator and transferred to the neonatal intensive care unit (nicu) and placed in an airborne infection isolation room (aiir) with negative pressure ventilation. it was given preterm formula supplemented with intravenous glucose infusion. venous blood gas was analysed at 4 h of age: ph 7.27, po2 5 kpa, pco2 7.3 kpa, be -1.8, hb 184 g/l and lactate 2.7 mmol/l. after parental consent, formula was changed to donated breast milk. the mother was supplied with a breast pump and instructed in its use. after the initial need of breathing support and supplemental oxygen during day one of life the baby has enjoyed an uneventful clinical course. nasopharyngeal swabs for sars-cov-2 detection were collected at 48 and 96 h of life and were found to be negative in both instances. we report a case of severe covid-19 during in third trimester pregnancy, which led to an emergency caesarean section and preterm delivery at 32 + 6 weeks of gestational age. this case adds to the growing body of evidence which raises concerns about the possible negative maternal outcomes of covid-19 infection during pregnancy [1, [5] [6] [7] [8] . whilst most mothers with covid-19 infection have mild symptoms which resolve without treatment, a number of cases have now emerged in the literature, where mothers have required intensive care admission, and one case requiring invasive ventilation with extracorporeal membrane oxygenation [8] . it has been suggested that pregnancy induced immune responses and cardio-vascular changes can exaggerate the course of the covid-19 infection [1] . mothers with prior medical conditions may be at higher risk for poor outcomes. in this case, there were several risk factors for preeclampsia such as somalian origin and high bmi [9] . our patient was severely obese (bmi 47 kg/m 2 ) and her condition deteriorated drastically 12 days after her initial symptoms. atypical hellp (haemolysis, elevated liver enzymes, and low platelet count) syndrome or mississippi class 3 [10] can present itself with platelets between 100 and 150 × 10 9 /l, aspartate aminotransferase (asat)/ alanine aminotransferase (alt) ≥ 0.68 µkat/l and lactate dehydrogenase (ldh) > 10.2 µkat/l but it is seldom associated with coagulation impairment [10] . in addition, the patient had remarkably high levels of asat (28 µkat/l), as compared to the other liver tests, which is not consistent with the generalized liver dysfunction seen in hellp or acute fatty liver of pregnancy [11] . liver injury has been reported by a number of studies in patients with severe covid-19 [12] [13] [14] [15] making this organ the most commonly affected besides the respiratory system. transient elevation of serum aminotransferases is often seen and a number of factors have been implicated for acute liver damage in severe covid-19, including severe hypoxemia due to acute respiratory failure, drug interactions, septic shock and multiorgan dysfunction [12] . on readmission, the plasma paracetamol concentration was well within the therapeutic reference interval (table 1 ) and liver injury secondary to a paracetamol overdose was therefore not suspected. although there is insufficient evidence for direct sars-cov-2 virus-related hepatocyte injury, liver dysfunction has been continuingly related to severe covid-19 infection [15] and intensive care admission [16, 17] . the patient also presented with elevated concentrations of several active-phase proteins (apps) including il-6, procalcitonin and ferritin, which may have indicated concomitant bacterial infection or severe systemic inflammation due to covid-19. subsequent blood and urine cultures were negative but the patient still received prophylactic treatment with broad spectrum antibiotics intravenously from day 1-5 of the readmission. the hyperactive immune responses characteristic of severe covid-19 can lead to stress-induced tissue injury and multiorgan impairment [18] . ruan et al. [19] found that elevated levels of il-6 were associated with a significantly increased risk of mortality in covid-19 patients but in this case the inflammatory markers (and liver enzyme tests) decreased significantly after the caesarean section. we can only speculate whether this indicated that severe covid-19 in pregnancy may improve promptly by the termination of the pregnancy or that the patient's condition was a combination of atypical hellp and covid-19 which subsequently improved after the caesarean section. considering the latter option, the patient showed some typical characteristics of hellp syndrome; including right upper abdominal pain, epigastric pain, nausea and vomiting but a normal blood pressure [20] . furthermore, she had no past obstetric history of preeclampsia or hellp. her platelet count was only marginally decreased and although subtle signs of haemolysis were present (decreased haemoglobin concentration and elevated ldh), haptoglobin concentration was not decreased [10] ( table 1 ). given that pregnancy itself is a hypercoagulable state, it has been suggested that covid-19 infection during pregnancy is associated with high risk of maternal thrombotic complications [15] [16] [17] . high levels of d-dimer in combination with elevated liver enzymes supports the likelihood that the patient's liver injury and coagulation dysfunction were secondary to the severe covid-19 infection. considering the neonate, we found no evidence for any vertical transmission of covid-19 between mother and the baby. however, there have been reported cases of early covid-19 detection in newborns, implying the potential risk of vertical transmission [21] , although in the vast majority of cases, no such evidence has been identified [22, 23] . since only a handful of cases have been reported in the literature, vertical transmission cannot be ruled out until systematic studies have been undertaken. our report has some limitations. we were unable to report a complete panel of coagulation tests due to errors in lab analysis. similarly, the presence of antiphospholipid antibodies to rule out antiphospholipid syndrome were not investigated. we did not evaluate the presence of sars-cov-2 in amniotic fluid, cord blood, or placental tissue which could further clarify the possibility of vertical transmission. in summary, we describe a severe case of maternal covid-19 during the third trimester of pregnancy which led to liver and coagulation impairment and preterm delivery. we believe these findings have important public implications both due to the severity of the disease progression but also due to the rapid nature of the improvement after delivery. atypical presentation of hellp could not be ruled out and the importance of a multidisciplinary team in the treatment and management of severe covid-19 during pregnancy is critical for positive patient outcome. pregnant women should be considered a vulnerable group in the population in which exposure to covid-19 should be avoided at all costs. maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? coronavirus (covid-19) infection in pregnancy. information for healthcare professionals version 7: published thursday 9 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? potentialmaternal and infant outcomesfrom (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records covid-19 in pregnancy: early lessons clinical manifestations and outcome of sars-cov-2 infection during pregnancy maternal deaths with covid19: a different outcome from mid to low resource countries? the spectrum of severe preeclampsia: comparative analysis by hellp (hemolysis, elevated liver enzyme levels, and low platelet count) syndrome classification acute fatty liver of pregnancy: pathophysiology, anesthetic implications, and obstetrical management characteristics and mechanism of liver injury in 2019 coronavirus disease coronavirus disease 2019 (covid-19): a perspective from china implications of covid-19 for patients with pre-existing digestive diseases clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china on the alert for cytokine storm: immunopathology in covid-19. arthritis rheumatol. 2020. accepted author manuscript clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intens care med revisiting hellp syndrome novel coronavirus infection in newborn babies under 28 days in china clinical characteristics and risk assessment of newborns born to mothers with covid-19 clinical characteristics of 19 neonates born to mothers with covid-19. front med publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors want to thank daisey lee for her contribution in editing fig. 2 authors' contributions lr: planning, data collection, analyzing and writing the manuscript. jkl: planning, data collection, writing the manuscript. ov: conception, planning, data collection and revising the manuscript. srh: conception, planning, data collection and revising the manuscript. ah: conception, planning, data collection and revising the manuscript. mz: conception, planning, data collection, writing and revising the manuscript. all authors have read and approved the manuscript. ethics approval and consent to participate ethical approval was not sought due to the nature of the case report. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. the patient provided written informed consent for the publication of potentially identifying images and clinical details on behalf of themselves and their child. the datasets used and analysed during the current study available from the corresponding author on reasonable request. a.h. is a board member and shareholder in amniotics ab, a start-up company developing treatment for severe lung disease in covid-19. s.r.h. holds patents for the diagnosis and treatment of preeclampsia. s.r.h. is also a cofounder of the company guard therapeutics international formerly named a1m pharma ab (https://guardtherapeutics.com). key: cord-348024-n8wn4och authors: lei, fang; liu, ye‐mao; zhou, feng; qin, juan‐juan; zhang, peng; zhu, lihua; zhang, xiao‐jing; cai, jingjing; lin, lijin; ouyang, shan; wang, xiaoming; yang, chengzhang; cheng, xu; liu, weifang; li, haomiao; xie, jing; wu, bin; luo, huiming; xiao, fei; chen, jing; tao, liang; cheng, gang; she, zhi‐gang; zhou, jianghua; wang, haitao; lin, jun; luo, pengcheng; fu, shouzhi; zhou, jihui; ye, ping; xiao, bing; mao, weiming; liu, liming; yan, youqin; liu, ling; chen, guohua; li, hongliang; huang, xiaodong; zhang, bing‐hong; yuan, yufeng title: longitudinal association between markers of liver injury and mortality in covid‐19 in china date: 2020-05-02 journal: hepatology doi: 10.1002/hep.31301 sha: doc_id: 348024 cord_uid: n8wn4och coronavirus disease 2019 (covid‐19) is a new infectious disease. to reveal the hepatic injury related to this disease and its clinical significance, we conducted a multicenter retrospective cohort study that included 5,771 adult patients with covid‐19 pneumonia in hubei province. we reported the distributional and temporal patterns of liver injury indicators in these patients and determined their associated factors and death risk. longitudinal liver function tests were retrospectively analyzed and correlated with the risk factors and death. liver injury dynamic patterns differed in alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and total bilirubin (tbil). ast elevated first, followed by alt, in severe patients. alp modestly increased during hospitalization and largely remained in the normal range. the fluctuation in tbil levels was mild in the non‐severe and the severe group. ast abnormality was associated with the highest mortality risk compared to other indicators of liver injury during hospitalization. common factors associated with elevated liver injury indicators were lymphocyte count decrease, neutrophil count increase, and male gender. conclusion: the dynamic patterns of liver injury indicators and their potential risk factors may provide an important explanation for the covid‐19‐associated liver injury. because elevated liver injury indicators, particularly ast, are strongly associated with the mortality risk, our study indicates that these parameters should be monitored during hospitalization. this article is protected by copyright. all rights reserved coronavirus disease 2019 (covid-19) is a viral respiratory illness caused by a novel coronavirus (ncov). covid-19 is highly contagious, with more than 2.3 million cases and nearly 163 thousand deaths worldwide on april 21, 2020. (1, 2) the targeting organs of this coronavirus are believed to be the lungs and airways. however, patients often have evidence of damage to other organs, which significantly increases their mortality. (3, 4) the pathophysiological foundation of multiorgan damage may be associated with organ-specific immune response to disseminated coronavirus or secondary to hypoxemia, systemic cytokine storm, and medication. (5) in an attempt to determine the distributional and temporal patterns of liver injury in patients with covid-19 with a focus on clinical significance and determinants of this change, we conducted a multicenter retrospective cohort study that included 5,771 patients in hubei province. in this multicenter retrospective cohort study, patients with covid-19 were admitted to the following in the study. exclusion criteria, which applied to 1,258 patients, were as follows: age younger than 18 or older than 75 years; pregnancy; severe medical conditions, including liver cirrhosis (10 cases), chronic renal dysfunction (above chronic kidney disease stage 3 [ckd 3]), leukemia, cancer, acquired immune deficiency syndrome, acute myocardial infarction during hospitalization, acute pulmonary embolism (due to a long-term pulmonary embolism disease history, long-term bed rest, and coagulopathy) stoke, and acute pancreatitis; or transfer to other hospitals (fig. 1 ). chest computed tomography (ct) or throat-swab specimens were obtained from all patients upon admission. covid-19 was diagnosed by clinical manifestations, chest ct, or real-time polymerase chain reaction (rt-pcr) according to world health organization (who) interim guidance and the new coronavirus pneumonia prevention and control program (5th edition) published by the national this article is protected by copyright. all rights reserved health commission of china. at the time of admission, patients with fever or suspected respiratory infection, plus one of the following clinical manifestations including respiratory rate greater than 30 breaths/minute, severe respiratory distress, or oxygen saturation (spo 2 ) less than 90% on room air, were classified as severe cases. (6) staging of covid-19 and evaluation of organ damages were conducted by a team of physicians. this study was approved by the central institutional ethics committee and ratified by each hospital. a waiver of the requirement for documentation of informed consent was granted for analyzing existing data without interfering with patient treatment. the demographic, clinical characteristics, medical history, laboratory tests, radiological reports, therapeutic intervention, and outcome data were obtained from patients' electronic medical records. clinical symptoms, including fever, cough, fatigue, dyspnea, and comorbidities, were extracted. the laboratory examination included a complete blood count, c-reactive protein (crp), procalcitonin, ddimer, and serum biochemical tests for liver, kidney, heart, and coagulation dysfunction. medical history comprised coexistence of chronic obstructive pulmonary disease (copd), type 2 diabetes mellitus (t2dm), hypertension, coronary heart disease, cerebrovascular disease, chronic liver disease, ckd, cancer, and autoimmune diseases. the unilateral and bilateral lesions in chest ct scan images were recorded and analyzed. personal identification information (e.g., name and id) of the study subjects was anonymized and replaced with a coding system before data extraction. data were reviewed and confirmed by experienced physicians and double-checked to ensure accuracy. acute lung injury (ali), acute respiratory distress syndrome (ards), and septic shock were defined according to the who interim guideline for "clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected." (4) acute kidney injury was defined as an elevation in serum creatinine level ≥26.5 μmol/l within 48 hours. (7) acute liver injury was defined as the levels of serum alanine aminotransferase (alt) above 3-fold of the upper limit of normal (uln). increase in liver enzyme levels was defined as the levels of serum liver enzyme above the uln. cardiac injury was defined as the serum level of cardiac troponin i/t or hypersensitive troponin i/t above the uln. (8) clinical disseminated intravascular coagulation (dic) was defined according to the international society on thrombosis and haemostasis (isth) criteria for diagnosing this article is protected by copyright. all rights reserved dic. (9) the primary endpoint was recorded and evaluated in this longitudinal cohort, which was 28day all-cause death. data were reviewed and confirmed by two certified physicians to ensure accuracy. categorical variables were presented as frequency. continuous variables were described as median (interquartile range [iqr]). means for continuous variables were compared using independent group t tests when the data were normally distributed; otherwise, the mann-whitney test was used. proportions for categorical variables were compared using the chi-square test. proportions for categorical variables were compared using the chi-square test or fisher exact test. distribution of highest liver enzyme levels (e.g., alt, ast, alp, and tbil) by the severity of covid-19 was presented using kernel density estimation. dynamic changes of liver enzyme levels by the severity of covid-19 were presented using locally weighted scatterplot smoothing (loess). site was modeled as a random effect in the mixed-effect cox model. the mixed-effect cox proportional hazards regression models were used to investigate the relationship of liver enzyme levels with all-cause mortality among patients. the mixed-effect cox models were adjusted for age, gender, and coexisting chronic diseases, including hypertension, coronary artery disease, cerebrovascular disease, t2dm, and ckd. ordinal logistic regression analysis was applied to evaluate the association of baseline characteristics and medications happened before peaking of liver enzymes with the peak levels of inhospital liver enzymes in the longitudinal cohort, where the liver function markers were trichotomized. the parallel lines assumption of the model was also tested and met. the p values were 2-sided, and an alpha level of 0.05 was used to define statistical significance. all analyses were conducted using r version 3.6.3 (r foundation for statistical computing, vienna, austria) or spss version 23.0 (ibm, armonk, ny, usa). the selected baseline liver characteristics of the enrolled patients are shown in table 1 . a total of 5,771 adult patients with covid-19 were included in the analysis. the median age was 56 (iqr, 43-accepted article (79.4%) were non-severe cases and 1,186 (20.6%) were classified as severe cases. a total of 81 (1.4%) patients reported chronic liver disease. among these patients, 4 recorded fatty liver diseases and 77 viral hepatitis (supporting table s3 ). the median day for acute liver injury (alt >3 uln) occurs at day 17 (iqr, 13-23) after symptom onset (fig. 1 ). to determine the distribution and trajectory of parameters indicating liver functions in patients enrolled in the study, multiple results from liver function tests were recorded during hospitalization. less disperse in non-severe cases. these levels increased and grew more disperse as the disease became more severe. non-severe and severe case distribution had widest separation by ast level (fig. 2b ). loess models illustrated trajectory of alt, ast, alp, and tbil in non-severe and severe patients during hospitalization (fig. 2c) . the models suggested a significant elevation of ast level upon admission and was maintained at higher levels in the severe group than the non-severe group. although there was a significant difference in alt between the non-severe group and the severe group, the magnitude of increase in alt was not as dramatic as ast upon admission. however, it rapidly increased, surpassed the uln value, and reached its peak within 10 to 15 days after admission in the severe group. alp level was higher in the severe group than the non-severe group. both groups rose gradually during hospitalization but largely stayed in the normal range. the fluctuation in tbil levels was mild in the non-severe and the severe group. the dynamic changes in indicators suggested potential mechanisms of covid-19-associated liver injury. impaired liver function is closely related to mortality in covid-19 patients. the associations of increased alt, ast, alp, and tbil levels with all-cause mortality were assessed using mixedeffect cox model. hospital sites were treated as a random effect. age, gender, and comorbidities were adjusted as confounders. hazard ratios for the associations between alt, ast, alp, and tbil and this article is protected by copyright. all rights reserved all-cause mortality are depicted in table 2 . the kaplan-meier survival curves with elevation in alt, ast, alp, and tbil levels for all-cause mortality are illustrated in fig. 3 . among these liver enzymes, elevated ast was associated with the highest mortality risk. compared to the patients with ast in the normal range, all-cause mortality risk significantly increased 4.81-fold (95% confidence interval [ci], 3.38-6.86; p < 0.001) in patients with ast between 40 and 120 u/l and increased 14.87-fold (95% ci, 9.64-22.93; p < 0.001) in patients with ast above 120 u/l after adjusting for age, gender, and comorbidities. moreover, elevation of other enzymes and tbil were also significantly associated with adverse outcomes of covid-19 (table 2 and supporting table s4 -s5). given discharge might be a competing risk for death, we further analyzed the association of liver function with 28-day mortality of covid-19 using cumulative risk analysis, to further avoid the potential over-estimation of risk in the hazard estimates. in this model, ast ranging from 40 to 120 u/l and above 120 u/l were associated with significantly increased the risk of all-cause mortality (adjusted hr, 6.00; 95%ci, 4.29-8.39; p < 0.001 for 40-120 u/l; adjusted hr, 17.05; 95%ci, 11.21-25.93; p < 0.001 for ast >120 u/l) (supporting table s6) the ordinal regression analysis revealed the effects of age, sex, and coexisting disease-adjusted baseline characteristics and hospital medication on peak alt, ast, alp, and tbil levels in covid-19 patients from the longitudinal cohort (tables 3) . male gender, systemic corticosteroids application, lymphocyte count decrease, neutrophil count increase, and fever were factors positively associated with elevated alt levels ( table 3) . use of antifungal drugs, lymphocyte count decrease, chronic liver disease, systemic corticosteroids use, and male gender were the leading factors positively associated with elevated ast levels. alp levels were tightly associated with antifungal drug use, neutrophil counts increase, chronic liver disease, and male gender. antifungal, antiviral, systemic corticosteroids use, and platelet count reduction were main factors positively correlated with increased tbil levels. neutrophil count increase, lymphocyte count decrease, and male gender were common factors positively associated with elevated alt, ast, alp, and tbil levels during hospitalization (table 3) . this article is protected by copyright. all rights reserved multiorgan injury, which significantly increases mortality, is often evident in patients with covid-19. (10) this study presents trajectories of liver enzyme levels during hospitalization and depicts their clinical significance in a multicenter retrospective cohort-derived data set of 5,771 individuals. liver injury was mild and transient in non-severe patients. the major finding is that elevation of ast level was more frequent and significant than the increase of alt in severe patients on hospital admission, and ast levels had the highest correlation with mortality compared to other indicators reflecting liver injury. this is contradictory to other hepatitis-induced liver injury. increase of alp was more significant toward the latter phase of the disease but mainly stayed within the normal range. elevated alt, ast, alp, and tbil levels were associated with increased risks of mortality and, among these liver enzymes, elevated ast was associated with the highest mortality risk. common factors associated with elevated liver injury indicators were lymphocyte count decrease, neutrophil count increase, dyspnea, and male gender. the liver biopsy specimens of patients with covid-19 showed moderate microvascular steatosis and mild lobular and portal lesion. (5, 11) liver damage may be associated with organ-specific immune response to coronavirus or secondary to hypoxemia, systemic inflammation response, and medication. a previous study showed the angiotensin-converting enzyme 2 (ace2) receptor expression is very low in hepatocytes and only expressed in cholangiocytes in the liver, (12) suggesting that cholangiocytes might be a direct target for severe acute respiratory syndrome (sars)-coronavirus 2 (cov-2) invading the liver. however, in our study, alp, an index of cholangiocytes injury, mainly stayed within the normal range during hospitalization. elevation in alt and ast, the indicators of hepatocyte injury, was more common and severe in patients with covid-19. pathological analysis of liver tissue from a patient who died from covid-19 failed to demonstrate cholangiocyte damage and viral infiltration in the liver. the elevations of lactate dehydrogenase (ldh) and gamma-glutamyl transpeptidase (ggt), which also reflect bile duct injury, were not significant. (5) thus, the hypothesis of cholangiocytes mediating viral-associated injury needs further investigation. in this study, we observed ast elevation upon admissions, followed by an increase in alt. previous studies also reported that ast increase is more frequent than alt in severe patients upon admission. (3, 13) this article is protected by copyright. all rights reserved in which alt elevation is the primary manifestation of liver injury. (14) (15) (16) ordinal regression analysis also showed that ast elevation is positively correlated with the increase of neutrophil counts and the decrease of lymphocyte counts at baseline-pathological alterations that are proven indicators of disease severity. (17, 18) early elevation in ast and its association with indicators for disease severity suggest immune-mediated inflammation may play a critical role in liver impairment in severe patients with covid-19. mechanisms that mediate the early ast elevation in severe patients warrant further study. the combination of antibiotics, antivirals, and systemic corticosteroids is widely utilized in covid-19 treatment. (13, 19) antifungal medication is more likely to be applied in weaker patients. in this study, use of antifungal drugs, antibiotics, antivirals, and systemic corticosteroids exhibited a positive correlation with elevated liver enzymes. although these data could not prove the causal effect of drugs on liver damage, drug hepatotoxicity during the treatment of coronavirus infection needs to be considered. due to this emergent situation, there is an insufficient report and diagnosis on chronic liver disease. only 81 patients recorded history of chronic liver disease (4 with nonalcoholic fatty liver disease [nafld] and 77 with viral hepatitis), which are much lower than the prevalence in china. (20, 21) we are unable to assess whether coexistence of chronic liver comorbidities increases susceptibility to liver injury in sars-cov-2 infection. previous studies have shown that sars-cov-2 may aggravate liver injury in patients with viral hepatitis. (22, 23) therefore, liver injury in patients with chronic liver disease needs to be investigated and monitored. limitations exist in our research. this study was retrospective, and some cases did not have sufficient history of illness. the multiple tests for liver function were carried out at different time intervals for each patient. bias may occur due to the increased number of tests in patients with liver dysfunction. observational studies demonstrate association, not causation. whether abnormal liver function is caused by sars-cov-2 or inflammation needs to be further investigated by direct clinical evidence. in conclusion, the dynamic patterns of liver injury indicators and their potential risk factors may provide an important explanation for the covid-19-associated liver injury. because the increase of liver injury indicators is strongly associated with the risk of mortality, these parameters, especially ast, may need to be monitored during hospitalization. this article is protected by copyright. all rights reserved a novel coronavirus from patients with pneumonia in china coronavirus disease (covid-2019) situation reports clinical characteristics of coronavirus disease 2019 in china association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 pathological findings of covid-19 associated with acute respiratory distress syndrome clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: world health organization (ncov)-infection-is-suspected kidney disease: improving global outcomes (kdigo) acute kidney injury work group. kdigo clinical practice guideline for acute kidney injury accf/aha guideline for the management of heart failure: a report of the american college of cardiology/american heart association task force on clinical practice guidelines and the heart failure society of america towards definition, clinical and accepted article this article is protected by copyright. all rights reserved laboratory criteria, and a scoring system for disseminated intravascular coagulation clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china changes of serum hbv-dna in relation to serum transaminase level during acute exacerbation in patients with chronic type b hepatitis sars-associated viral hepatitis caused by a novel coronavirus: report of three cases sequential changes of serum aminotransferase levels in patients with severe acute respiratory syndrome clinical and immunologic features in severe and moderate coronavirus disease dysregulation of immune response in patients with covid-19 in wuhan, china intensive care during the coronavirus epidemic epidemiological feature of nafld from 1999 to 2018 in china accepted article this article is protected by copyright. all rights reserved unexpected rapid increase in the burden of nafld in china from liver injury during highly pathogenic human coronavirus infections clinical best practice advice for hepatology and liver transplant providers during the covid-19 pandemic: aasld expert panel consensus statement author names in bold designate shared co-first authorship a/g ratio, median (iqr) abbreviations: a/g, albumin to globulin key: cord-355395-rckzi8vz authors: tian, dandan; ye, qing title: hepatic complications of covid‐19 and its treatment date: 2020-05-21 journal: j med virol doi: 10.1002/jmv.26036 sha: doc_id: 355395 cord_uid: rckzi8vz covid‐19 is highly contagious and has a variety of clinical manifestations, it can affect a number of other organs in addition to the lungs, and liver injury may occur. sars‐cov‐2 can cause liver injury through systemic inflammatory response syndrome (sirs), cytokine storms, ischemia‐reperfusion injury, side effects of treatment drugs, and underlying liver disease and can attack liver cells directly via ace2. clinical studies have found that liver injury in covid‐19 patients mainly manifests as abnormal liver biochemical indicators, but there have been no reports of liver failure caused by this disease. the number of covid‐19 patients with liver injury is increasing, and the incidence of liver injury in covid‐19 patients with severe disease are higher than in patients with mild disease. liver injury may be a risk factor for progresses and worsens in patients with covid‐19, and it is necessary to pay attention to the occurrence of liver injury in the diagnosis and treatment of covid‐19. this article is protected by copyright. all rights reserved. coronavirus disease 2019 (covid19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 1-3 . because it is highly contagious, people are generally susceptible, and it is mainly transmitted via respiratory droplets, covid-19 is spreading all over the world. it has been reported in more than 200 countries, and the number of deaths worldwide is also increasing. sars-cov-2 infection mainly affects the respiratory tract, and the main clinical manifestations are fever, dry cough, rhinorrhea and fatigue. many cases of covid-19 are acute and resolve quickly, but the disease can also be fatal, with a mortality rate of approximately 3% 4 . the fatality rate of critically ill patients admitted to the intensive care unit (icu) is relatively high, the results from a study of 52 critically ill patients with covid-19 who were admitted to the intensive care unit (icu) of wuhan jin yin-tan hospital (wuhan, china) between late december, 2019, and jan 26, 2020 showed that 32 (61.5%) patients had died within 28 days, and the median duration from icu admission to death was 7 (iqr 3-11) days in the non-survivors 5 . many clinical studies have also suggested that patients with covid-19 have multiple organ damage, including the circulatory system, urinary system, nervous system, and digestive system, among others. the presence and number of these complications are high-risk factors that aggravate a patient's condition and correlate with a poor prognosis. the whole genome sequencing results showed that sars-cov-2 shares 82% genome sequence similarity to sars-cov, and 50% genome sequence homology to middle east respiratory syndrome coronavirus (mers-cov) 22 sars-cov, middle east respiratory syndrome coronavirus (mers-cov) and sars-cov-2 are all coronaviruses and known to cause severe respiratory symptoms 11 . as many as 60% of patients infected with sars-cov have liver damage 23 , and some patients infected with mers-cov also have liver damage 24 19 . the above indicate that the incidence of liver injury in severe or critically ill cases is significantly higher than that in mild cases of covid-19, and the probability of patients with liver function impairment requiring icu treatment is increased. 19 . another single-center study of 138 patients with covid-19 showed that the levels of alt and ast in patients who required icu treatment were significantly higher than those in patients who did not require icu treatment 10 . moreover, in a large cohort of 1099 patients from 552 hospitals in 31 provinces or provincial municipalities, the total incidence of elevated total bilirubin was approximately 10%, of which the incidence in severe and nonsevere patients was approximately 13.3% and 9.9%, respectively; the incidence of increased total bilirubin in patients who needed to be moved to the icu, needed mechanical ventilation, or died was higher than in other patients (20.8% vs 9.8%) 5 . the above studies suggest that liver biochemical indicators may be used as predictors of severity and prognosis of covid-19 patients. thus, clinicians should pay more attention to changes in liver biochemical indicators and also identify patients with liver damage in a timely manner. the results from biopsies in a death covid-19 patient showed moderate microvascular steatosis and mild portal and lobular activity in liver tissue 25 . in another four autopsy in a death covid-19 patient showed mild zone 3 sinusoidal dilatation, patchy hepatic necrosis, mild increase in sinusoidal lymphocytes are the main pathologic changes of liver in case 3 and case 4, and rt-pcr showed direct evidence of the sars-cov-2 rna sequence in the liver tissues in case 1 26 . through learning of tian et al report 26 , li et al observed foci of hepatic necrosis in adjacent to terminal hepatic veins and peri-portal area, and found no significant surrounding inflammatory cellular infiltration, which consistent with the pattern of acute liver injury 27 . the above studies may indicate that hepatic injuries may be related to direct viral attack. it has been confirmed that sars-cov-2 enters host cells through binding of its s protein to angiotensin-converting enzyme 2 (ace2) on the surface of the host cell. ace2 is the most important cell receptor that mediates the entry of sars-cov-2 into target cells [28] [29] . however, based on single-cell sequencing and animal model analysis of liver tissue, chaix's team found that the expression level of ace2 in liver tissue was only approximately 0.31% and that specific expression of ace2 in bile duct epithelial cells was 20 this article is protected by copyright. all rights reserved. times higher than that in hepatocyte 30 . considering limited number of autopsy cases in patients with covid-19 studied and the relatively low expression of ace2 in liver, liver damage directly caused by sars-cov-2 infection of hepatocytes deserves further investigation. moreover, biomarkers for cholangiocyte injury, such as gamma-glutamyl transferase (ggt) and alkaline phosphatase (alp) have also been seen in some patients 31 , and consistent with injury to biliary epithelial cells, and about 10% of covid-19 patients have elevated total bilirubin level.these might suggest that sars-cov-2 might directly bind to cholangiocytes expressing ace2 result in cholangiocyte injury. huang c 18 and colleagues found that liver injury in covid-19 patients differed from that in sars patients. liver injury as the first manifestation in covid-19 patients was very rare, with most being secondary liver injury. it was speculated that in addition to the virus itself causing liver injury, immune injury, systemic inflammatory response syndrome (sirs), cytokine storms, ischemia and hypoxia reperfusion injury, and drug-induced injury may be the main mechanisms that cause secondary liver injury in patients with covid-19 [11] [12] 14, 27 . in addition to receptor-mediated viral infection, antibody-dependent enhancement of infection (ade) may occur in patients with sars 32 . ade refers to the interaction of a virus-specific antibody with fc receptor (fcr) and/or complement receptor (cr) to enhance the ability of the virus to enter granulocytes, monocytes, and macrophages. the virus constantly replicates in the above cells, resulting in increased virus production and aggravating infection. previous studies have reported that antibodies against the sars-cov spike protein trigger ade, causing sars-cov to enter immune cells that do not express ace2 and immune damage 33 . the liver contains a large number of cells related to the immune response. whether ade can also mediate sars-cov-2 to this article is protected by copyright. all rights reserved. infect immune cells by a non-ace2-dependent pathway and participate in liver injury caused by sars-cov-2 is a concern. systemic inflammatory response syndrome (sirs) and cytokine storms( as the liver, myocardium and kidney. these results suggest that the sirs and cytokine storms caused by sars-cov-2 infection may be one of the important mechanisms of liver injury. patients with covid-19 have varying degrees of hypoxemia, with more than 40% requiring oxygen therapy 5 drug hepatotoxicity( figure 2) in china, the incidence of drug-induced liver injury is second only to viral hepatitis and fatty liver disease (including alcoholic and non-alcoholic). drugs commonly causing liver injury include traditional chinese patent medicines containing components of saikosaponins [38] [39] , antitumor drugs, antituberculosis drugs, antimalarial drugs and antibiotics [40] [41] . most patients with covid-19 have fever, and many of them use antipyretic and analgesic drugs, which contain this article is protected by copyright. all rights reserved. acetaminophen and are known to cause liver damage, overdose of these drugs can cause liver damage. currently, there is no clearly effective antiviral drug, and many patients in the outbreak were given lopinavir, abidor, ritonavir and other antiviral drugs. the latest study published in jci 42 reported that cyp3a4 plays an important role in ritonavir's mediating of hepatotoxicity, and the cyp3a metabolic pathways can produce electrophilic content, oxygen free radical, etc, they can be covalent binding with macromolecular substances within the liver cells, cause system membrane lipid peroxidation, destruction of membrane integrity and membrane ca 2 + -atpase, disrupt cell internal and external ca 2 + homeostasis, influence function of critical organelles such as mitochondria, endoplasmic reticulum, and eventually lead to liver cell damage and even death. moreover, the combination of overdose of lopinavir and ritonavir can activate the endoplasmic reticulum stress pathway in the liver, inhibit the proliferation of hepatocytes, induce apoptosis of hepatocytes through the caspase cascade system, induce inflammatory reactions and accelerate liver injury by aggravating oxidative stress [43] [44] . some scholars believe that hiv protease inhibitors can effectively suppress sars-cov-2 replication, nevertheless, shen's team confirmed that the risk of liver damage was increased in patients taking both hormones and hiv protease inhibitors 45 . the incidence of liver injury caused by different drugs is varies, but the incidence increases with the increase in drug types. the diagnosis of drug-induced liver injury requires the combination of medical history and relevant tests to exclude other liver diseases and assess the association between liver injury and suspected drugs by causality. chronic liver disease is a major disease burden globally. liver diseases, including chronic viral hepatitis, alcohol-related liver disease, non-alcoholic fatty liver disease, and autoimmune hepatitis, affect approximately 300 million people in china. a retrospective analysis of 333 covid-19 inpatients 19 showed that 12 accepted article had a history of hepatitis b and that 2 had a history of hepatitis c. in another study of baseline liver biochemical parameters in 324 cases in the shanghai area, the hbsag-positivity rate in covid-19 patients reached 6.5% 46 for covid-19 patients with mild liver biochemical abnormalities, the primary disease should be treated actively when antiviral and supportive treatments are given to inhibit viral replication, reduce inflammation and improve immunity, and preventive application of liver-protecting and enzyme-lowering drugs is not recommended. with acute liver injury, clinicians should analyze and judge the causes of liver injury and take appropriate measures, while closely monitoring alt, ast, total bilirubin, direct bilirubin, albumin, and pta (inr). the occurrence of acute liver failure should also be identified, and liver-protecting and enzyme-lowering drugs, for which the composition and mechanism of action are relatively clear, should be chosen. nonetheless, too many drug types should not be administered (generally no more than 2), and the dosage should not be too large. for patients with severe and critical covid-19 disease with liver injury, which should be considered to be caused by cytokine storms and microcirculation ischemia and hypoxia, respiratory and circulatory support should be strengthened. if necessary, extracorporeal membrane oxygenation should be performed to improve the blood oxygen saturation of patients. patients with acute liver failure should be given intensive monitoring and symptomatic and supportive treatment, and hypoproteinemia should be corrected. in cases of liver injury caused by drugs, in addition to conventional anti-inflammatory liver protection treatment, stopping or reducing the amount of the suspected drugs should be considered, and the degree of liver damage should be assessed, followed by adjustment of the treatment plan. anti-hbv or anti-hcv treatment should not be discontinued, but large doses of hormones should not be used simultaneously. overall, the prevention and treatment experience of liver injury in sars patients can provide some reference for covid-19 patients with liver injury. for example, active prevention and control of the inflammatory response in the early stage of the disease is not only conducive to reducing the nonspecific inflammation of the liver this article is protected by copyright. all rights reserved. but also to preventing the occurrence of systemic inflammatory response syndrome to reduce the probability of mild disease developing into severe or critical disease. covid-19 combined with liver injury is very common, and the incidence of liver injury in patients with severe or critical covid-19 disease is higher than that in patients with mild disease. liver injury in patients with covid-19 may be caused by a variety of mechanisms, such as direct virus infection, immune injury, drug-induced liver injury, systemic inflammatory response, ischemia and hypoxia, and recurrence or exacerbation of the underlying liver disease, among others. since the liver is an important processing organ of the body, impaired liver function seriously affects the body's anabolism and prognosis of the disease. 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covd-19 with liver injury key: cord-292501-2jv7xkfn authors: jiang, saiping; wang, rongrong; li, lu; hong, dongsheng; ru, renping; rao, yuefeng; miao, jing; chen, na; wu, xiuhua; ye, ziqi; hu, yunzhen; xie, minghua; zuo, minjuan; lu, xiaoyang; qiu, yunqing; liang, tingbo title: liver injury in critically ill and non-critically ill covid-19 patients: a multicenter, retrospective, observational study date: 2020-06-23 journal: front med (lausanne) doi: 10.3389/fmed.2020.00347 sha: doc_id: 292501 cord_uid: 2jv7xkfn background: liver injury commonly occurs in patients with covid-19. there is limited data describing the course of liver injury occurrence in patients with different disease severity, and the causes and risk factors are unknown. we aim to investigate the incidence, characteristics, risk factors, and clinical outcomes of liver injury in patients with covid-19. methods: this retrospective observational study was conducted in three hospitals (zhejiang, china). from january 19, 2020 to february 20, 2020, patients confirmed with covid-19 (≥18 years) and without liver injury were enrolled and divided into non-critically ill and critically ill groups. the incidence and characteristics of liver injury were compared between the two groups. demographics, clinical characteristics, treatments, and treatment outcomes between patients with or without liver injury were compared within each group. the multivariable logistic regression model was used to explore the risk factors for liver injury. results: the mean age of 131 enrolled patients was 51.2 years (standard deviation [sd]: 16.1 years), and 70 (53.4%) patients were male. a total of 76 patients developed liver injury (mild, 40.5%; moderate, 15.3%; severe, 2.3%) with a median occurrence time of 10.0 days. critically ill patients had higher and earlier occurrence (81.5 vs. 51.9%, 12.0 vs. 5.0 days; p < 0.001), greater injury severity (p < 0.001), and slower recovery (50.0 vs. 61.1%) of liver function than non-critically ill patients. multivariable regression showed that the number of concomitant medications (odds ratio [or]: 1.12, 95% confidence interval [ci]: 1.05–1.21) and the combination treatment of lopinavir/ritonavir and arbidol (or: 3.58, 95% ci: 1.44–9.52) were risk factors for liver injury in non-critically ill patients. the metabolism of arbidol can be significantly inhibited by lopinavir/ritonavir in vitro (p < 0.005), which may be the underlying cause of drug-related liver injury. liver injury was related to increased length of hospital stay (mean difference [md]: 3.2, 95% ci: 1.3–5.2) and viral shedding duration (md: 3.0, 95% ci: 1.0–4.9). conclusions: critically ill patients with covid-19 suffered earlier occurrence, greater injury severity, and slower recovery from liver injury than non-critically ill patients. drug factors were related to liver injury in non-critically ill patients. liver injury was related to prolonged hospital stay and viral shedding duration in patients with covid-19. clinical trial registration: world health organization international clinical trials registry platform, chictr2000030593. registered march 8, 2020. the mean age of 131 enrolled patients was 51.2 years (standard deviation [sd]: 16.1 years), and 70 (53.4%) patients were male. a total of 76 patients developed liver injury (mild, 40.5%; moderate, 15.3%; severe, 2.3%) with a median occurrence time of 10.0 days. critically ill patients had higher and earlier occurrence (81.5 vs. 51.9%, 12.0 vs. 5.0 days; p < 0.001), greater injury severity (p < 0.001), and slower recovery (50.0 vs. 61.1%) of liver function than non-critically ill patients. multivariable regression showed that the number of concomitant medications (odds ratio [or]: 1.12, 95% confidence interval [ci]: 1.05-1.21) and the combination treatment of lopinavir/ritonavir and arbidol (or: 3.58, 95% ci: 1.44-9.52) were risk factors for liver injury in non-critically ill patients. the metabolism of arbidol can be significantly inhibited by lopinavir/ritonavir in vitro (p < 0.005), which may be the underlying cause of drug-related liver injury. liver injury was related to increased length of hospital stay (mean difference [md]: 3.2, 95% ci: 1.3-5.2) and viral shedding duration (md: 3.0, 95% ci: 1.0-4.9). since december 2019, a newly recognized acute respiratory illness, now officially named coronavirus disease-19 (covid19) , has become widespread globally and accounts for considerable human morbidity and mortality over 200 countries, areas, and territories worldwide (1) (2) (3) (4) . the novel coronavirus is identified and designated as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (5) by the international committee on taxonomy of viruses. as of may 30, 2020, more than five million covid-19 cases had been diagnosed worldwide, and almost 360,000 deaths had been reported (2) . covid-19 has been officially declared a pandemic by the world health organization (6) due to the ongoing outbreak globally. the common clinical manifestations of covid-19 include fever, cough, and shortness of breath (4, 7, 8) . according to the latest epidemiological studies, ∼16-53% of patients with covid-19 experienced different degrees of liver injury (4, (7) (8) (9) (10) (11) (12) (13) , and some patients have developed severe liver injury. the coagulant function abnormality induced by liver injury may cause serious bleeding, especially in critically ill patients who are receiving continuous renal replacement or extracorporeal membrane oxygenation. liver function deterioration can lead to liver failure and even death. therefore, liver injury in patients with covid-19 needs close attention. although some studies have reported the incidence of liver injury in patients with covid-19 (8, 14, 15) , there are limited data describing the course of liver injury occurrence, such as liver injury onset, progression, and recovery, during an entire hospitalization period, particularly in patients with different disease severity. studies on the causes and risk factors of liver injury during sars-cov-2 infection are still limited and controversial. according to current research, liver injury in covid-19 is associated with several main factors, such as sars-cov-2 infection, treatment with potentially hepatotoxic drugs, virally induced cytotoxic t cells, and dysregulated innate immune response (16) . moderate microvesicular steatosis and mild lobular activity are observed in the liver tissue of patients with covid-19 (17) . a preliminary study indicates that sars-cov-2 may directly bind to ace2-positive cholangiocytes to dysregulate liver function. however, systematic study of the causes and the abbreviations: covid-19, coronavirus disease 2019; sars-cov-2, severe acute respiratory syndrome coronaviruses 2; dili, drug-induced liver injury; rucam, a roussel uclaf causality assessment method; alt, alanine aminotransferase; ast, aspartate aminotransferase; alp, alkaline phosphatase; tbl, total bilirubin. risk factors of liver injury in specific populations, such as critically ill and non-critically ill patients with covid-19, is still lacking. additionally, little is known about the correlations between liver injury and some important clinical outcomes, such as length of hospital stay and duration of sars-cov-2 shedding. therefore, a further in-depth study is needed. here, we conducted a multicenter, retrospective, observational study to explore liver injury in critically ill and non-critically ill patients with covid-19 in zhejiang province, china. this study aims to reveal the course of occurrence, risk factors, and correlations with clinical outcome of liver injury in specific covid-19 populations. our study may be helpful in understanding the pathogenesis of liver injury in patients with covid-19, preventing liver injury, and optimizing individual therapeutic treatment. this retrospective observational study was conducted in three tertiary hospitals designated to treat patients with covid-19 in zhejiang province, china. the study was launch by the first affiliated hospital of zhejiang university, which is a university-affiliated tertiary hospital with 2m500 beds and over 100,000 discharged patients per year. sars-cov-2 infection was confirmed using real-time polymerase chain reaction (4, 7) by the local designated hospitals. from january 19, 2020, to february 20, 2020, patients diagnosed with covid-19 were enrolled in this study. patients were excluded on the basis of the following criteria: (1) pregnancy in women, (2) the clinical electronic medical records were reviewed, and epidemiological, clinical, demographic, laboratory, and outcome data were collected for all included patients. a standard case report form was used to record data, including sex, age, chronic medical illness, laboratory data, systemic antiviral agents (i.e., lopinavir/ritonavir, arbidol, fapilavir, and darunavir/cobicistat), potentially hepatotoxic concomitant drugs (18-21) (i.e., corticosteroids, quinolones, statins, immunosuppressive drugs, and non-steroidal anti-inflammatory drugs [nsaids]), number of concomitant drugs, length of hospital stay, and duration of viral shedding. clinical data were followed up until march 10, 2020. topical drugs were not included in concomitant medications. missing data were obtained by direct communication with doctors responsible for the treatment of the patient and their families. all data were verified by three researchers. the severity of covid-19 was defined as non-severe (mild or moderate pneumonia), severe (severe pneumonia), and critically ill during admission in accordance with the diagnostic and treatment guidelines for sars-cov-2 pneumonia of the chinese national health committee (version 6) (22) . in accordance with the severity of covid-19, the patients were divided into non-critically ill (non-severe and severe disease severity) and critically ill groups. non-severe cases included patients with mild and moderate covid-19. the clinical symptoms of mild cases were mild, and there was no sign of pneumonia on imaging. moderate covid-19 refers to fever and respiratory symptoms with radiological findings of pneumonia. severe covid-19 refers to cases meeting any of the following criteria: (1) respiratory distress, (2) oxygen saturation, and (3) arterial partial pressure of oxygen (pao2)/fraction of inspired oxygen (fio2) ≦300 mmhg or cases with chest imaging that showed obvious lesion progression within 24-48 h >50%. critically ill refers to cases meeting any of the following criteria: (1) respiratory failure necessitating mechanical ventilation, (2) shock, and (3) combination with organ failure and admission to an intensive care unit. liver injury was defined as any increase above the normal range for alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), or total bilirubin (tbl). the degree of liver injury was classified as mild, moderate, or severe ( table 1) in accordance with the common terminology criteria for adverse events (version 5) (23). mild, moderate, and severe liver injuries were defined as the occurrence of adverse event grades 1, 2, and 3 (i.e., the increase in alt, ast, alp, or tbl, table 1 ), respectively. the recovery rate of liver function was defined as the decrease in the number of patients with liver injury at discharge divided by the number of patients with liver injury during treatment. viral clearance was defined as the presence of two consecutive negative results with qpcr detection over an interval of 24 h. the metabolic interactions between lopinavir/ritonavir and arbidol was tested in human hepatic microsomes. the metabolic reaction was performed in 0.1 ml incubation mixture containing 0.5 mg microsome protein. the reaction was started by adding 1 mm nicotinamide adenine dinucleotide phosphate and statistical tests were performed using spss19.0 (www.spss.com) and r 3.5.1 (r core team, www.r-project.org). continuous variables were presented as mean (standard deviation [sd]) or median (interquartile range [iqr] ) and compared between and within non-critically ill and critically ill groups by using the student's t-tests or the mann-whitney u-test, as appropriate. categorical variables were presented as frequency (percentage) and assessed using the pearson χ 2 or fisher's exact test (cell size < 5). the occurrence time of liver injury was defined from the time a patient was admitted to hospital until liver injury occurred. the occurrence time of liver injury was portrayed by the kaplan-meier plot and compared between patients in critically ill and non-critically ill groups with a log-rank test. liver injury after admission and at discharge, abnormal liver function indicators, and recovery rate of liver function were compared between the critically ill and non-critically ill groups. univariable and multivariable logistic regression models were used to explore the risk factors for liver injury. demographic data, laboratory test indicators, disease severity, antiviral agents, and potentially hepatotoxic concomitant drugs were investigated. the factors that showed a significant association (95% confidence interval [ci]: does not include one) after univariate logistic regression analysis were entered into the multivariable logistic regression analysis. clinical outcomes (i.e., length of hospital stay and duration of viral clearance) were compared between patients with or without liver injury within each group. p < 0.05 was considered statistically significant. the demographic and clinical characteristics of the patients are shown in table 2 . nineteen ineligible patients were excluded, and the clinical data of 131 patients with confirmed covid-19 were collected (figure 1) . the sample sizes of included the baseline characteristics of patients in critically ill and noncritically ill groups were significantly different (p < 0.05) for the parameters age, prevalence of cardiovascular and cerebrovascular diseases, laboratory data (i.e., leucocytes, neutrophils, albumin, serum creatinine, and c-reactive protein), and treatments (i.e., number of concomitant medications and use of glucocorticoids and antiviral agents). the incidence of liver injury over the study period is shown in figure 2 . during the treatment, 76 patients (58.0%) had liver injury (mild, 40.5%; moderate, 15.3%; severe, 2.3%, table 3 ). the median occurrence time of liver injury was 10.0 days. the percentage of liver injury was reduced to 24.4% at discharge, and liver function returned to normal levels in 57.9% of patients with liver injury. however, none patients with severe liver injury returned to the normal liver function at discharge ( table 3) . the kaplan-meier survival curves for liver injury in different groups are presented in figure 2 . the incidence of liver injury was significantly different between patients in critically ill and non-critically ill groups (figure 2 , median: 12.0 day vs. 5.0 days, p < 0.001) over the study period. as shown in table 3 , 81.5% of the patients in the critically ill group developed liver injury, compared with 51.9% in the non-critically ill group. the severity of liver injury in the critically ill group was greater (p < 0.001) than that in the non-critically ill group. alt and ast levels were more commonly elevated in critically ill patients (p < 0.05) than in the non-critically ill group, whereas no statistical difference was observed in the abnormal alp and tbl levels ( table 3) . the recovery rate of liver function in the non-critically ill group was higher than that in the critically ill group (61.1 vs. 50.0%). univariable and multivariable logistic regression models were used to explore the risk factors for liver injury in the critically ill and non-critically ill groups. however, given the limited sample size in the critically ill group, the statistical power was insufficient, and the multivariable logistic regression model was not conducted in this group. the comparison between patients with or without liver injury in the critically ill group showed that the patients with liver injury had lower lymphocyte numbers, received more concomitant medications, and had higher serum creatinine levels on admission, but the differences were not statistically different (supplementary table s1 ). in the non-critically ill group, the univariate logistic analyses showed that the combination treatment of lopinavir/ritonavir and arbidol and the number of concomitant medications were significantly associated with liver injury ( table 4) . after the multivariable regression analysis, the combination treatment of lopinavir/ritonavir and arbidol and the number of concomitant medications were determined to be independent risk factors for liver injury. the patients who received the combination treatment of lopinavir/ritonavir and arbidol had 3.58 times the odds (95% ci: 1.44-9.52) of liver injury than patients who did not receive the aforementioned treatment. for every increase in concomitant medication, the odds of liver injury increased by 12.1% (95% ci: 4.9%−21.2%, table 3 ). lopinavir/ritonavir combined with arbidol was shown to be a risk factor of liver injury. we inferred that the metabolic interaction between arbidol and lopinavir/ritonavir may increase drug concentrations and may thus lead to a higher risk of liver injury. the metabolic interactions were tested in human hepatic microsomes in vitro, and the results showed that the metabolism of arbidol can be significantly inhibited after exposure to different concentrations of lopinavir/ritonavir (p < 0.005, figure 3) , whereas arbidol had no effect on the metabolism of lopinavir/ritonavir (p > 0.05, figure 3 ). the average length of hospital stay was 16.6 (sd: 5.7) days and was statistically longer in patients with liver injury than in patients without liver injury (md: 3.2, 95% ci: 1.3-5.2). the mean duration of viral clearance in patients with liver injury was 13.6 days, which was 3 days longer than that in patients with normal liver function (95% ci:1.0-4.9). within the non-critically ill and critically ill groups, the length of hospital stay and duration of viral clearance tended to increase in patients developing liver injury ( table 5) . covid-19 is a newly identified illness that has spread around the world and has become a global health crisis (2, 4, 7, 24) . epidemiological studies have demonstrated that liver injury can occur in patients with covid-19 (7, 8, 10, 12) and may be related to sars-cov-2 infection or therapeutic drugs (9, 17) . here, the onset, progression, recovery, risk factors, and correlation with clinical outcomes of liver injury in patients with varying severities of covid-19 were investigated. the results show that the liver injury in critically ill patients with covid-19 occurred more frequently and earlier, developed more seriously, and recovered more slowly than that in non-critically patients. drug factors, including the combination treatment of lopinavir/ritonavir and arbidol and the number of concomitant medications were independent risk factors for liver injury in non-critically ill patients with covid-19, which may be due to drug interactions at the metabolic level. liver injury was found to be related to prolonged hospital stay and delayed virus eradication in all enrolled patients. consistent with previous studies (7, 8, 25) , this study has found that the high incidence of liver injury in patients with covid-19 is related to disease severity. additionally, the progression and the recovery process of liver injury during the entire hospital stay were investigated. our results indicated that the liver injury in critically ill patients with covid-19 happened earlier and recovered more slowly than that in non-critically ill patients. a higher rate of liver injury was presented in critically ill patients over the study period. our further data showed that the severity of liver injury was also related to covid-19 disease severity. the incidences of mild liver injury were similar in different groups, whereas moderate and severe liver injuries occurred more frequently in critically ill patients than in noncritically ill patients. sars-cov-2 may directly dysregulate liver function by binding to ace2-positive cholangiocytes (26) . our results demonstrated that rather than tbl, alt and ast were the most elevated indicators in critically ill patients with covid-19. hepatocyte injury can be caused by immune interactions that involve virally induced cytotoxic t and kupffer cells (16) . we speculated that besides the direct damage by sars-cov-2, virus-induced cytokine storm may also play an important role in critically ill patients with liver injury. particularly, unlike other studies, this study has found that drug factors rather than disease severity may play a more important role in the liver injury of non-critically ill patients with covid-19. the combination treatment of lopinavir/ritonavir and arbidol was an independent risk factor. lopinavir/ritonavir and arbidol are recommended as potential drugs for sars-cov-2 infection by the chinese national health committee. existing evidence shows that elevated serum aminotransferase and jaundice occur in patients receiving lopinavir/ritonavir containing antiretroviral regimens (18) and that arbidol may induce an increase in transaminase (27). lopinavir and arbidol are mainly metabolized by cytochrome p3a (cyp3a) (28) . ritonavir is a potent cyp3a inhibitor. our results demonstrated that lopinavir/ritonavir can significantly inhibit the metabolism of arbidol, thereby leading to increased arbidol serum concentration. in addition, tbl was the major elevated indicator in non-critically ill patients, indicating that arbidol may have novel adverse reactions in jaundice. therapeutic drug monitoring may be useful in optimizing the regimens in covid-19 patients receiving the combination treatment of lopinavir/ritonavir and arbidol. the number of concomitant medications was another independent predictor of liver injury. concomitant drugs can affect the metabolism of other drugs through induction, inhibition, or substrate competition (20) . we considered that an increased number of concomitant drugs may lead to complex drug interactions and can increase the risk of liver injury. our study also found that the liver injury in patients with covid-19 was related to prolonged viral shedding and hospital stay durations. we inferred that liver injury would lead to immune dysfunction, thereby causing a delay in virus clearance. the prolonged hospital stay can be explained by the need for increased time for liver function recovery or the failure of virus eradication. in the critically ill group, the length of hospital stay and the duration of viral shedding in patients with liver injury were only numerically but not statistically higher than those in patients without liver injury. we speculated that the durations of hospital stay and viral shedding should be influenced by complex factors in critically ill patients with covid-19, and liver injury may not be the only factor affecting the clinical outcomes. our study has some limitations. first, this study is a retrospective, non-randomized clinical observational trial. our cohort is a convenience sample of patients with covid-19 admitted to three hospitals in zhejiang, china. however, this study reflects real-world clinical practices and provides relevant data about liver injury in patients with covid-19. second, the treatments among the three centers were highly consistent because all patients were from zhejiang province. antiviral agents were administered to all patients with covid-19 but limited to a few kinds (e.g., lopinavir/ritonavir and arbidol). therefore, our results cannot be extrapolated to antiviral agents that are not involved in this study, and large controlled studies are necessary to explore the potential risks of liver injury by other antivirals. finally, univariable and multivariable logistic regression models were planned to be used to explore the risk factors for liver injury in the critically ill and non-critically ill groups. however, considering the limited sample size in the critically ill group, the statistical power was insufficient, and the multivariable logistic regression model was not conducted in this group. the trends in risk factors can be reflected through data comparison to a certain extent. focusing on such a population with an expanded sample size would be challenging but would be interesting future research. liver injury has occurred widely in patients with covid-19. critically ill patients suffered higher incidence, earlier occurrence, greater injury severity, and slower recovery from liver injury. drug factors were independent risk factors for liver injury of non-critically ill patients, and drug interaction based on the cyp450 enzymes and concomitant drugs should be closely monitored. liver injury was related to prolonged hospital stay and viral shedding duration in patients with covid-19. therefore, special attention to liver injury during sars-cov-2 infection is recommended. healthcare workers should closely monitor the medications used during hospitalization and adjust and optimize the drug treatment in a timely manner. all datasets presented in this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by the ethics committee of the first affiliated hospital, college of medicine, zhejiang university (reference number: 2020iit[71]). written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. this study followed the statement of strengthening the reporting of observational studies in epidemiology. tl, yq, and xl participated in the conception and design of this study. sj and xl were the project managers and coordinated patient recruitment. dh, ll, rw, rr, yr, jm, nc, xw, zy, and mz coordinated all analyses in the project. sj, ll, rw, and dh were involved in the acquisition, analysis, or interpretation of data. mz and yh conducted the in vitro experiments on drug interaction. data analysis was done by dh, jm, zy, and nc. drafting of the manuscript was done by ll, rw, and xw. all authors contributed to the critical review and final approval of the manuscript. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia map production: who health emergencies programme epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 available online at clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan liver injury in covid-19 management and challenges epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of covid-19 related liver damage liver injury during highly pathogenic human coronavirus infections covid-19 and the liver: little cause for concern pathological findings of covid-19 associated with acute respiratory distress syndrome available online at interaction of glucocorticoids with fxr/fgf19/fgf21-mediated ileumliver crosstalk easl clinical practice guidelines: drug-induced liver injury drug-induced liver injury national health commission and state administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 6). (2020) available online at covid-19 in a designated infectious diseases hospital outside hubei province pecific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection efficacy and safety of arbidol in treatment of naturally acquired influenza pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans we thank the medical and nursing staff at all collaborating centers and especially all patients participating in this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2020.00347/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 jiang, wang, li, hong, ru, rao, miao, chen, wu, ye, hu, xie, zuo, lu, qiu and liang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-016757-3d320c0a authors: nan title: acute and chronic liver insufficiency date: 2008 journal: hepatology textbook and atlas doi: 10.1007/978-3-540-76839-5_20 sha: doc_id: 16757 cord_uid: 3d320c0a the term “liver insufficiency” denotes a break down in the functions of the liver. the syndrome of functional liver failure covers a wide spectrum of clinical, biochemical and neurophysiological changes. in principle, liver insufficiency can occur without previous liver damage as well as with already existing liver disease. it is characterized by a deterioration in the synthesizing, regulatory and detoxifying function of the liver. this final stage of liver disease terminates in hepatic coma. the term "liver insufficiency" denotes a breakdown in the functions of the liver. the syndrome of functional liver failure covers a wide spectrum of clinical, biochemical and neurophysiological changes. in principle, liver insufficiency can occur without previous liver damage as well as with already existing liver disease. it is characterized by a deterioration in the synthesizing, regulatory and detoxifying function of the liver. this final stage of liver disease terminates in hepatic coma. serious liver disease can affect the 12 main metabolic functions of the liver, with their 60ϫ70 even more important partial functions, to widely differing degrees. (s. tab. 3.1) the result is either global insufficiency or partial insufficiency, each with very varied clinical and biochemy y ical symptoms. the failure of certain metabolic functions is responsible to a greater or lesser extent for the development and intensity of liver insufficiency. impairments in the functions of detoxification and protein metabolism are particularly significant in this respect. the compensated stage does not usually display any signs of liver insufficiency (except possibly jaundice), nor are there any typical ailments. functional parameters that can be quantified in routine laboratory tests (such as cholinesterase, albumin, quick's value, bile acids) may still be normal or only minimally impaired in the individual instance. in contrast, liver function tests (galactose, indocyanine green, megx, etc.) demonstrate a reduction of liver function which is already quite considerable. the decompensated stage, i. e. manifest liver insufficiency, can present as cellular decompensation (e. g. in the case of acute liver failure due to toxic or inflammatory mass necrosis) or be expressed only in the form of portal decompensation (e. g. in cases of postsinusoidal intrahepatic portal hypertension). • as a rule, chronic liver insufficiency is accompanied by a combined decompensation with a loss in function of the liver cells and, at the same time, the sequelae of portal decompensation (collateral varicosis, encephalopathy, ascites, hepatorenal syndrome, hepatopulmonary syndrome, variceal bleeding). (see chapters 15ϫ19 and 35) 380 depending on the time period involved in the course of the disease, acute liver failure without pre-existing liver disease can initially be differentiated by massive liver cell disintegration due to a variety of causes. • in contrast, chronic liver insufficiency with pre-existing liver disease is mostly found in advanced liver cirrhosis with a progressive loss of function. • a sudden necrotising episode can also precipitate the change from chronic and still compensated liver insufficiency into acute liver failure (i.e. "acute-on-chronic" insufficiency) in the same way that acute liver failure which has been overcome may develop into chronic liver insufficiency. (60) hepatic coma can be subdivided according to its aetiology as follows: (1.) hepatocyte disintegration coma (ϭ endogenous coma due to the loss of parenchyma), (2 ( ( .) liver cell failure coma (ϭ exogenous coma due to metabolic disorders, almost always in the presence of cirrhosis), (3.) electrolyte coma (ϭ so-called "false" coma due to dyselectrolytaemia, almost always iatrogenic), and (4.) mixed forms of coma. (s. pp 281, 284, 386) (s. tab. 15.5) 3 acute liver insufficiency ᭤ j. w. morgagni (1761) was probably the first to describe acute yellow atrophy of the liver, i. e. hepatic coma. acute liver failure can be seen as identical to the "acute yellow atrophy" described by k. rokitansky in 1842. this acute and severe clinical picture was subsequently termed "bilious dyscrasia" (p. j. horaczek, 1844), "icterus gravis" (c. ozanam, 1849), "acholia" (f. th. frerichs, 1858), "hepatolysis" (r. ehrmann, 1922), "hepatodystrophy" (g. herxheimer, 1935) r r or "liver dystrophy" (r. böhmig, 1949 ). the terms "hepatargia" (h. i. quincke, 1899) and "hepatic coma" were used to denote the final stage, which usually sets in at the end of acute or chronic liver failure. • acute liver failure in the course of acute viral hepatitis was termed "fulminant hepatitis" by w. lucké et al. (1946) , who also defined a subacute form with a less severe course. (36) acute liver failure (alf) is defined as an acute clinical picture with jaundice due to a most severe disorder in the liver function and/or massive liver cell necrosis which, without any pre-existing liver disease, culminates in hepatic coma (ϭ endogenous coma) within 8 weeks. potentially, the condition is fully reversible (c. trey et al., 1970) . • in addition, coagulopathy must also be present (d. f. schafer et al., 1989 ). clinically, there are three different courses of disease following the onset of jaundice: (1.) fulminant or hyperacute liver failure (ϭ occurrence of hepatic encephalopathy in the 1 st week), (2 ( ( .) acute liver failure (ϭ occurrence of hepatic encephalopathy between the 2 nd and 4 th week), and (3.) subacute liver failure (ϭ occurrence of hepatic encephalopathy between the 5 th and 8 th week). • surprisingly, however, it could be shown that 30ϫ40% of the hyperacute forms survived in spite of the development of hepatic coma and cerebral oedema. as opposed to this, the subacute forms displayed a survival rate of only 10ϫ20% despite a lower frequency of cerebral oedema and better liver function. (s. tab. 20 in the pathological-anatomical context, hepatomegaly due to hyperaemia is often found at the outset. during the further course of disease, this can develop into liver atrophy as a result of parenchymal loss. histologically, acute liver failure shows a wide range of uncharacteristic changes. (1.) depending on the underlying cause, the morphological picture of acute necrotizing hepatitis may develop, with extensive confluent cellular destruction. the extent of necrosis, measured by the morphologically evidenced hepatic volume fraction of the still functioning liver parenchyma, yields reliable information on the chance of survival (j. scotto et al., 1973) . given a normal value of 85% hepatic volume fraction (hvf) of intact liver cells for each volume unit of the total liver, a decrease to < 30% (threshold 28ϫ35%) would possibly mean that the patient is unlikely to survive. (2 ( ( .) in acute liver failure caused by toxins or hypoxia, massive fatty degeneration of the hepatocytes can vary substantially. in diffuse fatty degeneration featuring minute vacuoles and damage to the organelles, liver cell necrosis cannot, as a rule, be detected (e. g. acute fatty liver during pregnancy, reye's syndrome, in association with tetracycline or valproic acid). (3.) between these two "classical" morphological manifestations, there are also compound forms, i. e. 381 courses of disease with a variety of histological changes of different intensities and combinations. on occasions, it is also possible to identify histological findings which point to a certain cause of the disease. (41, 59) from a morphological point of view, acute liver failure is potentially reversible, so that even complete regeneration can be attained. precursory cellular necrosis is hence less of a determinant than the capacity to regenerate. there have been reports on the transition from virusinduced acute liver failure to chronic hepatitis. as the final stage of fulminant viral hepatitis (also known as acute liver dystrophy or submassive hepatitic necrosis), a postdystrophic scarred liver ("potato liver") can develop. (s. fig. 35 .14) cicatricial distortions with a continuing effect, regenerative processes, intrahepatic vascular disorders and hypoxia-related damage lead to the conclusion that a posthepatitic, postdystrophic scarred liver may well be a special form of cirrhosis. ᭤ neither the functional state of liver insufficiency nor hepatic coma can be recognized histologically. the common target structures for the various causes of acute liver failure are usually the cellular and subcellular biomembranes of the hepatocytes. among other things, any damage to these biomembranes causes a massive inflow of calcium into the liver cells, which results in a severe disorder of the cell milieu and ultimately in cell death. in oxygen deficiency, the oxidative stress is mainly localized in the extracellular spaces. this is where the kupffer cells and neutrophils are involved in complex self-stimulating mechanisms, which can lead to the formation of inflammatory mediators and cytotoxic substances. • an important pathogenetic aspect is the "priming effect", which generally results in the increased production of oxygen radicals. the complex process of lipid peroxidation likewise effects massive liver cell damage in the form of self-perpetuation. • excessive immunological reactions, which occur in acute liver failure due to viral hepatitis, halothane hepatitis, etc., are significant. there are also isolated cases in which biotoxometabolites are produced and may act as neo-antigens. (s. fig. 3 . 11) consequently, severe damage to liver cells and widespread necrosis are usually the result of a network of altered cellular and humoral reactions, which for their part are often the initial cause of acute liver failure due to their synergistic and interactive effects (h. popper et al., 1986) . systemic reactions are responsible for the fact that other organs and functional sequences are equally affected, thus creating a wide spectrum of clinical findings and complications. acute liver failure is a rare occurrence. about five cases are found out of 6,000 hospital admissions (in the usa a total of ca. 2000 patients per year, in germany ca. 150). however, there can be wide variations in frequency due to the effect of regional differences on individual aetiology. the causes of acute liver failure are numerous and varied. diabetes mellitus and overweight (12) are extremely high risk factors. • primary or secondary hepatitis viruses are deemed a frequent cause, although there are regional and individual variations (e. g. drug 382 dependence, pregnancy) regarding the predominant virus type. • a further common cause (ca. 20%) are drugs (particularly paracetamol, often taken with suicidal intent, and halothane), followed by mycotoxins, alcohol and carbon tetrachloride (such as can be found in cleaning agents or solvents, and also with "glue sniffers"), heat-stroke (up to 10% of cases), ecstasy, and vascular diseases. (12, 79) (s. tab. 20.2) paracetamol: the first report on acute liver failure due to paracetamol poisoning was published in 1966 (d. g. d. davidson et al.). due to induced cyp ii e2, paracetamol is metabolized to the extremely reactive molecule n-acetyl-p-benzoquinone-imine (napqi). this binds covalently to cellular proteins. a small amount of napqi is neutralized by glutathione; however, with a larger quantity of napqi (following an intake of > 10 g paracetamol), the hepatic glutathione supplies are used up, so that the napqi becomes highly toxic. the overall picture of acute liver failure is first and foremost determined by the clinical findings. the symptoms are dramatic and subject to swift change. the course of disease can advance within a matter of days or, in a subacute form, take several weeks. (12, 58, 62, 79) general symptoms: the acute clinical picture develops swiftly with conspicuous symptoms, such as fatigability, loss of appetite, nausea, weakness, lassitude, meteorism, apathy and disruption of the circadian rhythm. encephalopathy: rapidly, often within one or two days, there is evidence of dysarthria, muscle tremor, finger tremor, lack of concentration and asterixis. restlessness, hyperkinesis and hallucinatory experiences occur. even screaming attacks have been observed. these symptoms, which can still be classified under stages i and ii, are fully reversible. nevertheless, lethality of 30ϫ35% must be anticipated in stage ii. in contrast, stage iii is clearly less reversible. somnolence, stupor with confusion, deviant behaviour, hyperreflexia, babinski's reflex, clonus and spasticity as well as nystagmus are now observed. there is usually still a response to acoustic stimuli. the eeg shows a slowing down of basic activity (0.5ϫ3.0/ sec.) together with mainly biphasic and triphasic potentials. lethality rises to over 50%. in stage iv, the patient is in a deep coma. there is evidence of areflexia, an absence of any corneal reflex and loss of tonicity; the brain waves flatten out to an isoelectric line. irrespective of therapy, lethality is 80ϫ90%. (s. tab. 15.5) cerebral oedema: as from coma stage iii, cerebral pressure can increase (75ϫ80% of cases) owing to water retention and/or vasodilation with hyperaemia, yet with a subsequent reduction in cerebral perfusion and hypoxia. intracranial cerebral compression is > 20 mm hg. cerebral oedema is vasogenetic and/or cytotoxic, the latter feature appearing to predominate. clinical symptoms include disorders in the respiratory rhythm (in particular tachypnoea), hypertension, bradycardia and increased muscular tonus. singultus implies damage to and impending constriction of the brain stem. the pupils are dilated due to the pressure on the oculomotor nerve. chemosis can develop, which is a fatal prognostic sign. (2) intracranial blood circulation sinks rapidly. in 30ϫ60% of cases, cerebral oedema is fatal. (9, 10, 39, 64, 66, 75, 78) jaundice: with the foudroyant disintegration of liver cells, a comatose condition can set in within a few hours, even before jaundice is identified. in most cases, however, jaundice is already present. the intensity and time of onset vary. severe jaundice (> 20 mg/dl) is considered to be a poor prognostic sign. the sweet aromatic smell of the exhaled breath (mercaptan derivatives) is seen as a reliable sign of acute liver failure, but it is not always present. the administration of poorly absorbable antibiotics (e. g. paromomycin) improves the condition of hepatic foetor, and can even eliminate it temporarily. (s. pp 91, 275) fever: fever often occurs; it mostly remains at 38°c, but septic temperatures are possible. • in some cases, this may be a question of aetiocholanolone fever, whereby r r aetiocholanolone can also be quantified in the serum. • bacterial infections likewise cause fever and require appropriate treatment. toxins may also be responsible for the febrile condition (tissue toxins, endotoxins). (s. p. 761) liver size: the liver may have normal size or it can be enlarged due to hyperaemia or massive fatty infiltration. a rapid shrinking of the liver to less than 1000 ml in volume ("dystrophy", "acute atrophy") ϫ requiring sonographic or ct monitoring at the bedside ϫ is deemed to be a poor prognostic sign. at present, there is no specific laboratory investigation which facilitates the diagnosis of acute liver failure. in view of the severity of this clinical picture, there are, however, a number of laboratory parameters which show marked pathological changes and thus require full diagnostic clarification. activin a serum levels were elevated, especially in patients with acute liver failure, due to a paracetamol overdose. this did not affect the final outcome, but was possibly a factor in the inhibition of liver regeneration. serum follistatin was also increased in patients with fulminant liver disease. (27, 35) furthermore, the laboratory values allow an assessment of the complications involved and an evaluation of the prognosis. (7, 13, 14, 58, 62, 79) various laboratory values are indicative of severe complications and thus considered to be criteria pointing to a poor prognosis. (s. tab. 20 6. group-specific component protein: this substance (ϭ α 2 -globulin) is synthesized in the liver and binds actin. gcp is released upon hepatocyte decay; its pronounced reduction in the serum results from the decrease in synthesis in acute liver failure. (57) the course taken by acute liver failure varies in each case as a result of the respective complications, which also decidedly worsen the prognosis. close-meshed and targeted laboratory investigations can usually identify complications early enough, so that successful therapy might still be possible. coagulation disorders: some 35ϫ55% of patients with acute liver failure are in danger of suffering from serious gastrointestinal bleeding. extensive cutaneous haemorrhages also occur frequently. in addition, disseminated intravascular coagulation (dic) sometimes develops. as a result, bleeding and coagulation disorders number among the most frequent causes of death (20ϫ25%). pathophysiology is based on the diminished synthesis of coagulation and fibrinolysis factors and inhibitors as well as a decrease in the breakdown of activated factors, a functional disorder of thrombocytes or thrombopenia, and latent consumptive coagulopathy. it is of great help to determine ptt and factor v. a high level of the thrombin-antithrombin iii complex (tat) points to a poor prognosis. the simultaneous development of portal hypertension in individual cases promotes a tendency towards nasopharyngeal and gastrointestinal bleeding. ( acid-base disorders: initial metabolic alkalosis (resulting from decreased urea synthesis with reduced bicarbonate consumption) may be superimposed by respiratory alkalosis as an outcome of disorders in lung function. during the further course, metabolic acidosis (with renal insufficiency) and respiratory acidosis (with pulmonary insufficiency) can be expected. in advanced or severe stages of the disease, lactate acidosis may develop in some 50% of all comatose patients owing to restricted gluconeogenesis. circulatory disorders: in general, acute liver failure is initially accompanied by hyperdynamic circulation. during the further course, approximately 80% of patients develop hypotension, which above all results in a considerable reduction in hepatic, cerebral and renal perfusion. at the same time, peripheral vasodilation is usually evident. bradycardia, generally resulting from cerebral oedema, worsens the cardiovascular conditions and is considered to be a poor prognostic sign. ultimately, the patient does not respond to volume expansion and catecholamines. hypoglycaemia: in 25ϫ40% of cases, hypoglycaemia develops and can all too easily be overlooked. the cause is seen to be a reduction in liver glycogen content, diminished glycogen synthesis and gluconeogenesis as well as hyperinsulinaemia due to reduced degradation of insulin in the liver. (21) it is often difficult to eliminate such hypoglycaemia, even with i.v. glucose infusions. • furthermore, there is a danger of hypokalaemia and even hypophosphataemia, necessitating phosphate substitution with continuous monitoring of the serum values of phosphate and calcium (reactive hypocalcaemia is dangerous). the frequency of hyperamylasaemia is reported to be 55% of patients with acute liver failure; in 20ϫ30% of cases, pancreatitis could be identified clinically and sonographically. the cause is multifactorial. infections: because of their greater susceptibility, about 80% of patients with acute liver failure are subject to the threat of bacterial infection, which in 10% of cases is also the reason for their death. the typical signs of an infection, such as fever or leucocytosis, are often absent. increased levels of procalcitonin (> 0.58 ng/ml) are deemed to be a valid marker of bacterial infection. the respiratory tract and the urinary passages are most frequently affected. regular bacteriological examinations (sputum or urine as well as catheter after removal) should therefore be carried out. haemocultures have to be checked for both aerobians and anaerobians. multiple serological tests may be necessary for aetiological clarification. there is also a certain risk of fungal infections. (51) the survival rate in acute liver failure is 10ϫ40%. this rate varies widely owing to a number of reasons. there is a better prognosis for poisoning from paracetamol or amanita phalloides, since successful therapy procedures are already established for these forms of intoxication. younger patients (10 to 40 years) have a better prognosis. this also applies to hav infection. a poor outcome can be expected in obesity, wilson's disease or the budd-chiari syndrome as well as in coma stages iii and iv (lethality over 80%) due to various complications (e. g. bleeding, renal or respiratory insufficiency, infection) ϫ especially with younger (< 10 years) or older patients (> 40 years). acute liver failure which is due to halothane, the application of various medicaments or viral hepatitis (delta superinfection, hev in pregnancy) likewise has a less favourable prognosis. • laboratory parameters such as serum bilirubin, higher afp values (especially during the first three hospital days), coagulation factors, galactose test and cholinesterase have proved helpful in assessing the course of disease, liver function and prognosis. (14, 18, 22, 43, 46, 59, 62, 79) the regenerative ability of the liver is of utmost importance for overcoming such a severe disease. (40) after a regeneration period, an intact cell mass (hepatic volume fraction) of > 45% is required for survival. (41) various factors are indicative of good regeneration: rising values of α 1 -foetoprotein (and also γgt), hgf, egf, thfα, tnfα and interleukin-6 as well as a decline in serum phosphorous levels. (14) it was possible to improve regeneration by means of hepatotropic substances, such as insulin and glucagon, so that these substances are also referred to as "goodies" for the liver (s. sherlock, 1976). subsequent investigations proved to be contradictory. (23, 81) an increase in the regeneration rate of the liver cells can possibly be achieved either by hepatic arterial infusion of pge 1 (56) or by silymarin through stimulation of rna synthesis. (s. pp 44, 896) (s. fig. 3 .5) chronic liver insufficiency is due to the progression of an already existing chronic liver disease. this generally tends to be advanced cirrhosis of varied aetiology. basically, however, any liver disease can be a potential cause of chronic liver insufficiency. alcohol, infections and certain medicaments are also deemed to be common causes. thus a great number of substances and events can trigger liver insufficiency. the clinical picture of chronic liver insufficiency comprises both a compensated and decompensated form. these two stages of manifest chronic liver insufficiency affect the hepatocellular area or the portal system either exclusively or predominantly (ϭ cellular or portal compensation or decompensation); mostly they occur as a combined form of disease. the resulting spectrum of clinical and laboratory findings will reflect either a global or partial insufficiency of the liver. (s. p. 380) general manifestations of the disease: the clinical picture of chronic liver insufficiency is characterized by a number of symptoms such as fatigue, apathy, lack of appetite, lack of concentration, infirmity, sensation of repletion and meteorism. clinical findings: organ-related so-called "minor signs" of liver insufficiency can be observed over a certain period of time. (s. tab. 20.4) ϫ itching ϫ skin stigmata of liver disease ϫ tendency to "bruise" ϫ nasal haemorrhage and ulorrhagia ϫ tongue changes ϫ intermittent acholic stool ϫ intermittent dark urine ϫ anaemia ϫ thrombopenia ϫ leucopenia ϫ macrocytosis 6. fever 7. splenomegaly tab. 20.4: so-called "minor signs" of chronic liver insufficiency constant meteorism ("first the wind and then the rain " ") and intermittent changes in the colour of stools and urine are distinct signs of impending insufficiency. the "blossoming" of spider naevi, an intensification of palmar erythema and tongue changes (e. g. transition of the moist "scarlet tongue" into a dry "raspberry tongue") are common. obvious features of the blood count are: anaemia (due to bleeding of the skin or mucosa, folic acid deficiency, reduced erythrocyte survival time) and thrombopenia (due to consumptive coagulopathy, dilutional thrombopenia with plasma dilution, immunothrombopenia, sequestration in splenomegaly and toxic inhibition of the bone marrow). decompensation in chronic liver insufficiency is characterized by the development of severe, life-threatening complications: 1. ascites and oedema 2. coagulopathy and bleeding 3. hepatic encephalopathy 4. hepatorenal syndrome 5. hepatopulmonary syndrome 6. impairment of liver functions of particular significance is the serious impairment of essential tasks performed by the liver such as the detoxification function (ammonia detoxification, biotransformation, radical scavenger function, clearance abilities of the res, etc.), the synthesis of vital proteins and the regulation of biochemical systems and substances ϫ these are considered to be precursors of complicative developments. any insufficiency of bilirubin metabolism is reflected in increasing jaundice, likewise deemed to be an unfavourable sign with respect to prognosis. the term hepatic encephalopathy (he) describes the entire field of neuropsychiatric symptoms which can be found in patients suffering from acute or chronic liver disease. the term portosystemic encephalopathy (pse) stresses the presence of portosystemic shunts, which are as a rule associated with liver cirrhosis. • hepatic coma (in stages iii and iv) is the ultimate and total loss of consciousness (coma ϭ deep, sound sleep). in clinical terms, four or five stages can be defined, but the latent or subclinical stage as well as stages i and ii may progress so rapidly that only the comatose final stage is actually determined. generally, chronic liver insufficiency is seen as a liver failure coma, i. e. exogenous coma. recurrent hepatic encephalopathy points to the existence of a chronic liver disease, particularly liver cirrhosis. the serum levels of tnf correlate positively with the severity of he. (see chapter 15) ascites and oedema are also found in severe hepatic diseases, pointing to serious disorders in the water and electrolyte metabolism. these complications are signs of decompensation in liver cirrhosis or chronic liver insufficiency. pleural effusion may also be evident. cirrhosisrelated pleural effusion without concomitant ascites has been described as a rarity. (see chapter 16) all liver diseases resulting in liver insufficiency can also give rise to the hepatorenal syndrome. this syndrome is 386 most frequently found in decompensated liver cirrhosis ("renal insufficiency in the terminal stage of cirrhosis"). it involves massive vasoconstriction of the renal cortical vessels with a critical drop in the glomerular filtration rate (urine production < 500 ml/day, possibly developing into anuresis). at the same time, systemic vasodilation and hyperdynamic cardiac function are generally in evidence. the survival time is very short. lethality is approx. 95%. (see chapter 17) in 15ϫ30% of patients with liver cirrhosis, coagulopathy leads to clinically relevant haemorrhagic diathesis. dangerous and considerable bleeding may occur (nasal, gingival), and there may well be pronounced cutaneous haemorrhages; the latter occasionally occur as sugillations, ecchymoses and petechial haemorrhages (s. this condition describes acute liver failure in cases of hitherto well-compensated liver disease. the result is a sudden deterioration in clinical status accompanied by jaundice as well as hepatic encephalopathy and/or the hepatorenal syndrome. there are a number of causes including (1.) well-known hepatotoxic factors (e. g. superimposed viral infection, alcohol consumption, hepatotoxic drugs, intoxication) and (2 ( ( .) endogenous factors (e. g. sepsis, variceal bleeding, gastrointestinal haemorrhage, diarrhoea, hypoxia). acute liver failure is frequently the result of a chain of damaging events, like a vicious circle. the clinical and laboratory findings of this sudden deterioration largely correspond to those of acute liver failure (see above). this also applies to potential complications such as coagulopathy, he, ascites and/or hrs. except for the treatment of, for example, paracetamol intoxication and amanita phalloides poisoning, there is no causal therapy for liver insufficiency. all conservative treatment measures are based on four principles: 1. prevention and treatment of complications 2. substitution of substances which cannot be adequately produced in the liver as a result of hepatic synthesis disorders 3. bridging the period of time until toxins have been eliminated, liver functions and regenerative processes have improved or liver transplantation can be carried out 4. promotion of liver regeneration ᭤ intensive care: patients with alf or decompensated chronic liver insufficiency (e. g. coma stages iiϫiv, refractory ascites, hepatorenal syndrome, disseminated intravascular coagulation, gastrointestinal bleeding) require monitoring and treatment in an intensive care unit, preferably in a transplantation centre. (7, 13, 62, 79) intensive care involves monitoring the cardiovascular system (blood pressure, pulse, ecg) and respiratory frequency. the patient's y y temperature and urine excretion have to be recorded every hour. the body weight is documented every day using a weighing bed. the water equilibrium should be carefully monitored. consistent preventive measures against infection must be guaranteed for those patients who are particularly at risk. regular physical measures for the prevention of pneumonia are a necessity. a moderate head-up position (30ϫ40°) is recommended. • a central venous catheter (monitoring central venous pressure, parenteral feeding), a nasogastral tube and a suprapubic bladder catheter are positioned for supply and monitoring purposes. nasal oxygen supply is advisable. the insertion of an epidural intracranial pressure probe is essential for early identification of cerebral oedema. feeding: provided the patient does not have a paralytic ileus, enteral feeding via a nasogastral tube is advisable to prevent villous atrophy and thus reduce the risk of bacterial translocation. (s. p. 878) • parenteral feeding (1,600ϫ2,000 kcal/day) consists of a continuous intravenous supply of glucose and fat emulsions (mct). hypertriglyceridaemia may, in the case of lipid infusions, point to a lipid metabolism disorder, but it can also be due to increased glucose intake, which results in fatty degeneration of the hepatocytes and a corresponding reduction in liver function. fructose, sorbitol and xylitol must be avoided! the supply of either liveradapted amino acids or branched-chain amino acids is recommended for chronic liver insufficiency ϫ but not advisable in cases of acute liver insufficiency, because almost all amino acids are elevated in the serum in endogenous hepatic coma. a high daily dosage of watersoluble vitamins (possibly divided into two doses) is important. administration of zinc is recommended. electrolytes (na, k, ca, mg) and blood sugar must be carefully monitored, and any deviation from the norm should be corrected immediately. the risk of hypophosphataemia must be eliminated by early parenteral substitution. during refractory episodes, such as those involving the acid-base equilibrium and hyperhydration, haemodialysis is usually indicated. in hypoalbuminaemia, substitution with salt-free albumin is necessary. • with about 75% of patients, artificial respiration is called for, the aim being controlled hyperventilation. n-acetylcysteine is believed to promote the supply of oxygen to the tissues. (73) as a result, this substance, which is free from side effects, was also recommended for cases of ccl 4 intoxication (53) and is even considered helpful in acute liver failure with a different aetiology. h antagonists and omeprazole are recommended. • the timely and repeated administration of fresh plasma (ffb) as well as of antithrombin iii has proved to be the most effective measure for balancing plasmatic coagulation disorders. bacterial infections are extremely common as a result of serious impairment of the cellular and humoral resistance (ca. 80%). close-meshed bacteriological investigations are required in the frequent absence of clinical signs of infection. this leads to early antibiotic therapy based on an antibiotic sensitivity test. although an antibiotic prophylaxis is not actually recommended, it should nevertheless be considered in the individual case, since the spreading of an infection has a decidedly nega-tive impact on prognosis. • administration of selenite (i.v.) may be advisable. around 30% of patients develop a fungus infection, with a mortality rate of 50%. (s. p. 310) the administration of amphotericin b or fluconazol is an effective prophylactic measure. • bacterial or fungal infection can also be effectively suppressed by intestinal restimulating of the bacterial flora or intestinal sterilisation by means of neomycin (or paromomycin), a combination of nystatin and gentamicin, or lactulose. (51, 54) (s. pp 285, 288, 310) essential phospholipids (epl): in a pilot study, it was possible to achieve recompensation and lasting stabilization in nine out of ten patients suffering from severe liver insufficiency by i.v. administration of a new galenic form of polyenylphosphatidylcholine. (32) • this clinical result accords with other clinical studies and might be supported by the finding that a considerable deterioration in liver function was associated with a deficit of epl. (15) (s. p. 894) paracetamol intoxication: liver damage due to paracetamol (> 10 g) becomes manifest within ca. 48 hours after intake. (s. p. 382) for this reason, it is essential first of all to remove the non-absorbed fractions by gastric lavage and intestinal cleansing. as medicinal treatment, i.v. administration of the glutathione precursor nacetylcysteine is the therapy of choice (l. f. prescott et al., 1977) . dosage is 150 mg/kg bw with glucose as a rapid i.v. infusion (15ϫ20 minutes), followed by 50 mg/ kg bw over 4 hours and finally 100 mg/kg bw during the next 16 hours (ϭ about 300 mg/kg bw within 20 hours). this therapy has to be commenced as soon as possible (no later than 12ϫ15 hours after intoxication), even though a hepatoprotective effect can still be achieved up to 36 hours later. a serum concentration of < 200 μg/ml within 4 hours or < 60 μg/ml within 12 hours after intake can be considered prognostically favourable. (s. fig. 20 .3) there is no specific antidote for amanita toxins. given timely and appropriate therapy, morbidity and mortality are surprisingly low. • in cases of therapy failure or a critical course of disease, liver transplantation may be indicated. cerebral oedema: mannitol (0.5 g/kg bw or 100 ml, each as 20% solution) is used to treat the dreaded cerebral oedema. if renal function is sufficient, this course of therapy can be repeated every one to four hours, as required. serum osmolality should not exceed 320 mosm/l, and intracranial pressure should not go above 20 mmhg. when renal function is restricted, dehydration must be effected by haemofiltration. artificial respiration is required (often as peep ventilation). continuous monitoring of the intracranial pressure using an epidural intracerebral pressure probe is extremely helpful. frequently, there is increased susceptibility to cerebral convulsibility; therefore, phenytoin should be administered at an early stage. therapeutic application of thiopental (a. forbes et al., 1989) as i.v. solution (up to 150 mg/hour) calls for intracranial pressure probe monitoring. (22) other means of lowering the intracerebral pressure include the use of aminophylline, ranitidine, luxus oxygenation and semirecumbent positioning. (8, 17, 34, 65) a prophylactic reduction in pco 2 down to 25ϫ35 mm hg through hyperventilation can be advantageous in the initial stage of a brain oedema. (17, 65) moderate hypothermia (core temperature down to 32ϫ33°c, for 10ϫ12 hours) may be useful in reducing the intracerebral pressure and cerebral blood flow as well as the cerebral uptake of ammonia. (30) ornithine aspartate (40 g/8 hours as intravenous infusion) (52) and flumazenil are advisable for the treatment of hepatic precoma and coma. dopamine (2 to 4 μg/kg bw/hr) should be administered early on to stabilize the circulation and renal blood flow. • n-acetylcysteine can be applied during oxygenation due to its positive effect on stabilizing the blood circulation and improving the serum coagulation factors. • indomethacine reduces cerebral ammonia uptake. the positive results achieved by the application of pge 1 were reported in 1987 (m. abacassis et al.). according to a subsequent prospective study, 71% of patients with fulminant and subfulminant hepatitis survived. (56, 63) the effect is attributed to improved arterial flow and regeneration of the liver (0.1 to 0.6 μg/ kg bw/hr by means of perfusor for up to 18 hours, with the dosage gradually being phased out). lamivudine (100 mg/day) proved to be effective: it was possible to achieve a lasting improvement in liver function and to avoid liver transplantation. no side effects were observed. in view of the loss of complex biochemical liver functions, drug intervention in the metabolic processes of the liver should be as varied as possible ϫ even the use of therapeutic agents which are not clinically controlled may be biochemically or pharmacologically justified. the most important survival factor in acute liver failure is the patient's age. in the 15 to 25-year age group, 30ϫ50% of patients survive, whereas those older than 30 years have hardly any chance of survival. it would appear that the good regenerative ability of the liver in young people is the best guarantee for survival. • an attempt must be made at bridging the decompensatory phase by means of optimum intensive care and monitoring of the cerebral pressure as well as by applying clinically proven or indeed new therapeutic procedures or medication until the liver has adequately regenerated or until liver transplantation can be carried out. • basically, there are three techniques available for bridging the compensatory phase: (1.) extracorporeal systems (2 ( ( .) biosynthetic artificial livers or hybrid organs, and (3.) transplantation of hepatocytes. (86, 87, 97, 98, 108) • it has proved to be much more successful when the serum (ca. 3 l fresh frozen plasma/day) is infused into the femoral artery rather than into the vein. in 1974 patient plasma separated by plasmapheresis was for the first time passed through activated charcoal and artificial resin in order to absorb toxins. in this way, the patient's own purified plasma is reinfused together with the solid components of the blood. this procedure produces fewer side effects and is easy to carry out. 6 . total body wash-out: this technique is a modification of exchange transfusion. the circulatory system is washed out with electrolyte solutions and then refilled with donor blood whilst the patient is in a state of hypothermia (g. klebanoff et al., 1972). 7. haemodialysis: in 1968 temporary improvement could be achieved for the first time by means of haemodialysis in a patient presenting with fulminant hepatic failure (w. m. keynes). the procedure, however, is not generally recommended. it may be indicated in renal failure, acid-base disorders or with hyperhydration. following haemodialysis, substitution of reduced amino acids is necessary. this procedure turned out to be of more value than haemodialysis. no dialysate fluid is required. instead, a solution containing buffered bicarbonate is used to replace the ultrafiltrate. in fulminant hepatic failure, continuous venovenous haemofiltration is recommended because of its advantages for the circulation and metabolism. heparin or prostacyclin can be used as anticoagulants. 9. haemodiabsorption: the biologicdt system is a combination of haemodialysis and haemoadsorption (s.r. ash et al. 1992 ). (84) plasma separation was subsequently added to this system (s.r. ash et al., 1998) . (85) this newly developed biologicdtpf facilitates direct plasma contact with the haemodiadsorber. the system, which makes use of both a charcoal and a cation exchanger, dialyzes blood across a parallel plate dialyzer with a cellulose mem-brane. so far, results have been disappointing ϫ only lactate, creatinine and bilirubin were reduced. the aim of albumin dialysis is to remove both soluble metabolites and albumin-bound substances (abs) from the blood of patients with acute liver failure. (s. tab. 20.5) benzodiazepines fatty acids bile acids phenylalanin bilirubin several peptides carbon hybrids tryptophan copper etc. tab. 20.5: albumin-bound substances (abs) relevant in acute liver failure spad: single-pass albumin dialysis was the first method to be developed. the blood of the patient is extracorporeally dialyzed through an albumin-impermeable membrane against albumin in the secondary circuit. the loaded albumin is discarded. the spad method was further developed into a combination of dialysis, filtration and adsorption (ϭ molecular adsorbent recycling system). (105) . the patient's blood is fed through a hollow-fibre filter and dialyzed against an albumin dialysate. the abs (s. tab. 20.5) pass through the pores in the filter and become bonded. plasma proteins, hormones and vitamins are not lost. the albumin dialysate is recirculated in a closed circuit where it is fed through a second dialyzer and two adsorber columns which bind the abs. the albumin dialysate is returned to the hollow-fibre filter. it is dialyzed against a bicarbonate solution in order to remove the excess water and water-soluble substances (ammonia, creatinine, urea, iron, copper) as well as to stabilize the electrolyte and glucose levels and the ph value. the results obtained to date are promising. (95, 103, 106) fpsa: fractioned plasma separation and adsorption is a very efficient and multifactorial method, employing 390 membranes and adsorbants. (88) it is additionally characterized by the use of microparticles (2.0ϫ3.5 μm), which are recirculated in suspension using high-speed flow (2ϫ4 l/min) to optimize the in-line filtration process. in a further development, a special sulfone filter is applied. in the meantime, the prometheus method has been introduced. (99) here, the plasma is separated out by an albumin-permeable filter and cleaned in a secondary circuit via an adsorber together with conventional high-flux haemodialysis. direct contact between the albumin plasma and the adsorber helps to increase the efficiency of this method. these liver support methods serve to detoxify the organism for a limited period of time. they are regarded as supportive measures in intensive care. survival time has often been prolonged, yet only in isolated cases has the overall life-span of the patient been extended. these methods of treatment, which are costly and involve considerable resources, can only be carried out in medical units that are equipped with all the facilities of intensive care and thus in a position to effect epidural brain pressure measurement, blood purifying processes and liver perfusion methods. • only young patients between the ages of 15 and 25 have a real chance of survival (40 to 50%), provided they receive optimum intensive care. with patients over 30 years, supportive techniques should only be applied to bridge the time period until a liver transplantation can be carried out. however, conservative treatment may be attempted for four or five days under the following conditions irrespective of age: (1.) there is a chance of regeneration during this period that can be made use of; (2 ( ( .) this period of time does not preclude the patient's chances of liver transplantation (which calls for two to four days' preparation time); (3.) should there be no signs of recovery or regeneration, not even in younger patients (< 30 years), transplantation is nevertheless indicated. • after four or five days, however, severe complications develop, also in younger patients, which render transplantation difficult or even impossible. especially older patients (> 30 years) should undergo liver transplantation without delay. temporary substitution of the liver function using hepatocytes (e. g. in haemofiltration systems or bioreactors) is conceivable in acute liver failure, possibly in conjunction with activated charcoal filtration or with plasma separation. the importance lies in bridging the phase of acute liver failure until compensation of the liver function or liver regeneration is achieved. the bioreactor is filled with capillaries in which the patient's blood circulates; some of this blood has already been oxygenated extracorporeally. the efficacy of the system depends on an efficient exchange of the corresponding substances in both directions as well as stable hepatocyte functions. it is possible to use human (allogeneic) or animal (xenogeneic) hepatocytes as well as cell cultures (immortalized cells or tumour cell lines). if human cells are taken, 10 10 hepatocytes per patient are required ϫ as would be needed for a conventional liver transplant. regarding the use of animal hepatocytes, there is a possible risk in that no solution has yet been found to the question of zoonosis transmission and there may be an immune reaction to foreign antigens. bile flow also remains a problem. (92, 100, 101, 109, 110) 1. the binding of microsomal liver enzymes to synthetic carriers is a promising method of temporarily compensating important liver functions (g. brunner, 1981) r r . freshly isolated hepatocytes of pigs, immobilized on collagen-coated microcarriers, remained vital in-vivo and in-vitro over a longer period in a perfusion system; they were able to conjugate bilirubin and synthesize proteins. these results provided the basis for developing an extracorporeal bioartificial liver (a. a. demetriou et al., 1986) . in more advanced systems, plasma was perfused through an activated charcoal column and a fibre system with cultured pig liver cells. (92, 97, 100, 110) • using a bal, the plasma is separated by centrifugation and directed into a reservoir in order to increase both the plasma and metabolite flow. by integrating an activated charcoal column, it is possible to effect a greater elimination of toxins. the separated plasma reaches the hollow-fibre bioreactor, where it is perfused through the previously inserted hepatocytes (7 ± 1 hours). • such a system yielded increased production of coagulation factors in a patient with alcohol cirrhosis (d. f. neuzil et al., 1993). (s. fig. 20 .5) attention has recently focused on temporarily replacing the liver function with hepatocytes which have been cultured in the extracapillary space of a cellulose-acetate hollow-fibre unit. each unit contains ca. 200 g c3a cells, an amount which is necessary for successful perfusion. elad has proved efficacious in clinical use. (108) 5. blss: the bioartificial liver support system is made up of a blood pump, a heat exchanger to control the blood temperature, as well as an oxygenator and a bioreactor. the hollow-fibre bioreactor generally contains 70ϫ100 g of porcine liver cells. initial experience with blss is encouraging. (94) 391 6. bels: the berlin extracorporeal liver support system consists of a three-dimensional accumulation of approx. 500 g pig liver cells. these cells are linked by means of capillaries and provided with oxygen independently of the patient's blood, so that they function and stay vital for several weeks. (91) the modular extracorporeal liver support system was developed from bels. in contrast to bels, however, it consists of three modules: (1.) a cell module with human hepatocytes, (2 ( ( .) single-pass albumin dialysis, and (3.) a dialysis module for constant venovenous haemofiltration. (102) the clinical significance of bioartificial systems largely depends upon whether it is possible (1.) to keep functional hepatocytes alive in extracorporeal systems for an adequate period of time and (2 ( ( .) to make such systems available at short notice for use in emergencies. the idea of extracorporeal liver perfusion (eclp) for removing toxins by way of perfusion using an animal liver goes back to andrews (1953) (86, 107) although the procedure is relatively safe, the results obtained with perfused livers from humans or baboons would appear to be better than is the case with livers taken from pigs. (96) (121) . the transplanted split should be around 1% of the body weight of the recipient. slt has a higher complication rate than olt. ldlt: with regard to living donor liver transplantation, slt has become particularly important in cases where no cadaver organ is readily available. living donor liver transplantation was first carried out on children. the left lateral segment, usually segments ii and iii, of the donor's liver is used. around 5% of olt candidates are also suitable for ldlt. more than 2,500 living donor liver transplantations have been carried out worldwide. the donor mortality rate is 0.2ϫ0.3%. (118, 119, 125) in 1991 auxiliary partial orthotopic liver transplantation (apolt) was successfully carried out for the first time in acute liver failure, with the subsequent possibility of dispensing with the transplant after regeneration of the patient's own liver. (115) the corresponding part of a donor liver is transplanted orthotopically as left lateral segments ii and iii into the acutely diseased liver. the requisite partial resection of the liver is considered difficult. (124) a european multicentre study (12 centres) achieved equally good results in 30 patients compared to orthotopic liver transplantation with the removal of the native liver (m.-p. chenard-neu et al., 1996) . apolt is intended as a temporary measure in acute liver failure with the aim of discontinuing immunosuppressive therapy after the patient's own liver has regenerated. so far, results imply that more complications are experienced in apolt than in olt. the concept of heterotopic transplantation of a complete or even partial ("spliced") donor liver should also be pursued further. heterotopic transplantation involves placing an auxilliary (additional) organ in the right upper abdomen (o. t. terpstra et al., 1988). in surgical terms, this technique is considered to be demanding due to the application of the piggy-back method (ϭ anastomosis of the donor liver with the appropriately prepared ostium of the hepatic veins to the infrahepatic caval vein, generally cranial to the opening of the renal vein). • these two methods (apolt and auxiliary heterotopic liver transplantation) are particularly suitable for juveniles with acute liver failure because they bridge the critical time span preceding the regeneration of the diseased liver. immunosuppression is thus only required for a restricted period of time. the transplant shrinks or is surgically removed. acute liver failure induced by ecstasy was successfully overcome using this technique. (116) it allows the liver function to be compensated and gives the diseased liver time to regenerate. (120) pigs with human immune system genes are expected to facilitate the production of transgenic donor organs (d. white, 1992) . this is the basis of all endeavours to use transgenic pig liver for the purpose of xenotransplantation (j. platt, 1993). in the future, genetic engineering should make it possible to eliminate the immunobiological risk of complement-activated, hyperacute rejection. however, the problem regarding the transmission of zoonoses has not yet been resolved. to date, a survival period of 70 days has been achieved with three xenotransplants in alf and chronic liver insufficiency (j. fung et al., 1997). among the experimentally tested transplantation sites are the spleen, kidneys, lungs, pancreas, peritoneum, greater omentum and fatty tissue. up to now, the spleen has proved to be the most suitable site. the transplantation of foetal liver cells into the spleen may even culminate in a liver lobule-like formation with bile ducts and veins ϫ however, the functional results have (so far) been no better than with normal hepatocytes. the question of the required number of hepatocytes has still not been resolved: the collapse of a certain liver function (e. g. normalization of factor viii values in serum) can be compensated by a far lower number of hepatocytes than is the case with total liver failure (e. g. acute liver failure). calculations made up to now have claimed that there are at least 10 7 ϫ10 8 liver cells in partially resected liver parenchyma. indications for the transplantation of hepatocytes predominantly involve those liver diseases in which functional failures occur in the liver cells (not in the bile 393 ducts). • permanent transplantation would be indicated, for example, in order to eliminate congenital metabolic disorders of the liver cells. in this case, it is possible to use hepatocytes from the patient, with subsequent elimination of the defect by gene technology, as well as hepatocytes from healthy donors. a therapeutic effect 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acute liver failure liver transplantation for acute liver failure auxiliary partial orthotopic liver transplantation (apolt) for fulminant hepatic failure: first successful case report auxiliäre lebertransplantation bei akutem leberversagen nach einnahme von 3,4-methylendioxymetamphetamin prospects for xenotransplantation of the liver right-lobe live donor liver transplantation improves survival of patients with acute liver failure living-related liver transplantation for patients with fulminant and subfulminant hepatic failure heterotopic liver transplantation for fulminant hepatic failure: a bridge to recovery split liver/auxiliary liver transplantation for fulminant hepatic failure emergency liver transplantation for acute liver failure. evaluation of london and clichy criteria transplantation orthotopique du foie pour intoxication grave par amanite phalloide long-term followup of auxiliary orthotopic liver transplantation for the treatment of fulminant hepatic failure living donor liver transplantation for fulminant hepatic failure auxiliary versus orthotopic liver transplantation for acute liver failure liver transplantation for fulminant hepatic failure hepatocyte transplantation hepatocyte transplantation in acute liver failure human fetal hepatocyte transplantation in patients with fulminant hepatic failure hepatocyte transplantation in man transplantation of isolated hepatocytes. principles, mechanisms, animal models, clinical results alternatives to liver transplantation: from hepatocyte transplantation to tissue-engineered organs hepatocyte transplantation: a potential treatment for acute liver failure key: cord-339786-elrzlbsg authors: gurala, dhineshreddy; al moussawi, hassan; philipose, jobin; abergel, jeffrey r title: acute liver failure in a covid-19 patient without any preexisting liver disease date: 2020-08-26 journal: cureus doi: 10.7759/cureus.10045 sha: doc_id: 339786 cord_uid: elrzlbsg in december 2019, an outbreak of novel coronavirus started in wuhan, china, which gradually spread to the entire world. the world health organization (who) on february 11, 2020, officially announced the name for the disease as coronavirus disease 2019, abbreviated as covid-19. it is caused by severe respiratory distress syndrome coronavirus 2 (sars-cov-2). the who declared sars-cov-2 as a pandemic on march 11, 2020. sars-cov-2 mainly causes fever as well as respiratory symptoms such as cough and shortness of breath. gastrointestinal/hepatic sequelae such as diarrhea, nausea, vomiting, and elevated liver enzymes have been reported as well. studies and data so far on coronavirus infections from china, singapore, and other countries showed that liver enzymes elevation could be seen in 20-50% of cases. more severe disease can correlate with the worsening of liver enzymes. however, acute liver failure in patients with covid-19 has not been described. herein we report a case of acute liver failure in an elderly patient with covid-19 infection who did not have a history of preexisting liver disease. until recently, seven types of coronaviruses had been reported to cause infections in humans [1] . coronaviruses can use animal hosts and then can evolve to infect humans. this process is thought to explain the emergence of sars-cov (severe respiratory distress syndrome coronavirus) in 2003, mers-cov (middle eastern respiratory syndrome) in 2012, and sars-cov-2 in 2019. sars-cov-2 has 82% genome sequence similarity to sars-cov and 50% genome sequence homology to mers-cov. covid-19 symptoms range from mild (fever, cough, or dyspnea) to moderate (respiratory failure requiring oxygen support) and can progress to ards (acute respiratory distress syndrome) and multiorgan failure. in one of the earlier studies, 80% of cases were mild, but the mortality rate ranged from 1.86% to 9.86% [2] . higher mortality rates were reported in countries like italy, possibly secondary to resource depletion in an overwhelmed health care system. gastrointestinal symptoms such as diarrhea have been reported in approximately 2-10% of patients [3] , with a lower rate in china (3.7%) as compared to singapore (17%) [4] . liver injury has been reported in 60% of patients with sars-cov [5] and has also been reported in patients infected with mers-cov [6] . studies also suggest that sars-cov-2 can affect the liver [7] [8] [9] . in a recent study published in shanghai, 75 (50.7%) out of 148 patients were found to have elevated liver enzymes with sars-cov-2 [7] . in another study published in the lancet in february 2020 by huang et al., an increase in aspartate aminotransferase (ast) was observed in 62% in intensive care unit (icu) patients compared to 25% in non-icu patients, indicating that more severe disease correlates with worsening of liver enzymes [10] . several other studies showed that liver injury in the form of an increase in ast/alanine aminotransferase (alt) levels with a mild increase in bilirubin ranging from 14.8% to 53% [11] . in patients who died of sars-cov-2, liver injury was reported as high as 58.06% [12] . the highest levels recorded included an alt of 7,590 u/l and an ast of 1,445 u/l [13] . generally speaking, transaminase elevations are mild in patients with covid-19. here, we report a case of acute liver failure in an elderly patient with covid-19 infection who did not have a history of preexisting liver disease. an 80-year-old male with a medical history of diabetes, hypertension, dyslipidemia, asthma, coronary artery disease with bypass graft, atrial fibrillation on warfarin, and heart failure with preserved ejection fraction with an automatic implantable cardiac defibrillator and pacemaker presented to the emergency department (ed) in march 2020 with intermittent fever, productive cough, and shortness of breath (sob) for four to five days. he initially started noticing fever that was partially relieved by acetaminophen five days prior to presentation (maximum temperature of 102°f at home). this was associated with sob on exertion, which progressed to sob at rest and a productive cough. he denied any recent travel, contact with sick person, herbal medications use, or a recent change in home medications. his home medications included oral warfarin daily, oral metoprolol tartrate two times daily, oral metformin er daily, oral aspirin daily, oral atorvastatin, and budesonide-formoterol inhaler twice daily. the review of systems was otherwise negative. the patient did not have a history of smoking, alcohol consumption, illicit drugs, or high-risk sexual behavior. vitals at the time of admission showed a temperature of 101.6°f, heart rate of 80 beats/minute, blood pressure 140/70 of mm hg, respiratory rate of 20 breaths/minute, and oxygen saturation of 98% on room air. physical examination was positive for bilateral wheeze and rhonchi in all lung fields, 1+ pedal edema bilateral. his chest was without spider angiomas and abdomen with no hepatosplenomegaly, and he had no shifting dullness, with normoactive bowel sounds and no palmar erythema. on neurological examination, he was alert, oriented to time, place/person, followed commands, and had no focal deficits. laboratory examination results are shown in table 1 the patient had normal liver enzymes at presentation but had elevated transaminases on day 4. the examination at that time was negative for asterixis or encephalopathy. atorvastatin was stopped, and the recommendation was made to start n acetylcysteine (nac), and workup for acute and chronic liver disease was ordered. his respiratory status continued to deteriorate, requiring increased oxygen support. his radiologic findings worsened with enlarging infiltrates on a chest x-ray on day 4, as shown in figures 1-3 . the patient then developed cytokine release syndrome (crs) (elevated interleukin [il]-6 and il-10 as mentioned in table 5 ), and he expired on day 9. covid-19 is a pandemic illness that primarily affects the respiratory system with a wide spectrum of disease presentation that ranges from mild disease (fever, cough) to severe (ards, multiorgan failure). the gastrointestinal/hepatic systems are the next most commonly affected, with symptoms such as nausea, vomiting, diarrhea, and an increase in liver enzymes. currently, studies on the exact pathophysiology of liver injury in these patients are limited, but it is believed either to be a direct effect of the virus or immune-mediated inflammatory response, such as crs, hypoxemia, and failure of innate immune regulation, or to be drug-induced. 1) it is postulated that both sars-cov-2 and sars-cov bind to angiotensin-converting enzyme 2 (ace2) receptors to enter the target cell [14] where the virus replication begins and starts to infect cells of the upper respiratory tract. based on the scrna-seq data, chai et al. [15] found that ace 2 receptors also found in the hepatobiliary system (high in bile duct cells, cholangiocytes, when compared to liver cells). cholangiocytes play a critical role in liver regeneration and immune responses [16] . thus, the authors concluded that potential damage of cholangiocytes by 2019-ncov might lead to profound consequences in the liver rather than the direct effect of the virus on hepatocytes. 2) crs is a group of disorders caused by a wide variety of inflammatory etiologies, resulting in a profound increase in inflammatory markers such as il-2, il-7, il-6 granulocyte colonystimulating factor, interferon-γ inducible protein 10, monocyte chemo-attractant protein 1, macrophage, inflammatory protein 1-α, and tumor necrosis factor-α. this can ultimately lead to hemodynamic instability, multiorgan dysfunction, and death [17] . elevations in il-6, il-10, procalcitonin, and ferritin, as well as thrombocytopenia have been associated with severe covid and potentially severe liver injury as seen in our patient [18] . 3) ischemic hepatitis, also known as shock liver, is characterized by a significant increase in serum aminotransferases due to reduced oxygen delivery to the liver, usually seen in shock and thromboembolic disease [19] . 4) clinicians should also consider drug-induced liver injury due to hepatotoxicity associated with drugs used in treating covid such as lopinavir, ritonavir, and hydroxychloroquine that are recently approved by the fda for the treatment of covid. our patient, who had no previous history of liver disease and normal liver enzymes at presentation, developed elevated liver enzyme levels on day 4. initial differential diagnosis was broad, including ischemic hepatitis, drug-induced liver injury, viral hepatitis, cholestasis of sepsis, and autoimmune diseases. on laboratory workup, viral infections such as hepatitis (a, b, c), epstein-barr virus, cytomegalovirus, herpes simplex virus, hiv, autoimmune, and metabolic causes were ruled out. since his blood pressure was stable until day 8 of his hospitalization without any pressor support, ischemic hepatitis was unlikely [20] . tylenol toxicity was excluded (levels were less than 5 ug/ml). the remainder of the patient's medications were reviewed, and none of the patient's medications was likely to be the culprit. for example, the patient's home medication coumadin is a rare cause of acute liver injury and usually results in a cholestatic pattern rather than a hepatocellular one, which is what our patient demonstrated. aspirin has been associated with an increase in liver enzymes but usually only with dosages of more than 1,800 to 3,200 mg daily. metoprolol and metformin have been associated with only mild elevations in liver enzymes. hydroxychloroquine used to treat sars-cov-2 has been rarely associated with clinically apparent liver injury. a single case series (two cases) of acute liver failure attributed to hydroxychloroquine was published, but these patients took the medication for more than two weeks. in summary, we describe the first case of acute liver failure caused by the covid-19 infection. acute liver failure was diagnosed clinically by rising liver function tests and inr, as well as progressive encephalopathy. we could not conclusively prove that the covid-19 was the etiologic agent as the patient declined a liver biopsy. however, alternative causes of acute liver failure were effectively ruled out. bloodwork did not identify another etiology, and the patient's hypotension was too late in his course and too mild to cause ischemic hepatopathy. additionally, none of his medications was among the usual suspect for acute liver failure. as we learn more about this new infection, we expect to better understand the spectrum, pathophysiology, and treatment of the resultant liver injury. human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. sars-cov-2: an emerging coronavirus that causes a global threat clinical characteristics of 2019 novel coronavirus infection in china enteric involvement of coronaviruses: is faecal-oral transmission of epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore sars-associated viral hepatitis caused by a novel coronavirus: report of three cases histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study clinical features of covid-19-related liver damage epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study liver injury in covid-19: management and challenges clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020 liver injury during highly pathogenic human coronavirus infections clinical characteristics of 36 non-survivors with covid-19 in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the novel coronavirus 2019 (2019-ncov) uses the sars-1 coronavirus receptor2 ace2 and the cellular protease tmprss2 for entry into target cells specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection cholangiocyte pathobiology the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china thrombocytopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a meta-analysis hypoxic hepatitis: a review and clinical update athlete's hepatitis in a young healthy marathon runner key: cord-340710-dmow5p7k authors: lagana, stephen m.; kudose, satoru; iuga, alina c.; lee, michael j.; fazlollahi, ladan; remotti, helen e.; del portillo, armando; de michele, simona; de gonzalez, anne koehne; saqi, anjali; khairallah, pascale; chong, alexander m.; park, heekuk; uhlemann, anne-catrin; lefkowitch, jay h.; verna, elizabeth c. title: hepatic pathology in patients dying of covid-19: a series of 40 cases including clinical, histologic, and virologic data date: 2020-08-13 journal: mod pathol doi: 10.1038/s41379-020-00649-x sha: doc_id: 340710 cord_uid: dmow5p7k the novel coronavirus sars-cov-2 (coronavirus disease 19, or covid-19) primarily causes pulmonary injury, but has been implicated to cause hepatic injury, both by serum markers and histologic evaluation. the histologic pattern of injury has not been completely described. studies quantifying viral load in the liver are lacking. here we report the clinical and histologic findings related to the liver in 40 patients who died of complications of covid-19. a subset of liver tissue blocks were subjected to polymerase chain reaction (pcr) for viral ribonucleic acid (rna). peak levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were elevated; median alt peak 68 u/l (normal up to 46 u/l) and median ast peak 102 u/l (normal up to 37 u/l). macrovesicular steatosis was the most common finding, involving 30 patients (75%). mild lobular necroinflammation and portal inflammation were present in 20 cases each (50%). vascular pathology, including sinusoidal microthrombi, was infrequent, seen in six cases (15%). pcr of liver tissue was positive in 11 of 20 patients tested (55%). in conclusion, we found patients dying of covid-19 had biochemical evidence of hepatitis (of variable severity) and demonstrated histologic findings of macrovesicular steatosis and mild acute hepatitis (lobular necroinflammation) and mild portal inflammation. we also identified viral rna in a sizeable subset of liver tissue samples. the pandemic caused by the novel coronavirus sars-cov-2 (covid-19) has resulted in significant pulmonary morbidity and mortality, but has also raised many questions regarding involvement of other organ systems such as the liver. hepatologists and liver pathologists were therefore interested to find that researchers from china demonstrated liver function test (lft) abnormalities in~44% of covid-19 patients and that these abnormalities seemed to correlate with the severity of pulmonary disease [1, 2] . histologic reports from china were limited. a small case series reported the presence of fat in hepatocytes, with macrovesicular steatosis depicted in the accompanying photomicrograph (although described in the text as microvesicular fat) [3] . a more recent study performed core needle biopsies on the livers of four patients and reported nonspecific findings, attributed to preexisting disease or perimortem injury [4] . our group recently described moderate acute hepatitis in a liver allograft recipient with concomitant acute cellular rejection [5, 6] . nonetheless, the spectrum of histological liver injury in covid-19 remains unknown. a central question has been whether the virus specifically infects the liver, or is the liver injury entirely related to the "cytokine storm" that some patients exhibit, or is a combination of these factors present. the angiotensin converting enzyme receptor 2 (ace2) has been suggested to be the main mode of entry for the virus into the cell. it is found abundantly on cholangiocytes, but only rarely on hepatocytes; though it may be upregulated on hepatocytes at times of physiologic stress [7] . ace2 is also expressed on endothelial cells, such as those found in the portal vasculature [8] . following the sars-cov outbreak of 2002, virus was reported in liver and features of acute hepatitis (referred to as "bystander hepatitis") were described [9] . early in the course of the pandemic, new york city was the global epicenter for cases of, and deaths from, covid-19. our volume of autopsies increased sharply, and most of the cases were due to sequelae of covid-19. the lung findings predominated, as expected; however,~2/3 of covid-19 patients in our healthcare system had abnormal lfts [10] . therefore, we characterized the liver findings (clinically and pathologically) in 40 autopsy cases. we also investigated whether viral ribonucleic acid (rna) could be detected in liver tissue by polymerase chain reaction (pcr). following approval from the columbia university irving medical center institutional review board, we reviewed lung and liver sections from 40 consecutive autopsies from patients who died of illness related to covid-19. all liver slides were reviewed by an experienced liver pathologist (sml) and any findings (or potential findings), which are uncommonly identified in routine practice (e.g., phlebosclerosis), were reviewed amongst the liver pathology group for consensus. sections were typical autopsy sections (~2 cm × 2 cm) taken from representative appearing area(s), formalin fixed and paraffin embedded (ffpe). hematoxylin and eosin (h&e) was the only stain routinely employed, though if further stains were used for clinical purposes, they were reviewed as deemed relevant. pcr was performed on selected cases for which additional tissue blocks of liver were available. rna from samples was extracted using quick-rna ffpe miniprep (zymo research). quantitative rt-pcr was performed on extracted rna using taqman 4x master mix and sars-cov-2 primer/ probe sets (idt) to detect presence of virus per the cdc recommendations [11] . each assay included a standard curve to determine the viral load (log 10 copies/ml). demographic and clinical data were collected on all cases as available, specifically: age, gender, length of hospital stay, body mass index (bmi), hypertension, diabetes mellitus, chronic kidney disease, and chronic heart disease. in addition, charts were interrogated for evidence of known or suspected chronic liver disease, including viral hepatitis, alcohol-related liver disease, nonalcoholic fatty liver disease (nafld), autoimmune liver disease, transplantation status, and/or cirrhosis. laboratory values were also investigated (initial and peak), including aspartate aminotransferase (ast), alanine aminotransferase (alt), total bilirubin (tb), albumin (initial and nadir), creatinine, ferritin, ddimer, c-reactive protein (crp), and interleukin-6 (il-6). certain therapeutic interventions were investigated including administration of corticosteroids, hydroxychloroquine, and tocilizumab (il-6 inhibitor). continuous variables were expressed as median (25th-75th percentile). categorical variables were shown as fraction (percentage). clinical and pathologic features among those with and without detectable virus by pcr and among those with and without acute lung injury (ali) were compared using fisher's exact test for categorical variables and mann-whitney u test for continuous variables. in addition, differences in the type of therapeutic intervention and length of stay stratified by the distribution and extent of steatosis and lobular or portal inflammation were assessed using fisher's exact test or kruskal-wallis test as appropriate. patients with missing data were excluded from the analysis. all statistical analyses were performed using r (version 3.6.1). lung findings were simplified for the purposes of this report into "ali" and "no ali." ali comprised the histologic spectrum of diffuse alveolar damage (dad) with an acute (exudative) phase demonstrating hyaline membranes with or without an organizing (proliferative) phase exhibiting interstitial fibroblastic proliferation as well as a single case with predominantly fibrin, compatible with acute and fibrinous pneumonia. we sequentially examined the liver sections of the first 44 covid-19 autopsies at our institution; however, four were excluded for severe autolysis resulting in a cohort of 40 patients. the overall median (iqr) age was 70 (66-80) years and 29 (70%) were men. twenty-three patients were hispanic, five were african american, and two were caucasian (the remaining ten were unknown). seven of the patients had died on arrival, and had either no or limited clinical data. the median length of stay was 8.5 days. twenty-two patients (55%) received steroids during their admission, 19 (47.5%) received hydroxychloroquine, and six (15%) received tocilizumab (this cohort was encountered before remdesivir was widely used, and none of these patients received it). patient characteristics and known comorbidities are summarized in table 1 . there were two patients with evidence of chronic liver disease, one with alcohol-related cirrhosis and one with a history of liver transplant for autoimmune-related liver disease and acute cellular rejection at the time of admission. five patients had imaging evidence of nafld on admission. in addition, one patient with imaging evidence of steatosis also had an isolated anti-hepatitis b (hbv) core antibody positive with low-level hbv dna detected. initial and peak laboratory values including liver enzymes and inflammatory markers are displayed in table 2 . the median initial and peak ast and alt were 1-3 times the upper limit of normal, while median tb values were in the normal range. kidney dysfunction was common with a median peak creatinine of 2.64 mg/dl (upper limit of normal = 0.98 mg/dl for females and 1.30 mg/dl for males). median peak levels of inflammatory markers including crp (268 mg/l, upper limit of normal = 10 mg/l), ferritin (1810 ng/ml, upper limit of normal = 150 ng/ml for females, 400 ng/ml for males), d-dimer (9.6 μg/ml, upper limit of normal = 0.8 μg/ml), and il-6 (>315 pg/ml, upper limit of normal = 5 pg/ml) were all markedly elevated. there were no significant associations between laboratory values and any specific histological feature (data not shown). grossly, two livers showed fibrosis and one had abscesses, the remaining livers showed varying degrees of steatosis, congestion, and ischemia, but no other significant gross pathology. histologically, the most frequently encountered findings were macrovesicular steatosis, mild acute hepatitis, and minimal-to-mild portal inflammation. various less frequent findings were also observed. the findings are described below and major findings are summarized in table 3 . as the focus of this study is liver pathology, the lungs were considered only in the context of how the pulmonary findings may relate to liver injury. overall, 29 of 40 patients (73%) had evidence of ali histologically (the gross appearance of the lungs was not reviewed for this study). the patients with ali did not differ from those without with respect to any of the laboratory variables we cataloged, or to any histologic findings (data not shown). there was, however, a trend toward increased serum crp in the ali group (282 mg/l vs. 218 mg/l, p = 0.07). cardiac disease, n = 28 10/28 (36%) all statistics are presented as median (interquartile range) or n (%). bmi body mass index. a chronic liver disease n based on patients in whom we had preadmission data. all statistics are presented as median (interquartile range). alt alanine aminotransferase, ast aspartate aminotransferase, crp c-reactive protein, tb total bilirubin. a total of 20 cases (50%) showed features of acute hepatitis, defined as the presence of lobular necroinflammation, fig. 1b . these foci contained lymphocytes and rare histiocytes. plasma cells were rare. sixteen of these cases were of mild severity (80%) and four were of moderate severity (20%). no severe hepatitis (e.g., submassive necrosis, massive hepatic necrosis, bridging necrosis) was encountered. four patients without necroinflammation showed rare, individual apoptotic hepatocytes. given the focality of the finding (one or two such cells in one or two sections of liver), the significance of this observation is uncertain. six cases with lobular necroinflammation also demonstrated occasional individual apoptotic hepatocytes, fig. 1c . lobular mitoses were seen in three cases (8%). two of these cases also had acute hepatitis, whereas one occurred in an allograft which did not demonstrate active hepatitis. there were no associations between lobular necroinflammation and length of stay (p = 0.87), hydroxychloroquine treatment (p = 0.73), or tocilizumab administration (p = 0.17). twenty patients (50%) had portal inflammation. three cases had interface hepatitis (equivalent to batts-ludwig grade 2, fig. 1d) , whereas 17 had only minimally increased portal mononuclear cells (lymphocytes and few portal macrophages). one of the cases with interface occurred in an allograft with recent severe rejection with autoimmune features (acr-aih). eosinophils and neutrophils were rare, and not prominent in any case. an occasional plasma cell was present, but in none of the cases were they significant enough to invoke consideration of autoimmune hepatitis or drug induced liver injury with autoimmune features (dili-aih), with the exception of the acr-aih case. no case demonstrated fig. 1 common autopsy findings. a an example of marked steatosis involving all three zones (×100). this patient had a body mass index of 25. b a typical focus of lobular necroinflammation, comprised predominantly of lymphocytes with admixed apoptotic debris (×600). c a single apoptotic hepatocyte (arrow, ×600). panel d is taken from one of the rare cases with interface hepatitis (×200). all images depict hematoxylin and eosin stained slides. lymphoid aggregates. overall, 6 of the 20 patients did not have evidence of lobular/acute hepatitis. of these one was the acr-aih patient and two came into the emergency room in cardiopulmonary arrest, and without history in our system. there were no associations between portal inflammation and length of stay (p = 0.86), hydroxychloroquine treatment (p = 0.20), or tocilizumab administration (p = 0.10). in cases with acute, severe cardiac dysfunction areas of congestion (right heart failure) and ischemia (left heart failure) are common regardless of etiology. in our series, 32 (78%) patients exhibited congestion and 16 (40%) exhibited centrilobular ischemic necrosis. none of the cases had diffuse vascular pathology; however, focal findings were identified. phlebosclerosis, reminiscent of veno-occlusive disease (vod) was present in six cases, fig. 2a . in five cases, this occurred in a portal venule, but one case had involvement of the central vein. portal arterioles were abnormal in nine cases. three of these had arteriolar muscular hyperplasia, fig. 2b . each case with muscular hypertrophy of the portal arterioles also had venous phlebosclerosis. four cases had hyalinosis of the vessel wall (fig. 2b) . two cases had fibrinoid necrosis with endothelial apoptosis (fig. 2c) . sinusoidal microthrombi were present in six cases (fig. 2d) . c4d immunohistochemistry (ihc) was performed on ten cases and was negative (either entirely or almost entirely negative in all cases). three cases had granulomatous inflammation. one case had portal and lobular granulomas reminiscent of "fibrin ring" morphology, fig. 3a . this patient had been treated with a number of potentially hepatotoxic medications throughout the hospital course including hydroxychloroquine and tocilizumab for severe covid-19, as well as amiodarone for atrial fibrillation and ceftriaxone and piperacillin/tazobactam for infection. one patient had multiple necrotizing granulomas forming grossly appreciable abscesses and had structures suggestive of schistosoma eggs. the final case demonstrated portal nonnecrotizing granulomas in two portal tracts and resembled primary biliary cholangitis (pbc). however, the patient was male, had no known history of pbc, and had a normal alkaline phosphatase. two cases had pale ovoid sinusoidal inclusions, which at low power resembled apoptotic hepatocytes. upon close examination, it became evident that these inclusions were present in the sinusoidal spaces, fig. 3b . we stained these with cd61, which was positive (fig. 3b inset) , supporting that these aggregates were rich in platelets. we chose to refer to these structures as "thrombotic bodies." pcr was performed on 20 autopsy livers and was positive in 11 (55%) ranging from 10 copies to 9254 copies/μl rna. the longest interval between initial diagnosis by nasopharyngeal swab and death was 25 days and the median was 13 days. there was no relationship between time from initial diagnosis (positive nasopharyngeal swab) and likelihood of pcr positivity in the liver at autopsy (p = 0.51). we investigated whether pcr positivity was associated with any laboratory parameters (table 4 ). median peak ast was higher among patients with a positive pcr compared to negative (239 vs. 86 u/l, respectively) though this did not reach statistical significance (p = 0.063). in addition, peak ferritin (3623 vs. 1014 ng/ml, p = 0.048) and peak creatinine (4.50 vs. 2.02 mg/dl, p = 0.025) were significantly higher among pcr positive patients. there were no significant correlations between pcr positivity and any histologic finding including congestion (supplementary table 1 ). here, we present the hepatic findings in 40 autopsies of patients who died of complications of covid-19. excluding perimortem changes (congestion and ischemia) the main findings were hepatic steatosis (75%), mild acute hepatitis (50%), and portal inflammation (50%). we determined that 55% of patients who died of covid-19 had virus in the liver detected by pcr, albeit the majority at very low levels. alt alanine aminotransferase, ast aspartate aminotransferase, crp c-reactive protein, tb total bilirubin. *wilcoxon rank-sum tests were performed. macrovesicular steatosis was common (75%) of patients. this is consistent with other studies, for example, a study performed by a working group affiliated with the centers for disease control found steatosis in 50% of autopsy livers (4/8) [12] . a high proportion of our patients were diabetics, and that could account for some of the steatosis we encountered. however, the distribution of fat in our series was not typical of nafld. ten cases had only mild steatosis, but had appreciable periportal fat. only 2 of 32 patients in whom bmi was recorded had a bmi > 35 (median 26.5). a total of 2 of 40 patients had ballooning and mallory-denk bodies, indicative of alcoholic or nonalcoholic steatohepatitis. ischemia, viral hepatitis (e.g., hepatitis b, hepatitis c, human immunodeficiency virus), medications (corticosteroids), malnutrition, and various other acute insults are known to cause fatty liver [13] . steroid administration was frequent in our cohort, and this could lead to steatosis, though no direct association was seen in this cohort [14] . the facts that steatosis was not correlated to bmi or diabetes, and that there were many cases of "mismatched fat" are surprising if preexisting nafld explains the entirety of the histology. we did not have alcohol intake history for every patient, however alcoholic steatosis often demonstrates features of steatohepatitis with ballooning, neutrophilic satellitosis, and mallory-denk bodies. features of frank steatohepatitis were present in only two cases. therefore, it would seem likely that the steatosis in some of these cases developed during the course of their covid-19 illness. the etiology may be multifactorial with corticosteroid administration, hypoxia, malnutrition, and direct viral effects all plausible considerations (though neither corticosteroid use nor length of stay, a surrogate for malnutrition, was statistically associated with amount or distribution of steatosis). a series of over 300 covid-19 cases in china investigated the relationship between steatosis and neutrophil to lymphocyte ratio (nlr). a higher ratio suggests more severe systemic inflammation. this study showed that steatosis and nlr were associated with severity of illness, and also that the effects of steatosis and high nlr compounded to predict the most severe cases [15] . the exact mechanism/pathobiology of hepatic steatosis in covid-19 is worthy of further investigation. mild acute hepatitis, defined as lobular necroinflammation, was present in 50% of cases. this climbs to 60% if cases with no inflammation, but with focal apoptotic hepatocytes are included. in general, individual apoptotic hepatocytes are considered as evidence of acute hepatitis, but in this series, such cases showed such infrequent hepatocyte apoptosis that the significance is uncertain. the degree of necroinflammatory activity was generally mild, but was moderate in four cases. the necroinflammatory foci consisted of one to several dead and dying hepatocytes with few accompanying lymphocytes and histiocytes. plasma cells were only rarely encountered. the cohort of histologically defined acute hepatitis cases did not demonstrate higher serum ast/alt levels than the cohort without lobular necroinflammation. indeed, laboratory values were essentially identical in the histologic acute hepatitis and nonhepatitis groups. there was, however, a trend toward significance when we queried if those with pcr positivity were more likely to have evidence of acute hepatitis (44% of pcr negative patients vs. 64% of pcr positive patients did; p = 0.08). it will be interesting to investigate this variable in a larger number of cases. we have previously reported moderate acute hepatitis in a liver allograft recipient (distinct from the allograft recipient in this report) whose biopsy demonstrated large clusters of apoptotic hepatocytes with abundant lymphohistiocytic inflammation [5, 6] . none of the cases in this series were as profoundly affected as that case. the role of immunosuppression and allograft recipient status on liver injury associated with covid-19 will need further study. portal inflammation was generally minimal and was not suggestive of any specific etiology. this finding is similar to what was reported in a series of 12 autopsies in europe, which described "minimal lymphoplasmacellular portal infiltrate" [16] . approximately 70% of patients in our series with portal inflammation had mild acute (lobular) hepatitis. the few patients with portal inflammation, but without acute hepatitis, were a heterogenous group including an allograft recipient and several patients who arrested on arrival in the emergency room without available medical records, so it is difficult to interpret the significance of isolated portal inflammation. there was no evident bile duct injury, with the exception of the allograft with recent history of severe rejection. ace2 receptor is present on cholangiocytes, so we were certainly interested in identifying bile duct injury, but it was not observed [17] . cholestasis, however, was common (38%). four patients had ductular cholestasis, suggesting sepsis. lobular cholestasis without injury to the bile ducts is suggestive of a hepatocellular insult; e.g., at the bile canaliculus. it is, however, possible that the cholangiocytes are functionally impaired, but that the injury is not evident by h&e morphology. further investigation by electron microscopy or genomic/proteomic methods may prove enlightening. the potential role of endothelial injury in covid-19 continues to evolve. virus has been demonstrated in endothelial cells, and one paper reported lymphocytic endotheliitis in liver [8, 17] . in our series, however, few vascular abnormalities were histologically identified. a minority of our cases had a vascular abnormality (portal arteriole hyperplasia, vod-like phlebosclerosis, sinusoidal thrombi), and these changes were very focal and sparse in any one histologic section. in our patient cohort, therefore, vascular injury did not appear to be a major form of injury. while we cannot exclude these findings being meaningful (if they are present throughout the entire liver), we do not believe that vascular injury is the major mechanism of injury in the liver in covid-19 patients. this contrasts with a case series from italy, which describes vascular pathology not frequently encountered in our cohort [18] . the "thrombotic bodies" we observed are likely the same "microthrombi" identified in multiple organs in a recent series of seven autopsies [19] . this study includes the largest cohort of liver tissue subjected to pcr. the results are interesting for several reasons. at the most superficial level, it is valuable to know that slightly more than half of patients had detectable virus in liver. pcr positivity was not significantly associated with ast/alt elevations, though there was a trend for association between pcr positivity and higher peak ast (86 u/ l vs. 239 u/l, p = 0.063). as additional cases are subjected to pcr, further clarity may be obtained. there was a statistically significant elevation of ferritin and trends toward increased d-dimer and albumin nadir in the positive group. there was also statistically significantly increase in serum creatinine in the pcr positive cohort. the overall impression is that the pcr positive group had a slightly more severe inflammatory state. a detail worthy of further exploration is the variation of pcr positivity in liver. nine cases had low viral loads (and the possibility of serum positivity cannot be excluded), whereas two cases had high viral loads. time between initial diagnosis and death was not a factor associated with pcr positivity (p = 0.51). the peak creatinine elevation in the pcr positive group (4.50 vs. 2.02; p = 0.025) is a noteworthy finding, as it raises the question of whether some patients have a more disseminated phenotype and others perhaps a more anatomically (airway) restricted one. further studies will be useful to determine if this is true, and if so, if it is clinically relevant. this study has weaknesses, including that being an autopsy study, this paper describes patients with severe disease. as the epidemic evolves, it will be important to gather data on liver injury in patients with non-lethal covid-19. we did not investigate pathogenesis on the cellular level. we attempted to validate in-situ hybridization (ish) protocols and ihc for sars-cov-2, but nonspecific marking of lipofuscin, and reaction with endogenous alkaline phosphatase in liver tissue from non-covid-19 patients who died years ago was nearly ubiquitous (sometimes strong and diffuse). thus, for technical reasons, we cannot report these tissue preserving methods at present. in our clinical experience, as well as in the published literature, both ish and ihc can be used to detect virus in lung, particularly during the acute phase of dad, and occasionally in other organs, such as placenta [20, 21] . it will be important and interesting to attempt to identify virus in specific cells in the liver and to determine whether the ace2 receptor is upregulated in hepatocytes in patients with severe covid-19. nonetheless, based on the pattern of injury observed and the results of the pcr analysis, sars-cov-2 seems to involve the liver, and is associated with, possibly causal of, macrovesicular steatosis and acute hepatitis. here, we have provided a description of the histologic, clinical, and virologic characteristics related to the liver in patients who died of sequelae of covid-19. we have shown that patients dying of complications of covid-19 often have abnormal liver enzymes, steatosis, mild acute hepatitis, and viral rna in liver. while more work is needed to further elucidate the possibly multifactorial mechanisms of liver injury in patients with severe covid-19, given the lack of association between steatosis and known nafld risk factors, and between hepatitic findings and known drug administration, these data suggest virally mediated liver injury based on clinical and histologic observations. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study pattern of liver injury in adult patients with covid-19: a retrospective analysis of 105 patients pathological findings of covid-19 associated with acute respiratory distress syndrome pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies covid-19 associated hepatitis complicating recent living donor liver transplantation a case of an infant with sars-cov-2 hepatitis early after liver transplantation hepatic consequences of covid-19 infection. lapping or biting? endothelial cell infection and endotheliitis in covid-19 sars-associated viral hepatitis caused by a novel coronavirus: report of three cases acute liver injury in covid-19: prevalence and association with clinical outcomes in a large us cohort 2019-ncov) real-time rt-pcr diagnostic panel. cdc-006-00019, cdc/ddid/ncird/division of viral diseases pathology and pathogenesis of sars-cov-2 associated with fatal coronavirus disease, united states fatty liver and the forensic pathologist functional proteomic analysis of corticosteroid pharmacodynamics in rat liver: relationship to hepatic stress, signaling, energy regulation, and drug metabolism detrimental effects of metabolic dysfunction-associated fatty liver disease and increased neutrophil-to-lymphocyte ratio on severity of covid-19 postmortem examination of patients with covid-19 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis liver histopathology in severe covid 19 respiratory failure is suggestive of vascular alterations megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in covid-19: a case series in situ detection of sars-cov-2 in lungs and airways of patients with covid-19 detection of sars-cov-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents key: cord-029138-avfvpqs5 authors: croome, kristopher p.; taner, c. burcin title: the changing landscapes in dcd liver transplantation date: 2020-07-13 journal: curr transplant rep doi: 10.1007/s40472-020-00283-1 sha: doc_id: 29138 cord_uid: avfvpqs5 purpose of review: the transplant community continues to look for ways to help address the discordance between donor liver graft availability and patients on the liver transplant waiting list. donation after circulatory death (dcd) donor livers represents one potential means to help address this discordance. the present review describes the changing landscape of dcd liver transplantation (lt). recent findings: the number of dcd lts performed annually within the usa has continued to grow on an annual basis. importantly, national data has demonstrated that outcomes with dcd lt have been improving. this improvement has been driven by better understanding of how to successfully utilize these organs through better donor and recipient matching and careful evaluation of both hemodynamics during withdrawal of life support and the refinement of the procurement operation. summary: despite these improvements in outcome, ischemic cholangiopathy (ic) continues to be the achilles heel of dcd lt. emerging technologies such as various forms of machine perfusion may allow for reduction of complications and better prognostication of the risk associated with dcd liver grafts. the transplant community continues to look for ways to help address the discordance between donor liver graft availability and patients on the liver transplant waiting list. donation after circulatory death (dcd) donor livers represents one potential means to help address this discordance. initial reports examining the use of liver grafts from dcd described inferior longterm outcomes when compared with donation after brain death donors (dbd). these inferior results were ascribed to high rates of biliary complications, as well as increased rates of primary non-function and hepatic artery thrombosis [1] [2] [3] [4] . since those initial publications, there have been substantial developments in the understanding of how to effectively utilize dcd livers. more recent single center publications from high volume dcd programs have demonstrated equivalent outcomes between dcd and dbd liver transplantation (lt), with appropriate donor and recipient selection [5] [6] [7] . the present review describes the changing landscape of dcd lt. this article is part of the topical collection on liver transplantation prior to the development of the harvard criteria for brain death in 1968, all deceased organ donors were declared deceased using circulatory arrest criteria and thus represented the first dcd organ transplants performed [8] . following the acceptance of declaration of death according to neurologic criteria as a legal entity, most countries including the usa almost exclusively utilized dbd donors until the 1990s. resurgence of dcd lt was first described by groups from pittsburgh and wisconsin in the mid-1990s [9, 10] . excitement surrounded dcd lt resulted in rapid growth in the number of transplants performed from 1994 until 2007 (fig. 1 ). this initial excitement was tempered by multiple reports that described high rates of complications, specifically ischemic cholangiopathy (ic), resulting in graft failure or patient death following dcd lt [1] [2] [3] [4] . these results led to a contraction in the number of dcd lts performed from 2007 until 2012. as more data began to emerge demonstrating that acceptable results with dcd lt could be achieved with appropriate donor and recipient selection, the number of dcd lt performed annually has continued to grow from 2012 to 2018. in addition to increasing dcd lt volumes, a concomitant improvement in us national results with dcd lt has taken place. we previously demonstrated a sequential improvement in both graft and patient survival from 2003 to 2014 following dcd lt [11 •• ] (fig. 2) . moreover, changes in both recipient and donor characteristics consistent with published literature during the aforementioned time period were observed between eras. as with all innovations in transplant practice, there is undoubtedly a learning curve associated with the optimal utilization of liver grafts from dcd donors which has taken place as new data and analyses have become available. changes that were observed over the eras included: 1. decrease in proportion of dcd lt used for recipients: donor liver allocation in the usa has never been more contentious. following years of debate and legal challenges from all sides, a liver distribution system based on acuity circles went into effect on february 4, 2020 [12] . this acuity circle allocation system replaces donation service area (dsa) and regional boundaries previously used in liver organ distribution with a system based on distance between donor hospital and transplant hospital. this system will result in broader sharing of livers and substantially increase both the distances traveled and the costs for organ retrieval. it is also predicted that this system will reduce meld score disparity across the usa. in the newly adopted acuity system, livers from dcd donors are allocated earlier in the match sequence to transplant centers closer to the donor hospital compared with liver donor allocation for dbd donors < 70 years of age (fig. 3 the effects that allocation changes will have on dcd lts remain unknown. substantial variability in the utilization of dcd livers currently exists across the country [13 •• ] . this variability in dcd liver utilization has been shown to have no correlation with median meld score at transplant [13 •• ] . indeed, it is known that dcd utilization in the usa is driven by a few high-utilization centers [14] . likely, the new allocation changes will result in significant changes to behavior both from transplant centers as well as opos with regard to the pursuit and utilization of dcd livers. transplant center behavior has previously been shown to account at least partially for significant variability in median meld score at transplant both inter-and intra-regionally [15] . one question to be answered is whether transplant centers will be willing to travel substantial distances for dcd livers? a previous single-center study demonstrated that each successful dcd liver procurement required an average of 218 more miles of travel than each successful dbd liver procurement [16] . this difference was largely due to lower successful procurement proportion among dcd procurement episodes. only 49.7% of dcd liver offers resulted in a liver procured for transplant, compared with 92.3% of dbd. as a result, the authors of this paper suggested that current reimbursement policies poorly reflect increased surgeon travel (and time) expenditures between dcd and dbd liver offers. while several tools exist in order to predict the likelihood that a dcd donor will expire within 60 min, their predictive value is moderate at best, making the decisions on which dcd liver donors to pursue challenging [17] [18] [19] . historically, lt candidates with hcc have experienced a substantial advantage in deceased donor liver allocation with lower waitlist mortality/dropout within 1 year of listing compared with those candidates without a hcc diagnosis [20] . in addition to the recent implementation of the acuity circles system, deliberate steps have been taken to adjust hcc exception scores so that these transplant candidates are no longer so heavily advantaged. hcc patients no longer have a "ladder" model of increasing exception scores over time and instead are given an exception score of median meld at transplant minus 3 (mmat-3) for a 250 mile radius surrounding the listing center. undoubtedly this will result in decreased access to highquality dbd organs for patients with hcc. one potential option for this population may be increased utilization of dcd livers. this potential shift is congruent with the principle of using extended criteria organs in recipients with lower biological meld scores, such as many hcc patients, because of the perception that these recipients are better able to tolerate an extended criteria organ [21] . a previous publication demonstrated that after the implementation of the share 35 policy, more hcc patients have received livers from dcd donors [22] , potentially as the result of the highest quality organs being preferentially utilized by higher meld recipients with broader sharing. [12] wit and the importance of the procurement operation initial studies investigating lt using liver grafts from dcd donors linked prolonged donor warm ischemia time (dwit) to biliary complications and graft loss [3, 23, 24] . while there is a general consensus that prolonged dwit negatively effects outcome, there exists significant disagreement on what length of dwit is acceptable and even on how dwit is defined. as the collective experience with dcd lt increased, a concept of functional donor warm ischemic time (fdwit) arose from the notion that individual events during dcd procurement, such as variations in hemodynamics, mandatory wait period, time from incision to cannulation of the aorta and cross-clamp, all of which are included in total dwit, may have different impacts on the outcome of the liver graft [25, 26] . previous studies have defined the start of f-dwit based on different hemodynamic parameters (such as drop in mean arterial pressure (map) or sbp) or by a drop in oxygen saturation below a specific level. f-dwit terminates at the time of cold organ flush/cross-clamp. while the concept of f-dwit is ubiquitously accepted, no consensus on what parameters specifically define f-dwit exists [27] . previously published national and society guidelines for fdwit can be seen in table 1 [28] [29] [30] [31] . recently, a consensus conference on dcd lt transpired in venice, italy (january 30, 2020) in which attempts to more uniformly define and describe dwit and fdwit were made. the results of this consensus meeting have yet to be published. the importance of the procurement operation in order to achieve successful outcomes with dcd lt should not be understated. not only is it imperative to be able to quickly cannulate the donor vessels and begin cold perfusion of the liver, but judgement as to the timing and length of fdwit, flush quality, and overall liver appearance is paramount. judgement comes with experience and many of the high-volume transplant centers will only utilize dcd livers in cases where the procurement has been performed by a member (frequently an attending surgeon) of their team. this judgement on whether the liver is "usable" even more heavily relies on procuring surgeon experience when attempting to utilize extended criteria (ecd) dcd liver grafts. the fundamental techniques of the procurement operation have changed very little since the initial description of the "super rapid technique" [9] . many components of dwit are not modifiable since they occur prior to final pronouncement of donor death. alternatively, the time from final pronouncement of death until cold flush of the organs is largely within the procuring surgeon's control. the procuring surgeon should discuss the steps of the procedure with all assistants and operating room personnel prior to withdrawal of care. once the "no touch" time has transpired and the donor is declared deceased, efforts to cannulate and began cold flush in under 2 min should be made. donor hepatectomy time is the time from initiation of cold perfusion to the end of the hepatectomy and removal of the liver from the donor. as a general rule, extraction of the liver from the body of a dcd donor should be done as quickly and safely as possible. several recent reports have suggested that donor hepatectomy time (dht) may be associated with outcomes following dcd lt [32, 33] . in a study from the uk, dht > 60 min was associated with primary non-function (pnf) [32] . an abstract from the netherlands demonstrated that dht > 90 min was associated with both ic and early graft loss [34] .whether prolonged dht is itself a risk factor, or simply a proxy for donor surgeon inexperience, is unknown. it has previously been demonstrated that transplant center standardization of organ acceptance criteria (such as donor bmi, donor age, cit, dwit), procurement operation (such as acceptable f-dwit, asystole-to-cross-clamp time), and experienced procurement team help decrease complication rates related to dcd lt [26] . recipient selection is equally if not more important than donor selection in order to achieve optimal outcomes with dcd lt. previously published registry studies (both srtr and uk national registry) have demonstrated that the following recipient characteristics are associated with increased hazard of graft loss following dcd lt: recipient in the icu, recipient on ventilator, elevated meld score, advanced recipient age, recipient undergoing retransplantation [24, 35, 36••] . dcd liver grafts have by definition already undergone an ischemic insult through mandatory warm ischemia in the period between withdrawal of life support and cold perfusion in the donor. as such, the goal of the recipient implantation -functional dwit defined as spo 2 < 80% or map < 50 mmhg the spanish national transplant organization [31] -functional dwit defined as sbp < 60 mmhg -recommendation: functional dwit < 30 min procedure should be to minimize surgical complexity and recipient instability as much as possible to avoid a tenuous environment for an already marginal graft. in an effort to more objectively define recipient surgical complexity when using marginal grafts, the group from ochsner clinic developed an "abc" surgical risk score [37] . for this score, liver transplant candidates are assigned a surgical risk score of a (low), b (moderate), and c (high) at the time of pre-transplant surgical evaluation with re-review at the time of presentation of the patient to the transplant selection committee. at the time of organ offer, marginal liver grafts (including dcds) are primary utilized for a-b candidates with general avoidance of c candidates. at mayo clinic florida, we utilize a similar surgical complexity scoring system based on a 1-5-point scale and do not even list patients with a surgical score of 5 for dcd organs (tables 2 and 3 ). in selecting an appropriate recipient for a dcd liver graft, it is also important to choose a recipient that can tolerate early allograft dysfunction (ead). previous studies have demonstrated that ead has been shown to have a baseline rate of 23-27% in generalized cohorts of patients undergoing lt [38, 39] . with dcd lt, the rate of ead is significantly higher (34-40%) [40, 41] . recipients who have high meld scores and are in the icu, recipients with a significant cardiac history, or recipients with compromised renal function may not be ideal candidates since they may not be able to tolerate ead. some of the general principles to follow when selecting an appropriate dcd lt recipient can be seen in below: 1. minimize surgical complexity by avoiding: the appropriateness of utilizing dcd grafts for simultaneous liver kidney (slk) recipients has previously been debated. initial studies investigating the outcomes of simultaneous liver kidney (slk) transplant using grafts from dcd donors described inferior outcomes compared with those using grafts from dbd donors (dbd-slk) [42] [43] [44] . these studies demonstrated inferior patient survival as well as inferior liver and kidney graft survival with dcd-slk compared with dbd-slk transplant. more recently, a study investigating srtr data demonstrated that there has been a significant improvement in the results with dcd slks in the modern era [45] . in that study, patients undergoing dcd slk in the era 2011-2018 were able to achieve similar outcomes compared with matched patients undergoing dbd-slk transplant, with no differences seen in patient, liver graft, or kidney graft survival. it should be noted that donors used for dcd-slk were generally younger with relatively shorter dwit. in addition, a concomitant decrease in the proportion of patients in the icu prior to dcd-slk transplant was observed. it therefore seems that in select patients (not in the icu prior to transplant) requiring an slk, it may be appropriate to receive organs from younger dcd donors. another question that has frequently been raised is the appropriateness of dcd grafts for patients listed with hcc. with the changes to exception points for patients with hcc (mmat-3 with no escalading ladder), it may become increasingly difficult to transplant patients with hcc with standard liver grafts. as such, patients with hcc, who generally have preserved liver function, may represent ideal candidates for dcd grafts. it has previously been postulated that the rate of hcc recurrence could be elevated in patients receiving a dcd allograft [46] . this postulate was based on biologic plausibility rather than direct evidence, as non-transplant studies have suggested that ischemia reperfusion injury is associated with stimulation of growth in micromestastases and in increasing the adhesion of tumor cells [47, 48] . despite the biologically plausible mechanism, a large single-center study demonstrated no difference in the rate of recurrence of hcc between dcd and dbd lt (12.3% and 12.1% respectively) [49] . more importantly, if ischemia reperfusion injury in the dcd grafts was felt to be an important factor in recurrence, one would expect to see a higher proportion of initial site of recurrence to be the liver in the dcd lt recipients. in fact, in that study, the opposite was true as the liver graft represented the first site of recurrence in 65% of recipients in the dbd group and only 37% of recipients in the dcd group. specific donor-and-recipient risk factors have been demonstrated to significantly affect outcomes following dcd lt. as such, several donor and recipient risk matching scores have been developed. these risk scores may be particularly useful for centers that are in the process of expanding their utilization of dcd liver grafts. three different donor-recipient risk scores have been described [36, 50, 51] : the most recent of these scores, based on a large cohort of dcd lts, is the uk dcd-risk index (fig. 4) this score is based on 2 donor (donor age, donor bmi), 2 ischemic time (fdwit and cit), and 3 recipient (recipient age, recipient meld, and re-transplantation status) variables. with these variables, a score between 0 and 27 is generated. dcd lts in the low-risk group (score ≤ 5) had a 1-year graft survival rate of > 95%, compared with > 85% in the moderate risk group (score 6-10) and < 40% in the futile group (score > 10). the authors advocate avoidance of dcd lts for the futile group. as the collective experience with dcd lts has increased, several high-volume dcd centers have pursued ecd dcd lts with acceptable results. pursuit of these organs should be undertaken cautiously for centers with less experience with dcd lt or with less experience in the utilization of marginal grafts; however, in highly selected cases in experienced centers, they can be utilized with acceptable outcomes. recent publications have described the utilization of two categories of ecd dcd lts, namely elderly dcds and steatotic dcds. as the population in the usa continues to age, there will undoubtedly be an increase in the number of potential donors with advanced chronological age. multiple previous registry studies have demonstrated that advanced donor age dcds have a higher risk of graft failure [24, 35, 36••] . therefore, there is little disagreement that younger is "better"; however, old is not necessarily "bad." previously, a single-center study from the birmingham, uk group investigated the outcomes of dcd donor age > 60 years compared with those age ≤ 60 years and demonstrated no differences in graft or patient survival between the groups [52] . in this study, the rate of vascular, biliary, and overall complications was similar between the groups. more recently, our group published a multicenter study that found there was no difference in graft survival between a dcd donor age ≥ 50 group and a dcd donor age < 50 group [53] . while the rate of ic was not statistically significant different between the groups, there was a slight trend of increased ic in the dcd donor age ≥ 50 group (11.6%) compared with the dcd donor age < 50 group (7.6%). in that study, using cox regression analysis for national data, we evaluated predictors of graft failure in dcd donor age ≥ 50 years. significant predictors of graft failure included a calculated meld score ≥ 30, mechanical ventilation at the time of transplant, medical condition (in icu), and increasing cit. as has been mentioned above in the previous section, when utilizing elderly dcd donors, appropriate recipient selection is paramount. given the potentially additive risk from using donor livers that are both steatotic and from a dcd donor, there is a paucity of data on the outcome of dcd lt utilizing livers with macrosteatosis [54] . in a recent multicenter analysis, we investigated the outcome of utilizing dcd liver grafts with macrosteatosis [55] . in that analysis, we found that dcd donors with macrosteatosis < 30% had no increase in peri-operative complications and similar patient and graft survival compared with dcd donors with no steatosis. in contrast, dcd liver grafts with moderate macrosteatosis (30-60%) had higher rates of prs, pnf, post-reperfusion cardiac arrest, ead, and aki compared with dcd donors with no steatosis. the data on the utilization of dcd liver grafts with steatosis remains limited; therefore, extreme caution should be taken when utilizing these grafts, particularly when the degree of steatosis approaches 30% or greater. ischemic cholangiopathy (ic) represents the achilles heel of dcd lt. while severe cases of ic frequently require re-transplantation, ischemic biliary strictures can present with a spectrum of clinical and radiologic severity following lt [56] . several distinct radiologic patterns of ic have been described, which are associated with different clinical courses [57, 58] : recipients who develop diffuse necrosis rapidly develop abnormalities of nearly the entire biliary system, are identified soon after transplant, and almost uniformly require re-transplantation. patients with a bilateral multifocal pattern begin with mild to moderate stenosis of the second-order and peripheral ducts and progressively worsen over time. in the confluence dominant pattern, recipients develop strictures confined to the biliary confluence, with relative preservation of the second-order and peripheral ducts. in this pattern, biliary abnormalities progress in severity over time but geographically never expanded beyond the hilar confluence. many of these patients can be successfully managed long term with ercp and stenting and frequently do not go on to need re-transplantation. finally, in the minor form, patients may display mild radiologic abnormalities consistent with early ic, but never go on to develop more extensive strictures. while ic is observed as a complication of both dbd and dcd lt, initial series with dcd lt demonstrated ic rates as high as 30% with dcd lt compared with rates of 2-4% seen with dbd lts [1] [2] [3] [4] [5] 7] [1] [2] [3] [4] [5] 24] . more recent single-center series from north america providing era stratified data have described ic rates following dcd lt ranging from 2.6 to 5.3% (table 4 ) [5, 7, 59, 60] . these data suggest that with strict donor and recipient selection, rates of ic similar to that observed with dbd lt can be achieved. all dcd lt have some inherent risk of ic. this level of risk can be thought of as a "dial" that increases as one accepts riskier donor or recipient variables (fig. 5) . transplant programs must ask themselves what rate of ic is "acceptable" to them and this will guide which dcd organs they pursue for which recipients. this risk tolerance will influence what any transplant program deems an acceptable dcd donor and an acceptable dcd liver graft recipient. risk tolerance may vary substantially in different environments based on a multitude of factors. these could include donor availability, waitlist mortality, regulatory environment, cultural expectation, program experience, and ability to re-transplant a patient should significant ic develop. significant variability in acceptable dcd donor and recipient risk factors may exist between countries or even within a country based on some of the aforementioned factors. in addition, newer technologies such as machine perfusion may allow for modification or at minimum improved prediction of the level of risk associated with each dcd graft. during the past decade, there has been renewed interest in the use of machine perfusion instead of static cold storage (scs) fig. 4 the uk dcd-risk index [35] mayo clinic florida [5] 2010-2015 4 100 oschsner clinic [7] 2010-2015 3 100 university of toronto [58] 2009-2017 2.6 77 indiana university [59] 2011-2015 5. 3 38 as a preservation technique for donor livers. machine perfusion (mp) has several theoretical benefits including organ repair that may lead to improved organ quality, pretransplantation viability assessment of the donor organ, and extending the amount of time between organ recovery and lt. various strategies for the utilization of mp have been explored with a variety of temperature settings. mp is classified as hypothermic, subnormothermic, and normothermic, with the temperature maintained at 0-12°c, 25-34°c, and 35-38°c, respectively [61] . the more commonly described machine perfusion techniques as they relate to dcd lt are described below. normothermic regional perfusion (nrp) involves in situ perfusion of the subdiaphragmatic abdominal region after isolation from the remainder of the circulation. this in situ oxygenated perfusion occurs after declaration of circulatory death and prior to organ procurement, with the goal of resuscitating the organs and restoration of intracellular energy stores. nrp relies on cardiopulmonary bypass (cpb)/extracorporeal membrane oxygenator (echmo) technology to recover donor venous blood, deliver it to a membrane oxygenator, and then return the oxygenated blood to the subdiaphragmatic aorta. this form of mp has gained significant traction in europe, where in some countries, such as italy, the "no touch" time for dcd donors is 20 min following circulatory arrest. in the usa, nrp technology has been described for dcd donors by the university of michigan [62] . in their previous publication, they describe their experience with 21 dcd liver procurements of which 13 livers were ultimately transplanted. in that series, 1-and 2-year graft-survival rates were 85.7% and 71.4%, respectively, while ic and pnf were reported in one patient each. the majority of publications on nrp have originated from european series. a national study from spain compared n = 95 liver grafts from controlled dcd (cdcd) donors that underwent nrp with n = 117 non-nrp cdcd liver grafts [63] . the authors found that nrp was associated with a lower rate of ic (2% for nrp vs. 13% for non-nrp) and graft loss (12% for nrp vs. 24% for non-nrp). a study from the uk compared n = 43 liver grafts from dcd donors that underwent nrp with n = 187 non-nrp dcd liver grafts [64] . in that study, the use of nrp was associated with a reduction in ead (12% for nrp vs. 32% for non-nrp livers), 30-day graft loss (2% nrp livers vs. 12% non-nrp liver), and ic (0% for nrp vs. 27% for non-nrp livers). a study from italy investigated outcomes in n = 20 dcd donors that underwent nrp [65] . in italy, dcd donors experienced a prolonged dwit as a 20-min no-touch period is required following circulatory arrest. pnf was seen in 2 patients (10%), ic was seen in 2 patients (10%), and 1-year graft and patient survival were 85% and 95% respectively. normothermic mp is designed to maintain full physiological cellular metabolism by perfusing the liver with oxygenated blood-based solutions at body temperature. normothermic ex vivo machine perfusion (nmp) has the potential to restore normal metabolic physiology, recondition marginal livers, and allow for assessment of graft viability. [66] . in that study, the authors investigated 47 declined liver grafts (12 dbd, 35 dcd), of which 22 resulted in transplantation. the authors concluded that liver viability during nmp might be assessed using a combination of transaminase release, glucose metabolism, and lactate clearance, and the ability to maintain acidbase balance. a randomized clinical trial using the organox platform compared n = 121 grafts from donors that underwent nmp (dbd n = 87, dcd n = 34) with 101 scs livers used for lt [67] . in that study, the authors demonstrated a 50% lower level of hepatocellular enzyme release, a 50% lower rate of organ discard, and a reduction in post-reperfusion hypothermic machine perfusion (hmp) is designed to provide perfusion of the liver graft with perfusate temperature most commonly defined as 4°c. hmp has been well described in kidney transplantation, where large multicenter trials have demonstrated that hmp can improve early graft function and 1-year allograft survival [68, 69] . the first clinical series of liver hypothermic machine perfusion by guarrera et al. demonstrated post-lt shortened length of hospital stay, reduced peak serum ast levels, and an improved renal function, despite demonstrating no difference in primary nonfunction (pnf), ead, graft, and patient survival in a cohort of dbd lts [70] . whether hmp alone supplies sufficient oxygenation through oxygen dissolved in the perfusate under atmospheric pressure for a hypothermic liver with its lower metabolic rate has been questioned. in a 2014 study, hypothermic oxygenation machine perfusion (hope) was utilized for a cohort of dcd donors [71] . this study demonstrated that the outcome of dcd lt after hope conditioning was comparable with matched dbd liver grafts in terms of aspartate aminotransferase/alanine aminotransferase (ast/alt), intensive care unit admission, and hospital stay. more recently, the concept of dhope involving the hypothermic oxygenation through the hepatic artery in addition to the portal vein used in hope has been proposed. a recent case control study from the groningen group in 10 dcd lt patients found a higher graft survival and a lower peak in ast/alt and bilirubin in dhope treated liver grafts [72] . hmp devices currently in development include hypothermic lifeport liver transporter machine by organ recovery systems and the vitasmart hypothermic oxygenated machine perfusion platform by bridge to life. the landscape of dcd lt is changing on multiple fronts. these changes have been driven by the ongoing organ shortage and continued waitlist mortality in recipients awaiting lt. encouraging data has suggested that results with dcd lt have improved and that these liver grafts have been increasingly pursued by transplant centers in the modern era. while excellent results can be achieved with dcd lt, it should be stressed that dcd liver grafts should not be thought of as interchangeable with dbd liver grafts, despite highly selected single-center experiences suggesting that in ideal conditions equivalent results can be achieved. to utilize these grafts effectively, numerous aspects of the transplant process from procurement to recipient selection must be carefully controlled and standardized. while liver transplant professionals have made significant progress in the last decade, ic continues to be the achilles heel of dcd lt. in current practice, no matter how controlled the process or how experienced the transplant program, a certain percentage of patients will inevitably develop ic. when utilizing dcd liver grafts, it is imperative to understand the degree of risk associated with each individual organ offer and recipient situation and to balance this risk with acceptable rates of ic and outcomes based on the environment that transplant program is operating within. this level of risk may vary between countries, transplant centers, and even from recipient to recipient. as future technologies such as mp become increasingly available, the transplant community remains hopeful that the degree of risk associated with dcd lt will be able to be modified in the positive direction allowing for broadening of acceptable dcd donors. conflict of interest the authors declare that they have no conflict of interest. particular interest, published recently, have been highlighted as:•• of major importance donation after cardiac death: the university of wisconsin experience with liver transplantation the impact of ischemic cholangiopathy in liver transplantation using donors after cardiac death: the untold story liver transplantation using donation after cardiac death donors: long-term follow-up from a single center a comprehensive risk assessment of mortality following donation after cardiac death liver transplant -an analysis of the national registry comparison of longterm outcomes and quality of life in recipients of donation after cardiac death liver grafts with a propensity-matched cohort liver transplantation using grafts from donors after circulatory death: a propensity score-matched study from a single center safety and outcomes in 100 consecutive donation after circulatory death liver transplants using a protocol that includes thrombolytic therapy report of the ad hoc committee of the harvard medical school to examine the definition of brain death experience with liver and kidney allografts from non-heart-beating donors successful extrarenal transplantation from non-heart-beating donors improving national results in liver transplantation using grafts from donation after cardiac death donors new national liver and intestinal organ transplant system in effect feb noneligible donors as a strategy to decrease the organ shortage this article describes the wide varyance in dcd liver graft utilization and suggests potential reasons for why this variance in seen united states donation after circulatory death liver transplantation is driven by a few high-utilization transplant centers intraregional model for end-stage liver disease score variation in liver transplantation: disparity in our own backyard going the distance for procurement of donation after circulatory death livers for transplantation-does reimbursement reflect reality? report of a national conference on donation after cardiac death development of the university of wisconsin donation after cardiac death evaluation tool prediction of potential for organ donation after cardiac death in patients in neurocritical care state: a prospective observational study liver transplantation and waitlist mortality for hcc and non-hcc candidates following the 2015 hcc exception policy change the survival benefit of deceased donor liver transplantation as a function of candidate disease severity and donor quality effects of the share 35 rule on waitlist and liver transplantation outcomes for patients with hepatocellular carcinoma donor postextubation hypotension and age correlate with outcome after donation after cardiac death transplantation risk factors for graft survival after liver transplantation from donation after cardiac death donors: an analysis of optn/unos data asystole to cross-clamp period predicts development of biliary complications in liver transplantation using donation after cardiac death donors events in procurement as risk factors for ischemic cholangiopathy in liver transplantation using donation after cardiac death donors the super-rapid technique in maastricht category iii donors: has it developed enough for marginal liver grafts from donors after cardiac death? asts recommended practice guidelines for controlled donation after cardiac death organ procurement and transplantation transplantation from deceased donors after circulatory death: british transplant society guidelines eurotransplant manual -chapter 9 the donor protocolo nacional de donación y trasplante hepático en donación en asistolía controlada impact of donor hepatectomy time during organ procurement in donation after circulatory death liver transplantation: the united kingdom experience developing a donation after cardiac death risk index for adult and pediatric liver transplantation donation after cardiac death liver transplantation: predictors of outcome the uk dcd risk score: a new proposal to define futility in donation-after-circulatory-death liver transplantation pre liver transplant (lt) prediction of surgical complexity by recipient risk grading and optimizing outcomes: the ochsner experience validation of a current definition of early allograft dysfunction in liver transplant recipients and analysis of risk factors the impact of postreperfusion syndrome during liver transplantation using livers with significant macrosteatosis intraoperative events in liver transplantation using donation after cardiac death donors early allograft dysfunction in liver transplantation with donation after cardiac death donors results in inferior survival inferior long-term outcomes of liver-kidney transplantation using donation after cardiac death donors: singlecenter and organ procurement and transplantation network analyses the outcomes of simultaneous liver and kidney transplantation using donation after cardiac death organs simultaneous liver and kidney transplantation using donation after cardiac death donors: a brief report improved national results with simultaneous liver-kidney transplantation using donation after circulatory death donors inferior survival in liver transplant recipients with hepatocellular carcinoma receiving donation after cardiac death liver allografts ischemia/reperfusion accelerates the outgrowth of hepatic micrometastases in a highly standardized murine model hepatic ischemia-reperfusion promotes liver metastasis of colon cancer the use of donation after cardiac death allografts does not increase recurrence of hepatocellular carcinoma liver transplantation using organ donation after cardiac death: a clinical predictive index for graft failure-free survival developing a donation after cardiac death risk index for adult and pediatric liver transplantation impact of donor age in donation after cardiac death liver transplantation: is the cut-off "60" still of relevance? outcomes of donation after circulatory death liver grafts from donors 50 years or older: a multicenter analysis the "skinny" on assessment and utilization of steatotic liver grafts: a systematic review perioperative and longterm outcomes of utilizing donation after circulatory death liver grafts with macrosteatosis: a multicenter analysis waitlist outcomes for patients relisted following failed donation after cardiac death liver transplant: implications for awarding model for endstage liver disease exception scores radiologic characterization of ischemic cholangiopathy in donation-after-cardiac-death liver transplants and correlation with clinical outcomes classification and prognosis of intrahepatic biliary stricture after liver transplantation expanding the donor pool: donation after circulatory death and living liver donation do not compromise the results of liver transplantation expanding the donor pool with the use of extended criteria donation after circulatory death livers machine perfusion of donor livers for transplantation: a proposal for standardized nomenclature and reporting guidelines donation after circulatory determination of death: the university of michigan experience with extracorporeal support normothermic regional perfusion vs. super-rapid recovery in controlled donation after circulatory death liver transplantation in situ normothermic perfusion of livers in controlled circulatory death donation may prevent ischemic cholangiopathy and improve graft survival liver grafts from donors after circulatory death on regional perfusion with extended warm ischemia compared with donors after brain death observations on the ex situ perfusion of livers for transplantation consortium for organ preservation in europe. a randomized trial of normothermic preservation in liver transplantation machine perfusion or cold storage in deceased-donor kidney transplantation pulsatile perfusion reduces the incidence of delayed graft function in expanded criteria donor kidney transplantation hypothermic machine preservation in human liver transplantation: the first clinical series hope for human liver grafts obtained from donors after cardiac death dual hypothermic oxygenated machine perfusion in liver transplants donated after circulatory death publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-340576-dabcs3w5 authors: nishikawa, hiroki; enomoto, hirayuki; nishiguchi, shuhei; iijima, hiroko title: liver cirrhosis and sarcopenia from the viewpoint of dysbiosis date: 2020-07-24 journal: int j mol sci doi: 10.3390/ijms21155254 sha: doc_id: 340576 cord_uid: dabcs3w5 sarcopenia in patients with liver cirrhosis (lc) has been attracting much attention these days because of the close linkage to adverse outcomes. lc can be related to secondary sarcopenia due to protein metabolic disorders and energy metabolic disorders. lc is associated with profound alterations in gut microbiota and injuries at the different levels of defensive mechanisms of the intestinal barrier. dysbiosis refers to a state in which the diversity of gut microbiota is decreased by decreasing the bacterial species and the number of bacteria that compose the gut microbiota. the severe disturbance of intestinal barrier in lc can result in dysbiosis, several bacterial infections, lc-related complications, and sarcopenia. here in this review, we will summarize the current knowledge of the relationship between sarcopenia and dysbiosis in patients with lc. in the gastrointestinal mucosa, various immune cells including macrophages, dendritic cells, etc., are constantly present [1, 2] . paneth cells, which are a type of intestinal epithelial cell, secrete antimicrobial peptides and are responsible for intestinal innate immunity by eliminating pathogens and by symbiosis with resident bacteria [3] . however, the barrier mechanism formed in the mucus layer may be incomplete, and indigestible proteins, bacteria, viruses, etc., along with nutrient components, can always enter the tissue across the mucus barrier [1, 2] . in that case, a secondary barrier consisting of macrophages, t cells, and b cells present in the lamina propria will respond to their invasion (the biological barrier, table 1 and figure 1 ). the foreign substances passing through the lamina propria can enter the bloodstream and reach the liver via the portal vein. a large number of kupffer cells (liver macrophages) are present in the sinusoidal blood vessels of the liver (the final barrier) to create a unique immune system [4] . in this way, the gastrointestinal tract and liver cooperate to participate in biological defense (gut-liver axis) [4, 5] . on the other hand, it has been revealed that various intestinal bacteria inhabit the colon, and they play an important role in maintaining homeostasis of the human body (the environmental barrier, table 1 and figure 1 ) [6] . the majority of bacteria in the colon are tightly attached to the outer side of the mucus layer, and the inner side of the mucus layer forms a barrier which limits bacterial contact with the epithelium (the physical barrier, table 1 and figure 1 ) [7] . dysbiosis refers to a state in which the diversity of gut microbiota (gm) is decreased by decreasing the bacterial species and the number of bacteria that compose the gm [8] [9] [10] [11] . analysis of gm at the gene level using a next-generation sequencer has come to the fore, and thus gm in patients with various table 1 . three types of intestinal barrier. gut microbiota antimicrobial peptide immune cells mucus layer tight junction in individuals with chronic liver diseases (clds), metabolic or nutritional dysfunctions including protein-energy malnutrition (pem) or muscle abnormalities are frequently found, which can be related to disabilities, poor quality of life, or mortality [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . liver cirrhosis (lc) involves a hypermetabolic state with increasing demand for calories and protein [23] . in addition, the energy metabolism of lc patients is in a hypercatabolic state, and when fasting early in the morning, they are in the same degree of starvation as when a healthy person fasts for 2-3 days [31, 32] . when liver function worsens, the detoxification of harmful substances such as ammonia can be reduced [32] . branched-chain amino acids (bcaas) are often excessively consumed in skeletal muscles to detoxify harmful substances in patients with decreased liver function [20, 32] . in lc patients, it is difficult to adequately supplement bcaas with diet intake alone [32] . in lc patients, sarcopenia, which is defined by decline in muscle mass and strength and/or physical activity, can occur because the excessive consumption of bcaas makes it difficult to synthesize the protein required for muscle mass increase [20] . sarcopenia is one of the most common consequences seen in patients with lc [20, 27, [33] [34] [35] [36] [37] [38] [39] . in japan's aging population, cld is also a crucial public health issue because aging is also closely linked to sarcopenia [40] [41] [42] . how sarcopenia is related to adverse consequences requires looking at sarcopenia as a systemic disorder [22, 43, 44] . lc-related complications themselves such as hepatocellular carcinoma (hcc), ascites, spontaneous bacterial peritonitis (sbp), varices, hepatic encephalopathy (he), and acute or chronic liver failure (aclf) can cause sarcopenia [22, 40] . clinical and research interest in sarcopenia in clds has thus been growing internationally. in 2016, the japanese society of hepatology (jsh) created their own criteria for the assessment of sarcopenia in in individuals with chronic liver diseases (clds), metabolic or nutritional dysfunctions including protein-energy malnutrition (pem) or muscle abnormalities are frequently found, which can be related to disabilities, poor quality of life, or mortality [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . liver cirrhosis (lc) involves a hypermetabolic state with increasing demand for calories and protein [23] . in addition, the energy metabolism of lc patients is in a hypercatabolic state, and when fasting early in the morning, they are in the same degree of starvation as when a healthy person fasts for 2-3 days [31, 32] . when liver function worsens, the detoxification of harmful substances such as ammonia can be reduced [32] . branched-chain amino acids (bcaas) are often excessively consumed in skeletal muscles to detoxify harmful substances in patients with decreased liver function [20, 32] . in lc patients, it is difficult to adequately supplement bcaas with diet intake alone [32] . in lc patients, sarcopenia, which is defined by decline in muscle mass and strength and/or physical activity, can occur because the excessive consumption of bcaas makes it difficult to synthesize the protein required for muscle mass increase [20] . sarcopenia is one of the most common consequences seen in patients with lc [20, 27, [33] [34] [35] [36] [37] [38] [39] . in japan's aging population, cld is also a crucial public health issue because aging is also closely linked to sarcopenia [40] [41] [42] . how sarcopenia is related to adverse consequences requires looking at sarcopenia as a systemic disorder [22, 43, 44] . lc-related complications themselves such as hepatocellular carcinoma (hcc), ascites, spontaneous bacterial peritonitis (sbp), varices, hepatic encephalopathy (he), and acute or chronic liver failure (aclf) can cause sarcopenia [22, 40] . clinical and research interest in sarcopenia in clds has thus been growing internationally. in 2016, the japanese society of hepatology (jsh) created their own criteria for the assessment of sarcopenia in clds [40] . in the jsh criteria for sarcopenia, age limitation is excluded because clds can cause secondary sarcopenia due to pem, which can occur regardless of age. in addition, the measurement of walking speed was abolished due to the difficulty of measuring it in daily clinical practice, and it was decided to use only grip strength for the evaluation of muscle strength (cutoff values: <26 kg in males and <18 kg in females). furthermore, since computed tomography (ct) is frequently used in cld patients, a standard value for ct was set for measuring muscle mass, and it was decided to use the bioimpedance analysis method (cutoff values: <7.0 kg/m 2 in males and <5.7 kg/m 2 in females) and/or ct method at the l3 level (cutoff values: <42 cm 2 /m 2 in males and <38 cm 2 /m 2 in females) to evaluate muscle mass [40] . in japan, a lot of debate regarding sarcopenia in clds has taken place based on the jsh criteria. here in this review, we will summarize the current knowledge of the relationship between dysbiosis and sarcopenia in patients with lc. lc patients with hyperammonemia are often encountered in routine clinical practice. most of the ammonia produced in vivo is derived from the digestive tract. the organs that metabolize ammonia include the liver, skeletal muscles, brain, and kidneys. of these, the only organ with sufficient capacity to detoxify ammonia produced in the body into urea is the liver, which has a urea cycle [45] . lc patients manifest the characteristics of low levels of bcaas due to pem and elevated blood ammonia level due to an impaired urea cycle caused by zinc deficiency, etc. [46] . ammonia suppresses phosphorylation of eukaryotic initiation factor 2α and mammalian target of rapamycin complex1 (mtorc1) signal through general control nonderepressible 2 which is an amino acid deficiency sensor, and directly suppresses protein synthesis in skeletal muscles. bcaa suppresses these reactions and promotes muscle protein synthesis, but l-leucine (one of bcaas) is consumed due to ammonia metabolism in skeletal muscles [47] . when the l-leucine level is decreased, protein synthesis in skeletal muscles becomes unsuccessful [48] . in lc patients, muscle proteolysis is stimulated via the activation of the ubiquitin-proteasome pathway [49, 50] . persistent chronic inflammation in lc can cause the marked elevation of the pro-inflammatory cytokines including tumor necrosis factor-alpha (tnf-α) and il-1, -6, which in turn stimulate muscle autophagy [51] . the inflammation-inducible ubiquitin-proteasome system can be linked to muscle atrophy through activation of muscle atrophy-related genes [52] . myostatin suppresses muscle satellite cell proliferation and differentiation. elevated myostatin levels in skeletal muscles can cause sarcopenia in lc patients [53] . hyperammonemia has been demonstrated to elevate muscle myostatin expression via tlr-independent nuclear factor kappa beta activation in an animal model [54] . as serum and skeletal muscle ammonia levels are often elevated in lc because of portosystemic shunts or impaired ureagenesis, significant increase of myostatin expression in skeletal muscles can be observed [55] . the decrease in serum free testosterone levels, bcaa, and insulin-like growth factor-1 levels also result in elevated myostatin levels in lc [56, 57] . lc patients are often involved in gonadal dysfunction, which can also result in hypermyostatinemia in skeletal muscles [57] . in our previous report, we demonstrated that elevated serum myostatin level can be associated with hyperammonemia (correlation coefficients; r = 0.5856 in males and r = 0.3922 in females), hypoalbuminemia (correlation coefficients; r = −0.3844 in males and r = −0.3945 in females), and poor outcomes [58] . in addition, we found a close inverse correlation between serum myostatin level and psoas muscle mass as assessed by ct at the l3 level in lc patients (median psoas muscle index (high vs. low serum myostatin group); 4.84 cm 2 /m 2 vs. 6.37 cm 2 /m 2 (p < 0.0001) in males and 3.87 cm 2 /m 2 vs. 4.25 cm 2 /m 2 (p = 0.0175) in females) [58] . sarcopenia in lc patients can contribute to increase risk of minimal or overt he. hanai et al. reported that in their 120 lc patients sarcopenia (hazard ratio (hr) = 3.31, 95% confidence interval (ci) = 1.19-9.42; p = 0.02) and serum bcaa levels <327 nmol/ml (hr = 2.98, 95% ci = 1.08-8.34) were found to be independent adverse predictive factors for the incidence of minimal he [59] . a recent meta-analysis (6 studies, comprising 1795 patients) reported that sarcopenia was closely linked to the presence of he (hr = 2.74, 95% ci = 1.87-4.01) [60] . on the other hand, chronic use of proton pump inhibitors (ppis) can alter the gm and can be a risk factor for he in lc patients [61, 62] . skeletal muscle is considered to be a metabolic organ, which consumes a lot of energy and plays an important role in supporting exercise capacity and regulating body fat mass and blood glucose levels. skeletal muscles take up glucose and control blood glucose levels and play an important role in storing glucose as glycogen [63, 64] . skeletal muscle has also been found to have a role as an endocrine organ secreting myokine (physiologically active substance) and regulating organ function throughout the body [65, 66] . skeletal muscle decline can be associated with insulin resistance and disturbance of gm (muscle-gut axis) [66, 67] . microorganisms in the gut exert their functions mainly through enzyme pathways for the purpose of digesting complex dietary carbohydrates and proteins [68, 69] . gm provides the bcaas including valine, leucine, and isoleucine, and particularly glycine, which is necessary for the synthesis of glutathione. glutathione has an auxiliary role in protecting cells from reactive oxygen species such as free radicals and peroxides [68, 69] . intake of a high fat diet can cause dysbiosis, which may be linked to the development of colorectal cancer (crc) [70] . microbial metabolites from the intestines have been demonstrated to act as nutrients or metabolic modulators in skeletal muscles [71] . short-chain fatty acids (scfas) include organic acids such as butyrate (4 carbon atoms), propionate (3 carbon atoms), and acetate (2 carbon atoms), which are produced by the gm. acetate and propionate pass into the bloodstream and are taken up by the peripheral organs and the liver, where they can act as substrates for gluconeogenesis and lipogenesis [69, 72] . butyrate has a role in providing energy for cell metabolism and regulates apoptosis, cell differentiation, and chemical modification of nuclear proteins and nucleic acid through action on numerous cells [72, 73] . butyrate is the most important energy source for intestinal epithelial cells and exerts excellent physiological effects such as an anti-inflammatory action. butyrate has been reported to have a significant effect on skeletal muscles [74, 75] . butyrate can help prevent skeletal muscle mass loss and maintain skeletal muscle mass via anti-inflammatory effects and activation of regulatory pathways, leading to atp increase and suppression of muscle protein catabolism and apoptosis [73, 76] . the relationship between gm and lipid metabolism in clds depends on the degree of liver damage [77] . a negative correlation between hepatic venous pressure gradient (hvpg) and butyrate levels in lc patients can be found [78] . acetate has also been reported to have the following effects: (1) decrease in intestinal ph, (2) suppression of ammonia-producing bacterial growth, and (3) suppression of absorption of intestinal ammonia [79, 80] . these observations may account for the high prevalence of sarcopenia in lc patients. the frequency of sarcopenia in lc patients is reported to be 10-70% [40] . the function of gm and its associated immune regulation mechanism have been elucidated, and it has been revealed that the intestine, which acts as a barrier at the forefront of the living body, affects the whole body by controlling substance permeation from the gastrointestinal tract into the systemic circulation [71] . it has been revealed that the tight junction (tj) between intestinal epithelial cells regulates the immune response to invasion of bacteria and foreign antigens from the intestinal tract in cooperation with intestinal-associated lymphoid tissue and the intestinal neuroendocrine network (the physical barrier, table 1 and figure 1 ) [81] [82] [83] . the major role of epithelial cells in contact with the outside is to protect the host from foreign antigens by forming epithelial cell sheets (tightly adhering cells) with tjs [84] . furthermore, it has also been revealed that the gm is deeply involved in the development and maintenance of the intestinal epithelial barrier [81] [82] [83] . tj was identified as a 47kda protein and named zonulin. zonulin enhances intestinal permeability, is involved in innate immunity, and is strongly suggested to be associated with the development of autoimmune diseases such as type 1 diabetes [81] [82] [83] . cumulative evidence has highlighted the relevance of increase in intestinal permeability (i.e., leaky gut syndrome) and consequent bacterial translocation in the development of clds. particularly, in recent hypotheses regarding patients with non-alcoholic fatty liver disease (nafld), intestinal permeability impairment, dietary habits, and gut dysbiosis are considered to be the main pathogenic triggers [85] [86] [87] . leaky gut is associated with chronic inflammation [87] . in advanced liver diseases, intestinal permeability can be enhanced by portal hypertension, which consequently leads to increased bacterial translocation that further damages liver function. furthermore, these pathogenic mechanisms are implicated in most lc-related complications, such as sbp, hepatorenal syndrome, severe ascites, he, sarcopenia, and hcc [85] [86] [87] . in lc rats, intestinal bacteria such as gram-negative bacilli in mesenteric lymph nodes were more likely to be detected compared with control, and the same strain of bacteria was detected in ascites [88, 89] . therefore, bacterial translocation is considered to be an etiology of the early stage of sbp. sarcopenia could worsen as liver disease progresses. hanai et al. reported that in patients with child-pugh class a, b, and c, the rate of decrease of skeletal muscle per year was 1.3%, 3.5%, and 6.1% [90] . cirrhosis to dysbiosis ratio (cdr, described later) decreases with the worsening of liver function [91, 92] . considering this evidence, the severity of sarcopenia in lc can be closely associated with the severity of dysbiosis. dysbiosis in lc can cause: (1) decreased bacterial diversity [91] , (2) decreased scfa (energy source in human body) production [93] , (3) collapse of tj and subsequent increased intestinal permeability (leaky gut syndrome) [94] , (4) antioxidant dysfunction [95] , and (5) endotoxemia [96, 97] . these can be associated with anabolic resistance, chronic inflammation, mitochondrial dysfunction, oxidative stress, and insulin resistance, which can lead to lc progression and subsequent development of sarcopenia in lc patients [25] . the ratio of beneficial to potentially harmful bacterial taxa, or the cdr (autochthonous to non-autochthonous taxa ratio), has been proposed as an index of alterations in the gm [92] . examples of benign and autochthonous gut taxa include lachnospiraceae, ruminococcaceae, veillonellaceae, and clostridiales incertae sedis xiv, while pathogenic gut taxa include enterobacteriaceae and bacteroidaceae [92] . a low cdr may suggest a decrease in beneficial bacteria and/or an excessive abundance of harmful taxa. altered bacterial function has also been demonstrated in lc patients compared with healthy controls [2, 12] . in other words, a deficit of autochthonous non-pathogenic bacteria and an excessive growth of potentially pathogenic bacteria are common characteristics in lc patients [2, 12, 92] . progressive alterations in the gm were found in worsening lc, such that the cdr was significantly decreased with liver disease progression [45] . in contrast, cdr and the gm were unchanged in patients without disease progression (i.e., stable liver disease) [92] . bajaj et al. demonstrated that in hospitalized patients with lc (n = 180), dysbiosis of the gm as assessed by cdr, etc., on admission can be associated with elevated risk of extra-hepatic organ failure, aclf, and mortality, independent of baseline clinical characteristics [98] . another study reported a significant fungal dysbiosis in lc patients [99] . in their results, the gm in lc patients altered differentially with antibiotics and ppi use, and stool bacterial/fungal profiles predicted 90-day hospitalizations well in lc patients [99] . small intestine bacterial overgrowth (sibo) is common in lc patients as a result of intestinal motility disorders and delayed transit times, and exacerbation of lc is associated with sibo [100] . in a previous study, the multivariate analysis showed that sibo (hr = 8.10, p = 0.002) and ascites (hr = 4.56, p = 0.022) were independently associated with the occurrence of malnutrition [100] . the severity of sibo can be linked to the severity of lc status [101] . increased intestinal permeability may help bacteria move into the systemic circulation. sibo has been implicated as an important risk factor in the etiology of both sbp and he in lc patients [102, 103] . thus, sibo is deeply involved in the progression of cld, which may be linked to sarcopenia [102, 103] . bile acids (bas) are synthesized from cholesterol in the liver and metabolized by the gm into secondary bas (e.g., deoxycholic acid (dca)). there is a positive correlation between abundance of ruminococcaceae (benign bacteria) and dca [104] . most of the bas that reach the ileum are reabsorbed and repeat gut-liver circulation, but some bas reach the colon and are converted by the gm (secondary bas). secondary bas regulate functions related to glucose and fat metabolism in the liver [105, 106] . in mice with dysbiosis, the expression levels of proteins in the liver involved in glycogen metabolism, cholesterol biosynthesis, and ba biosynthesis were altered, and these changes were recovered by supplementation with secondary bas [105] . atrophic change of skeletal muscle was confirmed in rats lacking the ba receptor tgr5 expressed in skeletal muscle, indicating that tgr5 enhances skeletal muscle hypertrophy and skeletal muscle cell differentiation [107] . in lc patients, a decreased conversion of primary to secondary fecal bas due to dysbiosis can be found [104, 108] . preventive effects of secondary bas on sarcopenia in lc patients are currently unknown. inoue et al. demonstrated informative data with regard to dysbiosis in patients with hepatitis c virus (hcv) as summarized below [91] : (1) even in hcv carriers with normal liver function (persistent normal alanine aminotransferase (pnalt)), alterations in gm already appeared. (2) as the liver function worsened from pnalt or chronic hepatitis to lc or hcc, the proportion of resident bacteria in the gm decreased, the types of bacteria that compose the gm decreased, and the ph of feces increased. these results mean that dysbiosis of the gm was occurring. (3) as the liver function worsened, streptococcus salivarius, which is a genus of streptococci, increased abnormally in the gm. it is possible that these bacteria decomposed urea in the intestinal tract to produce ammonia, and the ph of feces elevated. avoiding proliferation of such ammonia-producing bacteria may lead to the prevention or treatment of hyperammonemia seen in lc patients. (4) early interventions for gm (administration of probiotics, administration of appropriate antibiotics, oral care, etc.) may suppress the progression of hepatitis c and the development of hcc [91] . a recent study reported an increase in potentially pathologic bacteria and a decrease in potentially beneficial bacteria or genes in patients with hepatitis b virus, which is similar to data in patients with hcv [109] . in recent years, the association between dysbiosis and alcoholic hepatitis associated with excessive drinking has been receiving more attention [110] . in alcoholic liver injury, intestinal permeability is increased (i.e., leaky gut), and pathogen-associated molecular patterns (pamps) represented by endotoxin (lipopolysaccharide (lps)) derived from bacteria reach the liver through the portal vein and cause liver damage by activating kupffer cells [110] . endotoxin is mainly present in the cell wall of gram-negative bacteria. intestinal sterilization with antibiotics and probiotics such as lactobacillus can suppress alcoholic liver injury, and in lps receptor cd14 and toll-like receptor (tlr) 4 knockout mice, the onset of liver injury by chronic alcohol administration is suppressed [111, 112] . in addition, it has been suggested that the onset and progression of non-alcoholic steatohepatitis (nash) are associated with intestinal endotoxin [113] . in patients with nafld, low dose endotoxin can overreact to cause nash [113] . gm can be affected by aging. odamaki et al. demonstrated using fecal samples from 367 healthy japanese persons between 0 and 104 years that certain transition types of gm were enriched in infants (e.g., bifidobacterium), adults (e.g., lachnospiraceae), elderly individuals (e.g., bacteroides), and both infant and elderly subjects (e.g., enterobacteriaceae) [114] . on the other hand, flemer et al. reported in their prospective study that the gm in crc patients differs significantly from that of healthy persons throughout the colon [115] . coronavirus disease-19 (covid-19) has been rapidly becoming a global challenge. a recent study reported that in patients with covid-19, fecal gm alterations were associated with covid-19 severity [116] . additionally, covid-19 is likely to be accompanied by liver damage, and caution is required especially in lc patients for the disease progression caused by covid-19 [117] . covid-19 patients with liver disease had significantly higher mortality rates than those without (hr = 3.0, p = 0.001) [117] . interestingly, ren et al. reported that gm markers validated strong diagnosis potential for the early stage of hcc (area under the receiver operating characteristic curve = 0.8064) [118] . one possible factor which alters gm is the taking of antibiotics. while antibiotics are effective for the treatment and prevention of bacterial infections, they can cause dysbiosis [119] . rifaximin, which is a poorly-absorbable rifamycin-based antibiotic, acts on gm that are a source of ammonia to reduce ammonia production, thereby improving hyperammonemia in he [120, 121] . rifaximin has an effect of inhibiting bacterial rna synthesis, and the antibacterial activity of rifaximin covers a broad spectrum of bacteria [120, 121] . rifaximin has a favorable safety profile for long-term administration compared with oral systemic antibiotics [121] . kaji et al. demonstrated in their 20 decompensated lc patients that 4 weeks rifaximin therapy improved hyperammonemia and reduced endotoxin activity in direct correlation with the decline in serum ammonia levels, without impact on the composition of gm [97] . rifaximin also acts favorably on the serum pro-inflammatory cytokine profile and fecal secondary ba levels [122, 123] . rifaximin seems to alter the secondary to primary ba ratio in compensated lc patients, which can be associated with reduction in endotoxemia and reduction in harmful metabolite levels [104] . in addition, rifaximin appears to be effective and safe for the treatment of sibo [124] . the clinical activity of rifaximin may be attributed to the effects on metabolic function of the gm, rather than an alteration in relative bacterial abundance [125] . a recent meta-analysis (5 studies, comprising 555 patients) reported that rifaximin therapy may be effective in preventing sbp in patients with lc and ascites compared with systemic absorbed antibiotics and compared with placebo [126] . flamm et al. demonstrated that in patients with model for end-stage liver disease score 12 or greater and international normalized ratio 1.2 or greater, rifaximin group (n = 140) reduced the relative risk of any first complication (he, sbp, variceal bleeding, acute kidney injury, or hepatorenal syndrome) experienced during the study period by 59% [hr = 0.41, 95% ci = 0.25-0.67; p < 0.001] vs. placebo group (n = 159) [127] . kumar et al. reported that in rats with port-systemic shunts, the increase in skeletal muscle myostatin expression, suppressed mtorc1 function, and hyperammonemia-related stress response (i.e., autophagy markers) were reversed by ammonia-lowering therapy, concluding that it can lead to the improvement in skeletal muscle phenotype and function [128] . however, the preventive effects of rifaximin on sarcopenia incidence or progression in lc patients remain unclear. table 2 demonstrates randomized controlled trials (rcts) published since 2010 regarding the effects of rifaximin on outcomes in patients with decompensated lc . rcts with the improvement of sarcopenia as a primary endpoint are not found. in our hypothesis, rifaximin treatment in lc patients with sarcopenia potentially has an impact on the improvement of sarcopenia through the improvement of hyperammonemia and subsequent hypermyostatinemia in skeletal muscles. further exams with regard to the effect of rifaximin on sarcopenia in lc patients will be required to confirm these results. l-carnitine therapy, which is also an ammonia-lowering therapy, can improve sarcopenia in lc patients [154, 155] . probiotics are defined as microorganisms that have positive effects on the human body, or drugs and foods containing them. probiotics may act on the gm, intestinal epithelial cells, immunocompetent cells present in the intestinal mucosa, etc. [85, 156] . many neurotoxic substances are derived from the gm, and the usefulness of probiotics for improving the composition of gm has been investigated as a treatment for he [85, 156] . probiotics enhance the expression of tj-related proteins with improvement of dysbiosis and improve the intestinal barrier function [157, 158] . a previous meta-analysis demonstrated that probiotics reduce the risk of hospitalization and the progression to overt he to the same extent as lactulose in patients with minimal he, but do not affect mortality [125] . on the other hand, another systematic review comparing probiotics with placebo or no treatment summarized as follows: (1) there was no significant difference in mortality from any cause. (2) the non-recovery rate and the incidence of adverse events including he were lower in the probiotics group, but the effect on hospitalization was unclear. (3) quality of life was slightly improved in the probiotics group. (4) in comparison of probiotics and lactulose, the effects on mortality rate from any cause, non-recovery rate, incidence of adverse events including he, hospitalization, and quality of life were unable to be assessed due to the low quality of evidence [159] . table 3 demonstrates rcts published since 2010 regarding the effects of probiotics on outcomes in patients with decompensated lc [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] . rcts with the improvement of sarcopenia as a primary endpoint are not found. the effects of probiotics on sarcopenia in lc patients are unclear as well as those of rifaximin. in mice given probiotics (lactobacillus paracasei ps23), aging-related muscle mass decline and muscle strength decline significantly improved [173] . across a range of chronic diseases, several guidelines recommend exercise training. it has been found that exercise can change the composition of the gm, which leads to an intestinal flora with a beneficial metabolic system [174] . clarke et al. showed that the gm of rugby players was highly diverse, clearly different from normal healthy subjects, and that there was a high positive correlation between the diversity of gm and protein intake [175] . athletes presented a higher level of scfa-producing bacteria and bacterial genes related to nutritional metabolism compared with sedentary controls [176] . aerobic and resistance training revealed improved gm composition and functionality in rats with nafld [174] . however, only a few reports have shown exercise-related alterations on the gm in humans, and most evidence comes from non-randomized studies. huber et al. reported that in 41 nafld patients receiving an 8-week exercise program, increased metagenomic richness of the gm (i.e., increased diversity) was observed [177] . to date, there are no clinical studies or rcts looking specifically at exercise and the gm in patients with lc with sarcopenia, although exercise can decrease hvpg in lc patients [178] . if the improvement of the gm in lc patients with sarcopenia by exercise is confirmed, future treatment strategies for lc patients with sarcopenia and dysbiosis will be changed. interactions between dietary nutrients and gm promote host nutrition and health via various signaling pathways, and to maintain and promote human health, beneficial bacteria should be dominant in the gm [7] . in lc patients, these interactions can be disturbed due to pem, amino acid imbalance, dysbiosis, etc., which can cause sarcopenia. sarcopenia is a public health problem that cannot be overlooked. as mentioned earlier, skeletal muscle is considered to be a metabolic organ. when understanding the pathophysiology of lc, we must always keep in mind the relationship between organs including skeletal muscles and the digestive tract, that is, the organ network. in this article, we overviewed the current knowledge of the relationship between dysbiosis and sarcopenia in patients with lc. a summarized scheme is shown in figure 2 . in the past decade, marked advances have been made in this research field. our current research questions are whether or not rifaximin, probiotics, or exercise training can improve sarcopenia in lc through the improvement of the gm. to the best of our knowledge, appropriate rcts to address these research questions cannot be found. future research is eagerly awaited. across a range of chronic diseases, several guidelines recommend exercise training. it has been found that exercise can change the composition of the gm, which leads to an intestinal flora with a beneficial metabolic system [174] . clarke et al. showed that the gm of rugby players was highly diverse, clearly different from normal healthy subjects, and that there was a high positive correlation between the diversity of gm and protein intake [175] . athletes presented a higher level of scfaproducing bacteria and bacterial genes related to nutritional metabolism compared with sedentary controls [176] . aerobic and resistance training revealed improved gm composition and functionality in rats with nafld [174] . however, only a few reports have shown exercise-related alterations on the gm in humans, and most evidence comes from non-randomized studies. huber et al. reported that in 41 nafld patients receiving an 8-week exercise program, increased metagenomic richness of the gm (i.e., increased diversity) was observed [177] . to date, there are no clinical studies or rcts looking specifically at exercise and the gm in patients with lc with sarcopenia, although exercise can decrease hvpg in lc patients [178] . if the improvement of the gm in lc patients with sarcopenia by exercise is confirmed, future treatment strategies for lc patients with sarcopenia and dysbiosis will be changed. interactions between dietary nutrients and gm promote host nutrition and health via various signaling pathways, and to maintain and promote human health, beneficial bacteria should be dominant in the gm [7] . in lc patients, these interactions can be disturbed due to pem, amino acid imbalance, dysbiosis, etc., which can cause sarcopenia. sarcopenia is a public health problem that cannot be overlooked. as mentioned earlier, skeletal muscle is considered to be a metabolic organ. when understanding the pathophysiology of lc, we must always keep in mind the relationship between organs including skeletal muscles and the digestive tract, that is, the organ network. in this article, we overviewed the current knowledge of the relationship between dysbiosis and sarcopenia in patients with lc. a summarized scheme is shown in figure 2 . in the past decade, marked advances have been made in this research field. our current research questions are whether or not rifaximin, probiotics, or exercise training can improve sarcopenia in lc through the improvement of the gm. to the best of our knowledge, appropriate rcts to address these research questions cannot be found. future 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treatment of patients with hepatic encephalopathy lactobacillus paracasei ps23 decelerated age-related muscle loss by ensuring mitochondrial function in samp8 mice beneficial effects of exercise on gut microbiota functionality and barrier integrity, and gut-liver crosstalk in an in vivo model of early obesity and non-alcoholic fatty liver disease the microbiome of professional athletes differs from that of more sedentary subjects in composition and particularly at the functional metabolic level improvement of non-invasive markers of nafld from an individualised, web-based exercise program changes in hepatic venous pressure gradient induced by physical exercise in cirrhosis: results of a pilot randomized open clinical trial key: cord-018801-amet0wx4 authors: park, caroline; clark, damon title: care of the patient with liver failure requiring transplantation date: 2018-05-04 journal: surgical critical care therapy doi: 10.1007/978-3-319-71712-8_55 sha: doc_id: 18801 cord_uid: amet0wx4 patients undergo liver transplantation to address chronic liver failure, acute fulminant liver failure, or primary liver cancer. depending on acuity, patients with decompensated chronic or acute fulminant liver failure generally require preoperative intensive care unit admission to manage organ dysfunction. those with chronic liver failure are allocated an organ based on waiting list position determined by their local organ procurement organization (opo). this position is dependent upon blood type and model for endstage liver disease (meld) score. these patients thus are critically ill and require preoperative icu monitoring and care. patients with hepatocellular carcinoma (hcc) who require liver transplantation are given a meld exception and rarely require preoperative icu care. the patient’s ability to undergo liver transplant in the setting of hcc is determined by the milan criteria or the university of california, san francisco (ucsf) criteria. patients undergo liver transplantation to address chronic liver failure, acute fulminant liver failure, or primary liver cancer. depending on acuity, patients with decompensated chronic or acute fulminant liver failure generally require preoperative intensive care unit (icu) admission to manage organ dysfunction. those with chronic liver failure are allocated an organ based on waiting list position determined by their local organ procurement organization (opo). this position is dependent upon blood type and model for end-stage liver disease (meld) score. meld is determined by a weighting of serum bilirubin, creatinine, and international normalization ratio (inr). those with a high meld score have a greater risk of mortality and thus are given priority to transplantation. patients with a meld score of 40 have a 75% chance of death within 3 months. this is particularly important given the transplantation is typically performed on those with high meld scores in large urban areas such as ours in the greater los angeles area. these patients thus are critically ill and require preoperative icu monitoring and care. patients with hepatocellular carcinoma (hcc) who require liver transplantation are given a meld exception and rarely require preoperative icu care. the patient's ability to undergo liver transplant in the setting of hcc is determined by the milan criteria or the university of california, san francisco (ucsf) criteria. hcc patients with a single tumor <5 cm, up to three tumors <3 cm, absence of macroscopic vascular invasion, and absence of extrahepatic spread may undergo liver transplantation based on the milan criteria. altered level of consciousness is common in patients with decompensated cirrhosis. this is a result of portosystemic shunting and hepatocellular dysfunction/toxin production that results in hepatic encephalopathy. most severe or accelerated changes in the level of consciousness are the result of a precipitating event such as a significant upper gastrointestinal bleed and resultant uremia or a new infection. encephalopathy can be exacerbated by the administration of medications such as benzodiazepines that are generally avoided in the routine treatment of this patient population. those with low-grade encephalopathy, manifested by mild confusion and tremors, are administered agents such as lactulose to decrease ammonia absorption by acidifying bowel content and increase transit of the bowel contents. rifaximin is also commonly used to diminish the presence of urease and protease-splitting bacteria. it is important to remember to discontinue lactulose therapy in advance of anticipated liver transplantation to avoid intraoperative diarrhea and possible bowel distention. systemic and splanchnic arteriolar vasodilation is a wellknown physiologic derangement in patients with end-stage liver disease. as a result, these patients have hyperdynamic and low systemic vascular resistance cardiac profiles and often require vasopressor therapy-whether oral (e.g., midodrine) or intravenous (e.g., norepinephrine)-to maintain adequate mean arterial pressures (map) of >65 mmhg. it is important to consider that hypotension may be multifactorial, resulting from active hemorrhage, infection, and/or systemic inflammatory response (sirs). further, cardiac contractile function may be impaired in patients with longstanding cirrhosis or in those with associated ischemic cardiac disease. routine use of beta blockade as prophylaxis in those with varices may also cause hypotension. if hemor-rhage and sirs response have been addressed and ruled out, refractory hypotension could be a result of adrenal insufficiency; this is a common pathophysiology in end-stage liver disease patients. there is little additional role for invasive cardiac monitoring in cirrhotic patients for the diagnosis and treatment of shock over echocardiography and other noninvasive means. in general, the treatment of shock does not depart much from that in other patients. patients should be approached similarly with crystalloid resuscitation and early goal-directed therapy; there is little evidence that albumin should be the resuscitative fluid of choice. monitoring base deficit and lactate is as prognostic and useful to guide resuscitative efforts in patients with decompensated liver disease as it is for the general icu population. patients with advanced liver disease may have other associated pulmonary disorders, including hepatic hydrothorax, emphysema from alpha-1-antitrypsin deficiency, hepatopulmonary syndrome (hps), and portopulmonary hypertension (pph). hydrothorax is typically the result of tense abdominal ascites and should be addressed by paracentesis, thoracentesis, and total body volume management. draining of pulmonary effusions should be limited to those with impending respiratory failure due to the risk of infection and hemorrhage associated with invasive procedures and re-accumulation. definitive treatment of pulmonary effusions is with liver transplantation. hps is caused by intrapulmonary shunting and does not result in right heart failure. patients with hps will classically experience positional hypoxia. pph on the other hand results from pulmonary vascular vasoconstriction and can ultimately lead to thrombosis and fibrosis. in contrast to hps, right heart failure is typical in pph. it is important to distinguish between hps and pph. in general, liver transplantation is curative of the former and, until recently, was contraindicated in the latter. however, the introduction of many new agents and classes of agents to treat pulmonary hypertension may render liver transplantation possible in centers of excellence with careful pre-and intraoperative monitoring. although noninvasive monitoring is helpful and new modalities are being developed, the most expeditious and accurate modality to differentiate between hps and pph is right heart catheterization. hps, pph, and chf can all cause elevated pulmonary artery pressures. however, pulmonary artery wedge pressure (pawp) is low in hps and pph (and elevated in chf). pulmonary vascular resistance (pvr) is normal to decreased in hps but elevated in pph. thus, cardiac output is elevated in hps and normal to decreased in pph. one of the major causes of aki in patients with advanced cirrhosis and ascites is the phenomenon of hepatorenal syndrome (hrs) or acute renal failure without other etiology. there are two subtypes of hrs, types i and ii. the first type generally progresses with a rapid decrease in renal function and is characterized with doubling in serum creatinine within that period of time. it may be precipitated by spontaneous bacterial peritonitis, gastroenteritis with high-volume diarrhea, volume loss from gastrointestinal bleed, or largevolume paracentesis without appropriate volume repletion. type i hrs can be fatal and often leads to multi-system organ failure. type ii hrs demonstrates a more indolent course of renal failure, often precipitated by ascites refractory to diuretic treatment [1] . hrs develops due to splanchnic circulation vasodilation, intravascular hypovolemia, and renal vasoconstriction and is most often a diagnosis of exclusion after investigating for other causes of renal failure. the treatment approach includes strict intake and output monitoring, serum creatinine monitoring, and following changes from baseline or within the past 48 h. if the patient develops oliguria with elevated serum creatinine greater than 50% from a reference value or baseline serum creatinine level, suspect aki. multiple diagnostic variable are used to diagnose hrs in patients with cirrhosis ( fig. 55.1 ). these patients may require combined liver and renal transplant and/or intraoperative renal replacement therapy to assist with volume status, correcting acidosis, and electrolyte abnormalities. the mainstay of treatment for hrs is liver transplantation, after which a majority of patients demonstrate a return to adequate renal function. consideration should be given to a combined liver/kidney transplantation in those who have required dialysis for greater than 2 months, although this time period is controversial. a variety of approaches can be used to increase intravascular volume and map including albumin and vasopressors (norepinephrine, terlipressin). intermittent paracentesis may be necessary to manage third spacing of fluids in hepatorenal syndrome prior to liver transplant [2, 3] . type 1 hrs requires multiple therapeutic strategies and therapies; please refer to fig. 55 .2 for a detailed algorithm. the presence of infection in a cirrhotic patient quadruples mortality and worsens liver function. those with chronic liver failure are functionally immunosuppressed and are often colonized with multiresistant organisms. the most common infection in these patients is spontaneous bacterial peritonitis. strict attention should be paid to removing unnecessary catheters and avoiding intubation/mechanical ventilation, when possible. patients require prophylactic antibiotics for spontaneous bacterial peritonitis after a variceal bleed; however, there is little need for general broadspectrum antimicrobial therapy. patients with fulminant liver failure receive a meld exemption in listing-the appropriateness of transplantation may be determined by king's criteria (table 55 .1). in general, patients experience toxic necrosis of the liver on a baseline of normal function, most commonly due to intentional or inadvertent ingestion of large doses of acetaminophen. thus, the abnormalities noted are lactic acidosis, marked elevation of inr, and high-grade encephalopathy. ascites and hepatorenal syndrome are often not present given the acuity of presentation. creatinine, however, is often elevated due to atn. if time of ingestion is known, and within 8 h, n-acetylcysteine should be administered to prevent further toxicity in acute liver failure patients. if the patient fails medical management with progression of liver failure, liver transplantation will be required. the most significant risk of mortality to a patient with fulminant liver failure is that of cerebral edema and subsequent death from cerebral hernia-tion. intracranial monitoring remains controversial given severe coagulopathy, thrombocytopenia, and risk of infection. serum sodium of 145-150 meq/l should be maintained with hypertonic saline to decrease the amount of cerebral edema. if renal dialysis is required, a continuous mode is preferred to avoid rapid fluid shifts that may exacerbate cerebral edema. patients having undergone liver transplantation will require postoperative intensive care unit (icu) admission. close communication and coordination of care between the surgeons, anesthesiologists, intensivists, and nursing staff are essential to the management of the patient in this setting. liver transplantation typically entails a lengthy surgical procedure requiring significant amounts of blood product transfusion and risk of postoperative respiratory insufficiency. preoperatively, many of these patients have neurologic, cardiopulmonary, and renal dysfunction requiring fentanyl, a narcotic, is the first-line agent for the treatment of pain and agitation given its rapid onset and short duration of action in postoperative transplant patient. those who require additional sedation for agitation not controlled with narcotic analgesia benefit from the use of dexmedetomidine over benzodiazepines given a decreased risk of iatrogenic delirium and decreased length of mechanical ventilation [5] . dexmedetomidine is an alpha-2 adrenoreceptor agonist and should be used in caution with patients with hypotension and baseline bradycardia as it can exacerbate both conditions. with the use of spontaneous awakening trials (sats), richmond agitation and sedation score (rass), and confusion assessment method for the icu (cam-icu), patients have decreased episodes of delirium, duration of mechanical ventilation, and icu and hospital length of stay [6] . in this particular population, however, sustained delirium and encephalopathy may be the result of poor functioning of liver transplant graft, infection, intracranial hemorrhage or cerebral ischemia, seizures, and/or immunosuppressant toxicity. there should be a low threshold to pursue diagnostic ct scan of head, cultures including cerebral spinal fluid, and electroencephalography (eeg) in the posttransplant patient with change in mental status. encephalopathy due to cerebral edema associated with fulminant liver failure and elevated ammonia levels in patients with end-stage liver disease should be corrected with an adequately functioning liver transplant graft. if an intracranial monitor was placed preoperatively, it should be maintained until the inr is corrected and the liver is functioning well. in the past decade, early recovery after surgery or "fasttrack" programs have been implemented in a variety of disciplines, including hepatobiliary and colorectal patients after elective surgery with no worsening of postoperative outcomes and improvement in patient satisfaction. liver transplant patients may be eligible for fast-track extubation immediately postoperative in the operating room and upon arrival to the icu. patients that successfully undergo fasttrack extubation have been shown to benefit from decreased rates of re-intubation and tracheostomy along with improved survival [7] . patients that are likely not candidates for fasttrack extubation include those with preoperative acute liver failure, re-transplantation, child's c cirrhosis, and intraoperative red blood cell transfusion >6 units [7] . patients that require continued mechanical ventilation upon arrival to the icu should be placed on ventilator settings of tidal volume 6 ml/kg and fio2 < 0.4 [8] . patients who may exhibit transfusion-related lung injury after receiving a significant amount of blood products require ventilation strategies similar to patients with acute respiratory distress syndrome (ards); in this case, target tidal volumes of 6 ml/kg with supplemental oxygen and positive endexpiratory pressure (peep) [9] . infections of the lower respiratory tract require broad-spectrum antibiotics and antifungals until species and sensitivities are established. the liver transplant patients who remain hemodynamically stable and require minimal mechanical ventilation settings with resolved encephalopathy should undergo daily spontaneous breathing trials (sbt) and subsequent evaluation for possible extubation to reduce the duration of mechanical ventilation and icu length of stay [10] . early mobilization and aggressive chest physiotherapy are performed to prevent complications of atelectasis and inadequate ventilation. centers may opt to monitor patients intraoperatively with pulmonary artery catheters and/or transesophageal echocardiography. once stable and resuscitated, patients should be liberated from these devices. steroids are routinely administered as a part of early immunosuppression regimen after liver transplant and may require a prolonged course in treating hypotension secondary to adrenal insufficiency. liver transplantation patients remain at risk for postoperative hemorrhage due to thrombocytopenia, fibrinolysis, and deficiency of coagulation factors. abnormal coagulation tests and platelet count are not good predictors of bleeding; thus aggressive correction of these coagulopathies should be avoided. therapy should also include practical measures as avoiding hypothermia and persistent acidosis. aggressive correction of coagulopathy and thrombocytopenia may also put patients at higher risk of hepatic artery, portal vein, and deep vein thrombosis. typical target ranges include hemoglobin of 8 g/dl and platelet count >20 × 10^9/l [11] . thromboelastography (teg) may be useful in dictating guided blood product resuscitation in the post-liver transplant patients to decrease blood loss and transfusion requirements [12] . liver transplant patients with hemorrhage that are undergoing appropriate blood product resuscitation and become hemodynamically unstable or develop abdominal compartment syndrome should return immediately to the operating room. patients with advanced cirrhosis are often malnourished and as such are at higher risk for infections, worsening encephalopathy, and decompensation. though these patients may appear grossly overweight, their usual or dry weight is often masked by massive ascites and edema secondary to hypoalbuminemia. the american and european society for clinical nutrition and metabolism and the european society for clinical nutrition and metabolism (aspen [13] and espen [14] , respectively) have compiled an extensive set of guidelines, both of which provide a subset of consensus statements for patients with hepatic failure. the primary goals of nutrition for patients with hepatic failure include (1) identifying and assessing patients at risk for undernutrition, (2) calculating nutritional needs and incorporating adequate protein and high-calorie formulas, and (3) considering dobhoff placement if encephalopathy precludes voluntary enteral nutrition or short-term parenteral nutrition if unable to provide enteral feeds secondary to ileus or malabsorption. dry or usual weight may be difficult to ascertain given the chronicity of liver disease, thus complicating calculations for caloric needs. poor oral intake may be a result of underlying encephalopathy, gastroparesis, and overall decreased gastrointestinal motility. in prior years, protein restriction was emphasized to mitigate the effects on worsening hepatic encephalopathy. however, given the already reduced lean muscle mass of this vulnerable patient population, proteinrestricted diets can worsen hepatic failure. recommended protein intake is 1.2-1.5 g/kg/day, with a total energy intake of 35-40 kcal/kg/day. dobhoff placement is recommended if the patient is unable to meet his/her caloric needs per os; percutaneous endoscopic gastrostomy or open gastrostomy tube is otherwise not recommended given an increased risk of complications [14] . post-liver transplant acute kidney injury (aki) is a frequent event with reports of up to 52% of patients developing aki [15] . factors such as increased child-pugh score, preexisting diabetes, and large number of intraoperative transfusions increase the risk of aki in the post-liver transplant. the development of post-liver transplant aki leads to prolonged icu and hospital length of stay, increased mortality, and decreased duration of liver graft function [15] . in patients that develop aki post-liver transplantation, treatment includes the prevention of hypotension and decreased use of unnecessary blood products. the use of renal replacement therapy is reserved for patients that develop significant volume overload, uremia, and electrolyte abnormalities. the most effective treatment of postoperative liver transplant aki is prevention. preventive strategies include delayed initiation of calcineurin inhibitors, avoiding nephrotoxic agents such as iv contrast, and ensuring adequate control of hyperglycemia [15] . the most common cause of morbidity and mortality after liver transplantation is infection, accounting for 60% of the deaths after liver transplantation [16] . prolonged and complicated operations, multiple catheter insertions, immunosuppression, and large quantities of fresh frozen plasma can all increase the risk of infectious complications [17] . diagnosis of infections in this patient population may be difficult due to the lack of signs and symptoms such as fever, chills, cellulitis, and leukocytosis due to an immunosuppressed status. early postoperative infections in liver transplant patients are typically bacterial and related to the donor's status (previous infections from advanced cirrhosis), the surgical procedure itself, prolonged use of invasive catheters, and duration of mechanical ventilation. perioperative antibiotics are typically broad spectrum and may include third-generation cephalosporins. early removal of invasive catheters, early mobility, pulmonary toilet, vigilant monitoring of patient's surgical wounds and drains, and early discharge from the icu may decrease these infectious complications. liver transplant patients are at risk of developing opportunistic infections given the initial burst of immunosuppression with high-dose steroid therapy and, as such, should be initiated on prophylactic trimethoprimsulfamethoxazole (tmp-smx) to prevent pneumocystis carinii pneumonia and ganciclovir to prevent cytomegalovirus infection. besides surgical and coagulopathic bleeding, other postoperative complications can occur; these include postoperative hepatic artery thrombosis (3%) or portal vein thrombosis (< 1%) [11] . the resulting lack of blood flow and developing ischemia and necrosis from hepatic artery thrombosis present with signs and symptoms similar to fulminant liver failure patients with elevated liver serum tests, coagulopathy, and severe metabolic acidosis. doppler ultrasound of the hepatic artery and portal vein is routinely employed within the first 24-48 h after liver transplant to diagnose possible vascular complications prior to the development of ischemia and necrosis of the liver transplant graft. these patients are at high risk for continued ischemia and necrosis of the graft with the need for urgent relisting and re-transplantation. compared to patients with hepatic artery thrombosis, those with portal vein thrombosis do not present with such critical signs and symptoms as a rapid rise in liver function tests and disruption in synthetic function. although portal vein thrombosis leads to elevation in liver serum tests, signs and symptoms are less dramatic and may consist of mesenteric venous congestion, gastrointestinal hemorrhage, and the development of ascites. although these patients may require retransplantation, they can typically be managed with thrombectomy, shunt, or revision of the portal vein anastomosis. biliary duct complications, which include anastomosis stricture or leak, affect 5-25% of liver transplant patients and are often delayed diagnoses [18] . thrombosis of the liver transplant hepatic artery can also lead to nonanastomotic stricture [18] . biliary duct complications can be evaluated with ultrasound of the liver transplant graft looking for biloma and biliary duct dilation. similarly, internal to external drains placed during the liver transplantation may show biliary drainage during the first several postoperative days. elevated serum liver tests specifically bilirubin will elevate or fail to appropriately decrease after liver transplant, and the patients may develop signs and symptoms of infection. magnetic resonance cholangiopancreatography (mrcp) may be used as a noninvasive diagnostic modality to look for biliary anastomosis complications. endoscopic retrograde cholangiopancreatogram (ercp) can be used for the diagnosis of biliary anastomosis leak and stricture, in addition to possible treatment with sphincterotomy and/or biliary stent [19] . endoscopic treatment is often preferred over percutaneous management of biliary leaks and stricture. treatment options include endoscopic dilation and stenting and have excellent success rates approaching 75% [20] . surgical revision of the biliary anastomosis due to stricture or leak may be required in 10-20% of patients [18, 21] . the use of broad-spectrum antibiotics for treatment or prophylaxis is recommended due to the high risk of cholangitis and intra-abdominal sepsis [22] . 30-50% of patients with biliary stricture will have to undergo re-transplantation due to chronic biliary cirrhosis due to obstruction even with adequate treatment [19, 22] . primary graft nonfunction and hyperacute rejection can occur in the immediate or acute postoperative setting. primary graft nonfunction occurs in 2-14% of orthotopic liver transplants and typically presents similar to fulminant liver failure with significant metabolic acidosis, elevated liver enzymes, coagulopathy, and lack of bile production [11, 23] . intraoperative hemodynamic instability, reperfusion injury of the liver transplant graft, marginal livers, and advanced age of donors and recipient are factors that may lead to primary graft nonfunction. once diagnosed, the only treatment indicated is for relisting and liver re-transplantation. development of hyperacute rejection (har) after liver transplant is a rare complication that may develop intraoperatively or in the immediate postoperative period, which is antibody-mediated and due to abo crossmatch incompatibility. patients with har typically present with progressive encephalopathy and weakness, elevated bilirubin, severe coagulopathy, thrombocytopenia, metabolic acidosis, and shock. diagnosis is confirmed with doppler ultrasound that displays portal vein thrombosis and absence of biliary duct stricture. along with critical care supportive therapy, patients can be managed with plasma exchange for antibody removal and intravenous immunoglobulin [11] . overall, patients that develop har will need immediate relisting and re-transplantation. acute cellular rejection after liver transplant may occur within the first 6-8 weeks, and the patients are often out of the icu and no longer critically ill. patients with acute cellular rejection typically are not critically ill and may present with fever, weakness, and elevated liver function tests. prior to treating the patients for acute cellular rejection, one must rule out all possible acute infections that could account for the signs and symptoms given that the treatment of acute cellular rejection requires immunosuppression with pulse-dose glucocorticoids and adjustment of other immunosuppression medications. glucocorticoids are the first-line therapy for the prevention and treatment of acute cellular rejection. common glucocorticoids used for liver transplant include prednisone, hydrocortisone, and methylprednisolone with the first dose given while in surgery. intravenous hydrocortisone is typically administered in the immediate postoperative period until the patient is taking enteral nutrition and can transition to oral prednisone. most patients will undergo a glucocorticoid taper and either transitioned off of glucocorticoids or to a low maintenance dose, typically over a 6-month to 2-year period [24] . glucocorticoids have a significant number of side effects including poor wound healing, increased infection risks, hyperglycemia, and hypertension; these patients may need judicious adjustment of insulin sliding scale for hyperglycemia. glucocorticoid-free immunosuppression is possible and may be of benefit in patients with cirrhosis due to hepatitis c virus. [25, 26] calcineurin inhibitors (cni), including cyclosporine and tacrolimus, are used to prevent and treat acute rejection and liver transplant graft loss. both provide immunosuppression by inhibiting interleukin-2 and interferon-gamma production and require monitoring of blood levels to reach appropriate therapeutic levels. potential side effects including altered mental status, seizures, neuropathy, renal failure, electrolyte abnormalities, and others should be monitored for and treated appropriately. tacrolimus is currently the cni of choice and has demonstrated superiority in preventing acute rejection and graft loss with decreased mortality [27, 28] . posterior reversible encephalopathy syndrome (pres) is a rare syndrome and side effect of cni that is diagnosed with clinical exam and ct or mri. patients with pres most commonly present with seizures but may also have symptoms such as headache, delirium, and visual changes. head ct or mri typically demonstrates vasogenic edema of the parietal or occipital lobes; however mri may be more sensitive in diagnosing pres. treatment of pres most often involves discontinuing the offending cni and supportive care. mycophenolate mofetil (mmf, cellcept) is an antimetabolite that inhibits purine and pyrimidine synthesis with the active by-product of mycophenolic acid (mpa). mpa ultimately inhibits the proliferation of t lymphocytes for immunosuppression. mmf is typically used long term to reduce the dose or replace glucocorticoids. as such, the use of mmf will avoid common cni side effects, such as nephrotoxicity and neurotoxicity, though it can cause other side effects, including abdominal pain, nausea, vomiting, anorexia, diarrhea, and bone marrow suppression. mmf as a monotherapy after the acute phase of liver transplant has shown similar results to glucocorticoids and cni for prevention of chronic rejection and mortality [29, 30] . mammalian target of rapamycin (mtor) inhibitors (everolimus and sirolimus) inhibit the proliferation of lymphocytes. the use of mtor inhibitors allows for immunosuppression while avoiding renal dysfunction and has shown potential benefit in patients undergoing liver transplant for hcv. common complications of dyslipidemia and oral ulcers are typically easy to manage. hepatorenal syndrome pathogenesis of hepatorenal syndrome: implications for therapy systematic review of randomized trials on vasoconstrictor drugs for hepatorenal syndrome management of the critically ill patient with cirrhosis: a multidisciplinary perspective improved analgesia, sedation and delirium protocol associated with decreased duration of delirium and mechanical ventilation dexmedetomidine vs. midazolam or propofol for sedation during prolonged ventilation fast tracking in liver transplanatation: which patient benefits from this approach? a trial of intraoperative low-tidal-volume ventilation in abdominal surgery the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome protocolized versus non-protocolized weaning for reducing the duration of mechanical ventilation in critically ill adult patients immediate postoperative management and complications on the intensive care unit methods to decrease blood loss and transfusion requirements for liver transplantation guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition espen guidelines on enteral nutrition liver disease acute kidney injury following orthotopic liver transplantation: incidence, risk factors and effects on patient and graft outcomes causes of death in autopsied liver transplantation patients risk factors for cytomegalovirus and severe bacterial infections following liver transplantation: a prospective multivariate time-dependent analysis management of biliary complications after liver transplantation biliary complications after liver transplantation: the role of endoscopy efficacy of endoscopic and percutaneous treatments for biliary complications after cadaveric and living donor liver transplantation anastomotic biliary strictures after liver transplantation: causes and consequences nonanastomotic biliary strictures after liver transplantation, part 2: management, outcome and risk factors for disease progression improving the diagnostic criteria for primary liver graft nonfunction in adults utilizing standard and transportable laboratory parameters: an outcome-based analysis influence of steroids on hcv recurrence after liver transplantation: a prospective study a randomized, multicenter study comparing steroid free immunosuppression and standard immunosuppression for liver transplant recipients with chronic hepatitis c randomized, multicenter trial comparing tacrolimus plus mycophenolate mofetil to tacrolimus plus steroids in hepatitis c virus-positive recipients of living donor liver transplantation randomized controlled trial of tacrolimus versus microemulsified cyclosporine (tmc) in liver transplantation: poststudy surveillance to 3 years cyclosporin versus tacrolimus for liver transplanted patients corticosteroid-free strategies in liver transplantation four-year follow-up of mycophenolate mofetil for graft rescue in liver allograft recipients key: cord-018225-dozmy3lb authors: hawker, felicity h. title: the liver in critical illness date: 2008 journal: classic papers in critical care doi: 10.1007/978-1-84800-145-9_7 sha: doc_id: 18225 cord_uid: dozmy3lb the liver is in some ways the forgotten organ in intensive care practice. very many more laboratory and clinical studies have investigated the role, function, and support of the lung, heart, brain, and kidney in critical illness than have studied the liver. nevertheless, in the time of the greek scholars, there was already acknowledgement of the role of the liver in non-hepatic diseases such as systemic sepsis, and an understanding that such involvement confers a poorer prognosis – hence the inclusion of the wisdom of hippocrates in this compilation of classic papers. in the review article by matuschak and rinaldo, the reasons why liver dysfunction is associated with a poorer outcome in critical illness are explored, and the concept of the liver being a ‘driving force’ in multiple organ dysfunction is developed. in addition, jaundice without significant liver dysfunction is associated with left ventricular dysfunction, at least in the dog model developed by professor otto better and his colleagues in israel. this observation is relevant to the progressive resistance to inotropic and vasopressor agents in jaundiced critically ill patients. the liver is in some ways the forgotten organ in intensive care practice. very many more laboratory and clinical studies have investigated the role, function, and support of the lung, heart, brain, and kidney in critical illness than have studied the liver. nevertheless, in the time of the greek scholars, there was already acknowledgement of the role of the liver in non-hepatic diseases such as systemic sepsis, and an understanding that such involvement confers a poorer prognosis -hence the inclusion of the wisdom of hippocrates in this compilation of classic papers. in the review article by matuschak and rinaldo, the reasons why liver dysfunction is associated with a poorer outcome in critical illness are explored, and the concept of the liver being a 'driving force' in multiple organ dysfunction is developed. in addition, jaundice without significant liver dysfunction is associated with left ventricular dysfunction, at least in the dog model developed by professor otto better and his colleagues in israel. this observation is relevant to the progressive resistance to inotropic and vasopressor agents in jaundiced critically ill patients. one of the most devastating insults that can occur in critical illness is acute liver ischemia, resulting in the clinical syndrome of ischemic or hypoxic hepatitis. although relatively common, this syndrome has been difficult to study prospectively. consequently, the paper by jean henrion from belgium offers new insights into the hemodynamic associations of ischemic hepatitis, which is, of course, a circulatory disease, not a liver disease. commonly used 'liver function tests' assess damage to the liver rather than its function. an understanding of the complex relationship between critical illness and liver dysfunction has been hampered by the absence of a simple test that assesses at least one of the many functions of the liver. the megx test, developed by oellerich and co-workers in germany, allows dynamic assessment of liver function, and may prove useful in the evaluation of therapies to support liver function in critical illness. the complex issues involved in the management of patients with acute liver failure exemplify the many important functions of the healthy liver. with the advent of liver transplantation as the principle treatment for patients with fulminant hepatic failure, these patients are almost always managed in icus servicing liver transplant units. consequently, it is extremely important that intensive care practitioners in non-liver units understand the need to refer patients to transplant units, as well as the role of transplantation in acute liver failure and its indications. the seminal paper by o'grady and the group from king's college hospital, london, develops criteria for liver transplantation in acute liver failure. that a successful outcome is possible after emergency liver transplantation for acute liver failure is shown in the early series of cases reported from the french group from clichy and villejuif. when transplantation is not indicated, or while awaiting a donor, the principal treatment is supportive. the paper by harrison and co-workers, again from the king's liver unit, investigates the effects of n-acetylcysteine in patients with acute liver failure, and the findings of this study have resulted in widespread use of this agent in this setting. currently, there is still no safe, effective means of artificial liver support for patients awaiting transplantation or liver regeneration. the case report from saunders and co-workers from cape town, south africa, describes an early attempt to develop such support. finally, patients with cirrhosis and other forms of chronic liver disease present different management challenges for the intensive care specialist. the randomized controlled trial from the barcelona group shows that in patients with spontaneous bacterial peritonitis and cirrhosis, the outcome is improved by infusing albumin in addition to antibiotic therapy. there are many classic papers that have not been included in this small selection. the majority of papers have been chosen for the underlying ideas and variety rather than for scientific rigor. they are aimed at clinical intensive care specialists with enquiring minds who work in general intensive care units, rather than experts in liver units or intensive care units servicing liver transplant units. i have found the liver a fascinating organ to study. i trust this selection of papers will likewise stimulate your interest. hippocrates examines a number of questions with respect to jaundice associated with systemic infection. he concludes that jaundice is a result of impairment of the separative faculty of the liver; that is, impairment of its ability to purify all the humors. he further states that this may occur as a result of obstruction of the biliary vessels or of seething inflammations, in which case the blood is distributed through the whole body with a strong admixture of bile. this review article explores the role of the liver in the pathogenesis of the adult respiratory distress syndrome (ards) and progressive multiple systems organ failure. it is argued that the normal liver plays a pivotal role in aspects of host defence that are crucial to the protection of the lung and other organs in systemic sepsis. the authors note that the fixed hepatic macrophages, or kupffer cells, constitute nearly 90% of the body's reticuloendothelial cell mass. they further note that these cells are the major mechanism for clearance of systemic endotoxin and bacterial products. the strategic location of the liver, immediately downstream from the large gastrointestinal reservoir of bacteria and bacterial products, normally constitutes an effective first line in host control of systemic endotoxemia and bacteremia. however, the balance may be altered by an excess load of bacterial products in the gut, mesenteric or hepatic ischemia, and kupffer cell dysfunction, so that the phagocytic capacity of kupffer cells is exceeded, resulting in systemic spread of bacterial products. moreover, the authors describe the activation of kupffer cells by the phagocytic burst, resulting in the release of an array of inflammatory mediators that are 'exported' systemically, and result in acute pathophysiological reactions in distant organs. these inflammatory mediators also have effects on adjoining hepatic parenchymal cells. evidence is presented that the resulting hepatocyte damage may result in decreased clearance of some of these mediators, resulting in heightened and prolonged effects, and consequent amplification of multiple organ damage. furthermore, these inflammatory mediators -particularly interleukin-1 -induce changes in hepatic protein synthesis, characterized principally by the 'acute phase response'. thus, this review acknowledges the role of the liver in host defence but also proposes ways in which liver dysfunction might fuel multiple organ dysfunction -particularly ards. the liver plays a major role in host defence, but also has the capacity to augment the host response, with consequent adverse effects on other organs in some circumstances. this has led to the concept of the liver as 'the motor of multiple organ failure'. this review was among the first to provide a plausible concept of organ system interactions that may result in ards and multiple organ failure in sepsis. although some of the detail has been refined, the general principles remain accepted 15 years later. this paper synthesizes experimental findings from a number of disciplines -intensive care medicine, hepatology, immunology, and thoracic medicine -to arrive at a concept that was new at the time of publication, and which continues to be supported in the main by more recent evidence. this paper is not a systematic review. the general concepts in this review remain valid, and have led to a more concentrated focus on the gut and liver in the critically ill patient, both experimentally and in the clinical setting. although there have been major advances in understanding of relevant factors such as splanchnic blood flow, no 'liver-orientated therapy' has yet been shown to be of value in ards and multiple organ failure, and the treatment of liver dysfunction remains supportive. the "jaundiced heart": a possible explanation for postoperative shock in obstructive jaundice abstract patients with obstructive jaundice are susceptible to postoperative shock and kidney failure. the cause of these potentially fatal complications has not been fully clarified. the present study was designed to assess the role of myocardial dysfunction in the hemodynamic disturbance of obstructive jaundice. we studied the effect of isolated cholemia on left ventricular performance in five conscious dogs, before and 2 weeks after choledochocaval anastomosis, by using measurements of systolic time intervals (stis) and maximal dp/dt. mean left ventricular ejection time (lvet) decreased after cholemia from 159 +/-2.8 msec to 139 +/-2.6 msec (p < 0.005), while mean preejection period (pep) and mean pep/lvet were increased from 41 +/-8.5 msec to 87 +/-14 msec (p < 0.05) and from 0.39 +/-0.06 to 0.62 +/-0.1 (p < 0.01), respectively. during cholemia, stis were unchanged after intravenous administration of ouabain, whereas in the control period, there was shortening of mean pep from 71 +/-8.8 msec to 58 +/-7.6 msec (p < 0.05), and of q-s2 from 257 +/-12 msec to 235 +/-14 msec (p < 0.005) in response to ouabain. maximal dp/dt decreased after choledochocaval anastomosis from 4543 +/-593 mmhg/ sec to 3666 +/-648 mmhg/sec (p < 0.025). we conclude that cholemia in the dog is clearly associated with impaired left ventricular performance. the present data also support a previously published in vitro study from our laboratory showing that cholemia blunts the myocardial contractile response to sympathomimetic agents. the cardiodepressor effect of cholemia may explain the increased tendency of patients with obstructive jaundice to postoperative shock and renal failure. the effect of deep jaundice on left ventricular function was studied in five dogs. each dog was studied before and 2 weeks after choledochocaval anastomosis (cdca), thus each dog served as its own control. cdca results in severe jaundice with minimal liver damage. mean arterial blood pressure, end-diastolic left ventricular pressure, and maximal rate of increase in left ventricular pressure (dp/dt) were obtained by retrograde catheterization of the aorta and left ventricle. systolic time intervals (pre-ejection period (pep) and left ventricular ejection time (lvet)) were measured with simultaneous ecg and aortic pulse tracings. systolic time intervals were measured before and after intravenous injection of ouabain. the findings revealed that deep jaundice was associated with impaired left ventricular performance. there was a decrease in dp/dt without changes to arterial pressure, heart rate, or preload. moreover, there was an associated increase in mean left ventricular pep, and a decrease in lvet when compared with predicted values. although administration of ouabain in normal dogs (i.e. pre-cdca) was associated with decreased pep, no effect was observed in deeply jaundiced animals. 48 deep jaundice may have hemodynamic consequences even when liver function is relatively intact. it is associated with impaired left ventricular performance that is refractory to cardiac glycosides in this animal model. the hemodynamic manifestations of advanced liver failure with jaundice are well described, and are basically a 'high output-low resistance' state. this paper suggests that jaundice has its own effects on cardiac function independent of liver failure. the authors are clearly original thinkers who have devised an animal model to shed light on a clinical problem. the model is relevant to patients with jaundice associated with multiple organ failure, as well as obstructive jaundice this is a small animal study. the methods of assessment of left ventricular function are probably valid, but are not conventional. patients with multiple organ failure are frequently jaundiced, and in this setting, plasma bilirubin concentrations may be grossly elevated. such patients often also have severe hemodynamic compromise that can be refractory to inotropic agents. although this paper does not offer a solution to this problem, it does propose a plausible mechanism. the incidence of hypoxic hepatitis was prospectively studied for 1 year in a group of high-risk patients suffering from low cardiac output in a coronary care unit. hypoxic hepatitis, defined as an increase in serum aminotransferase activity of at least 20 times the upper limit of normal without any other cause for hepatic necrosis, was observed in 20 patients. this represents 2.6% of the 766 patients admitted to the unit during this period, and 21.9% of the 91 patients suffering from low cardiac output. clinical, biological, and hemodynamic data were compared between 20 patients with low cardiac output and hypoxic hepatitis, and 48 patients with low cardiac output but without hypoxic hepatitis who survived more than 24 hour. in these two groups of patients, hepatic blood flow was measured by galactose clearance at low concentration. patients with hypoxic hepatitis exhibited a higher central venous pressure (90% versus 38%, p < 0.001), as well as a lower hepatic blood flow (867 +/-377 ml/min versus 1429 +/-644 ml/min, p = 0.001). in conclusion, although it is considered a rare hepatic disorder, hypoxic hepatitis is frequent in patients with low cardiac output admitted to the coronary care unit, and is associated with a decrease in hepatic blood flow, and passive hepatic venous congestion. over a 1-year period in a belgian coronary care unit, 91 patients were identified as having a low cardiac output using clinical criteria. hypoxic (ischemic) hepatitis (hh) was said to be present if there was a clinical setting of circulatory failure, a sharp but transient increase in serum aminotransferase activities of at least 20 times the upper limit of normal, and after exclusion of other causes of hepatic necrosis. of the 91 patients, 20 developed hh (2.6% of admissions to the coronary care unit; 21.9% of patients with low cardiac output), 48 did not develop hh, and 23 died within 24 hours. hepatic blood flow was measured in the 20 patients with hh, and in 41 of the 48 surviving patients without hh, using the method of galactose clearance at low concentrations. when compared with patients who did not develop hh, patients with hh had lower hepatic blood flow (867 ± 377 ml/min vs 1429 ± 644 ml/min), higher central venous pressure (22.5 cmh 2 o vs 14 cmh 2 o), and a trend toward lower cardiac output (3.4 l/min vs 4.4 l/min). interestingly, only approximately 50% of patients in each group had a systolic arterial pressure below 90 mmhg. the overall mortality was high -55% in the hh group and 46% in the group without hh. the findings of this study show that ischemic hepatitis is common in environments where there are patients with low cardiac output states, such as coronary care units and intensive care units. the classic hemodynamic pattern is low cardiac output (resulting in reduced hepatic blood flow), and high right atrial pressure. hypotension is not a prerequisite. the mortality rate is >50% this is the first prospective study to determine the incidence of ischemic hepatitis in patients with severe cardiac disease and low cardiac output. it is also by far the most comprehensive study of hemodynamic measurements in patients with ischemic hepatitis, and the only one to measure liver blood flow. this is a large, prospective, observational study of 766 patients admitted to a single intensive care unit over a 1-year period. exclusion of the 23 patients who died within 24 hours of the episode of low cardiac output, before measurements of hepatic blood flow had been made, may have altered the findings, particularly regarding the incidence of ischemic hepatitis. the validity of the hepatic blood flow measurements themselves are open to some dispute -firstly because of the use of galactose clearance rather than indocyanine green clearance, and secondly because they were performed subsequent to the episode of low cardiac output, and therefore may not reflect the hemodynamic state at that time. ischemic hepatitis occurs in up to 5% of admissions to intensive care units and coronary care units, and is consequently more common than a number of other conditions that have attracted massive research interest. this paper describes the clinical setting, hemodynamic characteristics, and outcome of ischemic hepatitis, and consequently makes a major contribution to the body of knowledge on the subject. ischemic hepatitis is not a rare hepatic disorder, but rather it is a circulatory disorder, and therefore of great relevance to intensive care medicine. abstract a novel quantitative liver function test is described which is based on monoethylglycinexylidide (megx) formation after lidocaine bolus injection. following the administration of small single doses of lidocaine hydrochloride (1 mg/kg), monoethylglycinexylidide serum concentration-time curves were determined by a novel, highly sensitive fluorescence polarization immunoassay (fpia) in healthy volunteers, liver donors, and patients with liver cirrhosis. the fpia allowed rapid and reliable monoethylglycinexylidide determinations in serum and urine (between-days coefficient of variation: < 10.3%, recovery: 80-113%). monoethylglycinexylidide concentrations measured by fpia in 32 serum samples from patients correlated well with those determined by hplc. the monoethylglycinexylidide concentration in serum determined 15 minute after a lidocaine bolus injection proved to be a highly sensitive and specific indicator of hepatic dysfunction. average monoethylglycinexylidide concentrations in serum obtained 15 minute after lidocaine injection were substantially lower in patients with liver cirrhosis than in healthy volunteers. the average monoethylglycinexylidide concentrations in serum were also substantially lower in liver donors with ballooning or fatty changes of hepatocytes than in donors without relevant alterations of liver histology. by means of monoethylglycinexylidide formation in the liver donors, primary function of the transplanted liver was correctly predicted in 32/37 cases, and initial non-function in 4/6 cases. monoethylglycinexylidide (megx) is the first metabolite of lignocaine formed by oxidative de-ethylation by the hepatic cytochrome p-450 system. in this study, the appearance of megx measured by a fluorescence polarization immunoassay after a test dose of intravenous lignocaine was investigated as a liver function test. the findings showed that there was a satisfactory correlation between the measurement of megx by the novel fluorescence polarization immunoassay and by high performance liquid chromatography. the formation and elimination kinetics of megx were investigated for healthy volunteers, patients with histologically confirmed liver cirrhosis, and liver donors after a single intravenous bolus injection of lignocaine (1 mg/kg). in healthy volunteers, the peak megx concentration occurred 15 minutes after the bolus of lignocaine, whereas in patients with cirrhosis, its rate of formation was markedly decreased, and maximum concentrations were observed at about 4 hours. in liver donors the 15-minute megx concentration was lower in donors, with altered liver histology than in those with normal histology. patients with cirrhosis had the lowest 15-minute megx concentration of all the groups. conventional 'liver function tests' measure liver damage rather than liver function. the megx test assesses the liver's capacity to metabolize drugs (in this case lignocaine), and thus is a true 'liver function test'. the data presented in this study suggest that the 15-minute megx concentration varies with the adequacy of liver function. this paper is the first to describe and assess the formation of megx after a standard dose of lignocaine as a dynamic liver function test. novel approach. the test is not well validated in this report. because of its ease of use and rapid turnaround, the megx test has found widespread application for the real-time assessment of hepatic function. in liver transplantation, the megx test has found a place in selection of transplant candidates, and in monitoring of liver recipients early after transplantation. in intensive care patients, a rapid decrease in megx test values is associated with increased risk of developing multiple organ failure, and a poor outcome, and consequently may have a role in investigation of the role of the liver in the multiple organ failure syndrome. the successful use of orthotopic liver transplantation in fulminant hepatic failure has created a need for early prognostic indicators to select the patients most likely to benefit at a time when liver transplantation is still feasible. univariate and multivariate analysis was performed on 588 patients with acute liver failure managed medically during 1973-1985, to identify the factors most likely to indicate a poor prognosis. in acetaminophen-induced fulminant hepatic failure, survival correlated with arterial blood ph, peak prothrombin time, and serum creatinine -a ph < 7.30, prothrombin time > 100 seconds, and creatinine > 300 µmol/l indicated a poor prognosis. in patients with viral hepatitis and drug reactions, three static variables [etiology (non a, non b hepatitis or drug reactions), age < 11 and > 40 year, duration of jaundice before the onset of encephalopathy > 7 days], and two dynamic variables (serum bilirubin > 300 µmol/l, and prothrombin time > 50 seconds) indicated a poor prognosis. the value of these indicators in determining outcome was tested retrospectively in a further 175 patients admitted during 1986-1987, leading to the construction of models for the selection of patients for liver transplantation. the records of 588 patients with fulminant hepatic failure (fhf) and grade iii or iv encephalopathy admitted to king's college hospital, london, between 1973 and 1985, were scrutinized to identify simple parameters that might prove to be prognostic indicators. these parameters were examined by univariate and multivariate analysis to determine the factors most likely to be associated with poor prognosis. the analysis was performed in two groups, paracetamol-induced and non-paracetamol-induced, because of the higher incidence of renal failure and metabolic acidosis in the former group. the prognostic indicators so determined were then examined retrospectively, in a second group of 175 patients with fhf admitted during 1986-1987, to determine their sensitivity and specificity in prediction of a fatal outcome. models were then constructed for selection of patients for liver transplantation on the basis of an extremely poor prognosis with medical management. the criteria adopted for paracetamol-induced fhf were ph < 7.30 (irrespective of grade of encephalopathy), or prothrombin time >100 seconds and serum creatinine >300 µmol/l in patients with grade iii or iv encephalopathy. for fhf unrelated to paracetamol, the criteria determined were prothrombin time >100 seconds (irrespective of grade of encephalopathy), or any three of (irrespective of grade of encephalopathy): age <10 or >40 years, etiology (non-a, non-b hepatitis or idiosyncratic drug reactions), duration of jaundice before onset of encephalopathy > 7 days, prothrombin time > 50 seconds, and serum bilirubin > 300 µmol/l. this study has developed criteria that predict death in patients with fhf who are managed medically. in the era of liver transplantation, these criteria are also used to select patients for liver transplantation. all patients with fhf should be discussed with a transplant unit. patients who meet these criteria should be transplanted. this paper has changed practice. it allows decisions to be made about transplantation in fhf so that unnecessary transplantation can be minimized. because the criteria involve simple, easy to apply parameters that are readily available, the algorithm also permits decisions to transplant to be taken early, maximizing the period of time available to find a suitable donor, and allowing transplantation to be undertaken before the development of cerebral edema. this study is based on the largest number of patients in a single unit ever reported. data were collected over a period of 13 years, and it is possible that one or more aspects of management may have changed in that time, and may independently affect outcome. various models have been used to predict outcome in fhf, but the 'king's criteria' identified in this study are the most widely used. these criteria are based on the study of a huge number of patients treated medically, and, because so many patients are now transplanted, it is extremely unlikely that the study could ever be repeated. consequently, the criteria identified in this paper are likely to be used widely to select transplant candidates for the foreseeable future. orthotopic liver transplantation was done in 17 patients with fulminant hepatitis. the cause of the liver disease was infection with hepatitis b virus, or co-infection with hepatitis b virus and hepatitis d virus, or infection with hepatitis a virus in 6 patients; drug hepatotoxicity in 5; and indeterminate in 6. grafts from incompatible blood groups, steatotic grafts, or reduced-size grafts were used in 5, 4, and 4 patients, respectively. of the 17 patients, 5 died: 2 of early liver failure due to the poor quality of the graft, 1 presumably of accidentally transmitted acute infection with the human immunodeficiency virus, and 2 of decerebration occurring during or immediately after surgery. the 12 other patients were alive 2 to 15 months after transplantation. this paper reports the clinical details and outcome of 17 patients with acute liver failure who underwent orthotopic liver transplantation in the mid-1980s, in the hopital paul-brousse, villejuif, and the hopital beaujon, clichy, france. six patients had documented viral hepatitis, five had drug hepatotoxicity, and the cause was undetermined in the remaining six. despite compromises necessitated by emergency transplantation (abo and size incompatibility, and steatotic grafts), 12 patients survived. deaths were due to cerebral edema (2), early graft failure (2), and graft-transmitted infection. why it's important this paper was the first large case study to document the feasibility and success of liver transplantation for acute liver failure. this is a succinct review of 17 patients with acute liver failure who underwent liver transplantation, with appropriate demographic, etiological, clinical, and outcome information that remains relevant many years later. no case control data are reported. although it is stated that transplantation was undertaken for a rapidly deteriorating neurological condition and severe coagulation disorders, the outcome of patients who did not meet these criteria, or who met the criteria and could not be transplanted, is not reported. hence, although this paper describes the feasibility of liver transplantation in this population, it does not assess the value. liver transplantation, as described in this case series, remains the best chance of survival for the majority of patients with acute liver failure. although criteria for transplantation, and aspects of the surgical technique and postoperative immunosuppression have developed over the intervening years, orthotopic liver transplantation has not been superceded, and should be considered as an option in all patients with acute liver failure. abstract background. when administered early after an overdose of acetaminophen, intravenous acetylcysteine prevents hepatic necrosis by replenishing reduced stores of glutathione. how acetylcysteine improves the survival of patients with established liver damage induced by acetaminophen, however, is unknown. this study was undertaken to determine whether the beneficial effect of acetylcysteine under such circumstances could be due to enhancement of oxygen delivery and consumption. methods. we studied the effect of acetylcysteine on systemic hemodynamics and oxygen transport in 12 patients with acetaminophen-induced fulminant hepatic failure, and 8 patients with acute liver failure from other causes. the acetylcysteine was given in a dose of 150 mg/kg in 250 ml of 5% dextrose over a period of 15 minutes, and then in a dose of 50 mg/kg in 500 ml of 5% dextrose over a period of 4 hours; measurements were made before treatment began, and after 30 minutes of the regimen. results. in the patients with acetaminophen-induced liver failure, the infusion of acetylcysteine resulted in an increase in mean oxygen delivery from 856 to 975 ml/min/m 2 (p = 0.0036), due to an increase in the cardiac index from 5.6 to 6.7 mm/m 2 (p = 0.0021). mean arterial pressure rose from 88 to 95 mmhg (p = 0.0054), despite a decrease in systemic vascular resistance from 1296 to 1113 dyn.sec.cm -5 per square meter (p = 0.027). there was an increase in oxygen consumption from 127 to 184 ml/min/m 2 (p = 0.0007) associated with an increase in the oxygen-extraction ratio from 16 to 21 percent (p = 0.022). the effects in the patients with acute liver failure from other causes were similar. conclusions. the increase in oxygen delivery and consumption in response to acetylcysteine may account for its beneficial effect on survival in patients with fulminant hepatic failure induced by acetaminophen. the effects of intravenous n-acetylcysteine on systemic hemodynamics and oxygen transport were studied in 12 patients with paracetamol-induced acute liver failure, and eight patients with acute liver failure from other causes. measurements were made before a bolus dose of 150 mg/kg of n-acetylcysteine given over 15 minutes, and 30 minutes after commencement of an infusion of 50 mg/kg of n-acetylcysteine given over 4 hours. the findings were that in patients with paracetamol-induced acute liver failure, infusion of n-acetylcysteine resulted in an increase in mean oxygen delivery from 856 to 975 ml/min/m 2 , resulting mainly from an increase in the mean cardiac index by approximately 20% (5.6 to 6.7 l/min/m 2 ). mean arterial pressure increased from 88 to 95 mmhg despite a decrease in the mean systemic vascular resistance from 1296 to 1113 dyn/sec/cm -5 . in patients with acute liver failure from other causes, the effects of n-acetylcysteine were similar but less marked, and there were significant changes (increases) only for oxygen consumption and oxygen extraction ratio. each patient had similar measurements performed before and during an infusion of prostacyclin at a rate of 5 ng/kg, and again when n-acetylcysteine and prostacyclin were infused simultaneously. the effects of prostacyclin were similar to those of n-acetylcysteine, but there was a decrease in mean arterial pressure rather than an increase, and a much smaller increase in oxygen extraction. four patients with acute liver failure secondary to paracetamol were studied after recovery. in this group, n-acetylcysteine had no effect on any hemodynamic or oxygen transport variable. however, with infusion of prostacyclin, there was an increase in cardiac index, decrease in systemic vascular resistance, and increased oxygen delivery without an increase in consumption. there is a characteristic high output-low resistance hemodynamic state in fulminant hepatic failure that may be associated with covert tissue hypoxia. intravenous infusion of n-acetylcysteine was associated with an increase in oxygen delivery and consumption, and also in tissue oxygen extraction in patients with fulminant hepatic failure. it is postulated that this may account for the beneficial effect of n-acetylcysteine on survival in patients with acute liver failure caused by paracetamol, and further, may be of benefit in patients with multiple organ failure from other causes, such as sepsis. this study is the justification for the widespread use of n-acetylcysteine in patients with acute liver failure, and it has also provoked investigation of possible clinical benefits in other conditions, such as septic shock. this is a comprehensive clinical study of a relatively homogeneous group of patients. because the fick method was used to calculate oxygen consumption from the cardiac output and the arteriovenous oxygen content difference, it is possible that an artifactual relationship between oxygen delivery and consumption was shown because of mathematical coupling. it is now generally accepted that oxygen delivery and consumption should be determined independently. although undoubtedly a classic study, the findings presented in this paper have not been confirmed in a similar more recent study (see related reference no. 1). in the latter study, oxygen consumption was measured independently of oxygen delivery, using a method based on respiratory gas analysis, and patients were studied over a longer period. nevertheless, the paper under review has been responsible in part for a resurgence of interest in n-acetylcysteine as a therapeutic agent, not only in patients with acute liver failure, but also in other groups of critically ill patients, such as those with sepsis, acute lung injury, and multiple organ failure. as with acute hepatic failure, published studies of other groups of critically ill patients have shown inconsistent findings, and the role of n-acetylcysteine in critically ill patients remains unclear. this paper marks the beginning of a long and still-continuing struggle to find a clinically relevant artificial liver device that can be used as a bridge to regeneration or transplantation in patients with acute liver failure. this is a report of original and far-sighted ideas that must have stretched the boundaries of knowledge and technology in the late 1960s. this is a case report only. it is possible that the improvement after cross-circulation may have been incidental, and the patient may have recovered spontaneously without it. the precise technique used in this report is most unlikely to be used again because of concerns about transmission of infection, and immunological considerations. however, more than 30 years later, there remains a need to develop a safe and effective artificial liver for use in patients with acute liver failure. abstract background: in patients with cirrhosis and spontaneous bacterial peritonitis, renal function frequently becomes impaired. this impairment is probably related to a reduction in effective arterial blood volume, and is associated with a high mortality rate. we conducted a study to determine whether plasma volume expansion with intravenous albumin prevents renal impairment and reduces mortality in these patients. methods: we randomly assigned 126 patients with cirrhosis and spontaneous bacterial peritonitis to treatment with intravenous cefotaxime (63 patients), or cefotaxime and intravenous albumin (63 patients). cefotaxime was given daily in dosages that varied according to the serum creatinine level, and albumin was given at a dose of 1.5 kg of body weight at the time of diagnosis, followed by 1 kg on day 3. renal impairment was defined as nonreversible deterioration of renal function during hospitalization. results: the infection resolved in 59 patients in the cefotaxime group (94%) and 62 in the cefotaxime-plus-albumin group (98%) (p = 0.36). renal impairment developed in 21 patients in the cefotaxime group (33%), and 6 in the cefotaxime-plus-albumin group (10%) (p = 0.002). eighteen patients (29%) in the cefotaxime group died in the hospital, as compared with 6 (10 percent) in the cefotaxime-plus-albumin group (p = 0.01); at three months, the mortality rates were 41 percent (a total of 26 deaths), and 22% (a total of 14 deaths), respectively (p = 0.03). patients treated with cefotaxime had higher levels of plasma renin activity than those treated with cefotaxime and albumin; patients with renal impairment had the highest values. conclusions: in patients with cirrhosis and spontaneous bacterial peritonitis, treatment with intravenous albumin in addition to an antibiotic reduces the incidence of renal impairment and death, in comparison with treatment with an antibiotic alone. the effect of intravenous albumin on renal impairment and mortality was studied in 126 patients with cirrhosis and spontaneous bacterial peritonitis. patients were randomly assigned to receive treatment with intravenous cefotaxime or cefotaxime and intravenous albumin. albumin was given at a dose of 1.5 g/kg body weight at the time of diagnosis, and 1 g/kg on day 3. the infection resolved in a similar number of patients in both groups. however, the incidence of renal impairment was very much lower in the cefotaxime plus albumin group (6 patients; 10%) than in the cefotaxime alone group (21 patients; 33%). furthermore, the mortality rates were lower in the cefotaxime plus albumin groups, i.e. 6 patients or 10% vs 18 patients or 29% for in-hospital mortality, and 14 patients (22%) and 26 patients (41%) at 3 months. the effects of whole bile and bile salts on the perfused heart severe sinus node dysfunction in obstructive jaundice obstructive jaundice blunts myocardial contractile response to isoprenaline in the dog: a clue to the susceptibility of jaundiced patients to shock? the megx test: a tool for the real-time assessment of hepatic function liver function and splanchnic ischemia in critically ill patients lignocaine metabolite formation: an indicator for liver dysfunction and predictor of survival in surgical intensive care patients assessment of prognosis in fulminant hepatic failure multivariate analysis of prognostic factors in fulminant hepatitis b early indicators of prognosis in fulminant hepatic failure: and assessment of the king's criteria liver transplantation in europe for patients with acute liver failure the effect of n-acetylcysteine on oxygen transport and uptake in patients with fulminant hepatic failure improved outcome of paracetamol-induced fulminant hepatic failure by late administration of acetylcysteine n-acetylcysteine increases liver blood flow and improves liver function in septic shock patients: results of a prospective, randomised, double-blind study molecular adsorbent recycling system (mars): clinical results of a new-membrane-based blood purification system for bioartificial liver support extracorporeal liver support: cell-based therapy for the failing liver key message complex forms of extracorporeal perfusion are possible in critically ill patients with acute liver failure and advanced encephalopathy. a focus on metabolic support (for example from functioning hepatocytes) rather than blood purification will most likely be successful spontaneous bacterial peritonitis bacterial infections in liver disease definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis. international ascites club the case is reported of a 29-year-old woman who developed presumptive viral hepatitis. her jaundice initially resolved after 6 weeks, but recurred soon after and was accompanied by a flapping tremor. her neurological state deteriorated markedly to grade iv encephalopathy. she was treated with a regimen of daily exchange transfusion for 6 days, but despite some apparent improvement related to these procedures, this was not sustained. she duly became apneic, and after an unsuccessful seventh exchange transfusion, she underwent cross-circulation with a baboon. the baboon had been especially prepared by washing out the blood volume with lactate solution under hypothermia on bypass, and then replacing the blood volume with human blood compatible with the patient. after about 12 hours, spontaneous ventilation returned, and there was an improvement in conscious state and the treatment was terminated. she remained neurologically impaired for two further days, and then improved markedly to become fully conscious with no abnormal neurological signs. her improvement persisted until publication of the case report. patients treated with cefotaxime had higher levels of plasma renin activity than those treated with cefotaxime and albumin, and patients with renal impairment had the highest values.the authors conclude that in patients with cirrhosis and spontaneous bacterial peritonitis, treatment with intravenous albumin in addition to an antibiotic reduces the incidence of renal impairment and death in comparison with an antibiotic alone. spontaneous bacterial peritonitis is a common complication in patients with cirrhosis and ascites, and may be accompanied by renal impairment, development or worsening of encephalopathy, and increased risk of death. treatment with non-nephrotoxic antibiotics has been associated with improved outcome, but volume expansion with albumin in addition to antibiotics results in further improvement in survival and a reduced incidence of renal impairment. the most likely explanation is that albumin infusion prevents hypovolemia and the subsequent activation of vasoconstrictor systems. however, mechanisms unrelated to plasma expansion cannot be ruled out. this study provides strong evidence that intravenous albumin therapy should be given, in addition to antibiotic therapy, to patients with cirrhosis and spontaneous bacterial peritonitis. the effects of albumin therapy are not known in patients with organic nephropathy and serum creatinine concentration >265 µmol/l because these groups were excluded from the study. this is a well-designed, randomized, controlled trial from the group who virtually singlehandedly have defined the management of ascites in cirrhosis. there are few. further studies should investigate the effect of different doses of albumin, and whether other plasma expanders are equally effective. this study does not specifically involve patients treated in an intensive care unit. nevertheless, spontaneous bacterial peritonitis is responsible for many admissions of cirrhotic patients to the icu, as it is sometimes a silent precipitant of gastrointestinal bleeding, encephalopathy, and renal impairment, as well as overt sepsis. indeed, it should be excluded in every cirrhotic patient admitted to the icu. consequently, advances in its treatment are very relevant to the icu clinician. furthermore, despite the current uncertainty as to the place of albumin therapy in critically ill patients, it seems clear that it confers a survival advantage to patients with cirrhosis and spontaneous bacterial peritonitis. key: cord-032131-ghgciqfk authors: ganschow, rainer; melter, michael; deutsch, johann title: lebertransplantation und leberversagen date: 2013 journal: p&#x000e4;diatrische gastroenterologie, hepatologie und ern&#x000e4;hrung doi: 10.1007/978-3-642-24710-1_19 sha: doc_id: 32131 cord_uid: ghgciqfk es kann geschätzt werden, dass etwa 2 von 10.000 neugeborenen oder 1–2 neugeborene pro 1 mio. einwohner jährlich auf eine lebertransplantation angewiesen sein werden. nachdem die erste lebertransplantation bei einem kind im jahre 1963 von starzl erfolgreich durchgeführt wurde (starzl 1969) , waren die ergebnisse in den folgenden zweieinhalb jahrzehnten zunächst aufgrund verschiedener ursachen noch mäßig. heutzutage hingegen stellt die lebertransplantation eine weit verbreitete kurative therapieoption beim chronischen und akuten leberversagen im kindes-und jugendalter dar, und die ergebnisse können in hinblick auf überleben und lebensqualität als sehr erfreulich gelten. in europa hat die lebertransplantation in den vergangenen 15 jahren ihren durchbruch gehabt, insbesondere durch entwicklung innovativer chirurgischer techniken, neue und verbesserte immunsuppressiva und nicht zuletzt durch den zugewinn an erfahrung der betreuenden teams. z indikationen es kann geschätzt werden, dass etwa 2 von 10.000 neugeborenen oder 1-2 neugeborene pro 1 mio. einwohner jährlich auf eine lebertransplantation angewiesen sein werden. die indikationen zur lebertransplantation bei kindern unterscheiden sich von denen erwachsener grundlegend. während bei erwachsenen patienten mit chronischem leberversagen vornehmlich infektionen (hepatitiden b und c), tumoren und äthyltoxische ursachen zur lebertransplantation führen, so sind es bei kindern und jugendlichen angeborene fehlbildungen des galleableitenden systems und familiäre cholestasesyndrome. eine übersicht über die zur lebertransplantation führenden grunderkrankungen gibt . tab. 19.1, wobei zu berücksichtigen ist, dass das indikationsspektrum von zentrum zu zentrum z. t. deutlich variieren kann. > die indikation zur lebertransplantation sollte bei chronischem leberversagen möglichst frühzeitig gestellt werden, sobald der krankheitsverlauf unzweifelhaft progredient ist, so dass mit einer späteren dekompensation zu rechnen ist. bei bestehendem akutem leberversagen ist die indikationsstellung jedoch selbst in erfahrenen zentren oftmals individuell und unter berücksichtigung diverser parameter und der dynamik der erkrankung zu stellen. im folgenden werden die charakteristika der wichtigsten zur lebertransplantation führenden krankheitsbilder kurz aufgeführt. k gallengangatresie bei der gallengangatresie (▶ abschn. 16.2) handelt es sich um eine erkrankung des frühen säuglingsalters, bei der insbesondere die großen gallengänge außerhalb der leber (extrahepatische gallengangatresie) zerstört werden, es können aber auch die intrahepatischen gallenwege mit betroffen sein. die zugrunde liegenden ursachen für diese morphologische fehlentwicklung sind letztlich noch unklar. es wer-den sowohl infektiologische als auch immunologische bzw. autoinflammatorische, aber auch genetische ursachen diskutiert. klinisch fallen betroffene kinder bereits in den ersten lebenswochen mit einem ikterus prolongatus mit einer hyperbilirubinämie durch direktes bilirubin sowie acholischen stühlen und gedeihstörung auf. je nach ausprägungsgrad der gallengangatresie sind laborchemisch im serum erhöhte cholestaseparameter (alkalische phosphatase, lipoprotein x, γ-glutamyltranspeptidase -γ-gt) nachweisbar. für die diagnosestellung ist der sonographisch fehlende nachweis einer gefüllten gallenblase darüber hinaus von besonderer bedeutung. sofern eine leberbiopsie realisiert werden kann, finden sich typische histologische befunde, welche die verdachtsdiagnose bestätigen. bei einigen patienten ist die portoenterostomie nach kasai (▶ abschn. 16.2) erfolgreich, sofern die leber noch nicht zirrhotisch umgebaut ist. es zeigt sich jedoch immer deutlicher, dass diese operation nur in sehr erfahrenen zentren durchgeführt werden sollte und selbst dort nicht immer den gewünschten erfolg erbringt. k familiäre cholestasesyndrome bei den familiären cholestasesyndromen (▶ abschn. 16.3) erfolgt die leberzellschädigung in der regel langsam und nicht selten über mehrere jahre, bis die indikation zur lebertransplantation gestellt werden muss. ursächlich liegen verschiedene defekte von gallensäurentransportproteinen vor, wobei für die klassischen formen korrespondierende gendefekte identifiziert werden konnten. unter dem begriff der progressiven familiären intrahepatischen cholestase (pfic) werden bislang 3 typen (pfic 1-3) unterschieden, welche sich klinisch nur geringfügig unterscheiden, wobei für den typ 3 eine erhöhte γ-gt-aktivität charakteristisch ist. der früher verwendete begriff "m. byler" entspricht der pfic 1 und sollte in der nomenklatur keine verwendung mehr finden. bei einigen patienten mit pfic der typen 1 und 2 kann die sog. partielle diversion, eine form der externen galleableitung, das fortschreiten der leberschädigung aufhalten und klinische symptome wie juckreiz bessern. k alagille-syndrom beim alagille-syndrom (▶ abschn. 16.3.2) handelt es sich um eine autosomal-dominant vererbte erkrankung mit sehr unterschiedlicher expressivität. es besteht ein defekt eines differenzierungsproteins aus der gruppe der notch-proteine, welcher sich klinisch in form verschiedener fehlbildungen an diversen organen manifestiert (ganschow et al. 2001c ). so können betroffene patienten beispielsweise eine periphere pulmonalstenose, wirbelkörperfehlbildungen ("schmetterlingswirbel"), ein sog. embryotoxon posterior an den augen, gefäßfehlbildungen und weitere anatomische normabweichungen zeigen. typisch für das alagille-syndrom ist eine charakteristische fazies (. abb ein entscheidender durchbruch gelang durch die entdeckung von ciclosporin a, welches seit 1982 auch zur immunsuppression bei kindern eingesetzt wird. es ist seither zum wichtigsten basisimmunsuppressivum in der pädiatrischen transplantationsmedizin geworden und hat zu einer dramatischen verbesserung der transplantationsfunktionsraten geführt. über eine hemmung des calcineurins wird eine reversible inhibition der nf-("nucleotide-binding-factor"-) κb-und -at-abhängigen gentranskription erreicht, so dass die zellen die zur proliferation erforderlichen zytokine (interleukin 2 und α-interferon) nicht mehr ausreichend bilden können. es resultiert eine inhibierung immunkompetenter cd4 + -t-lymphozyten mit konsekutiver störung der cd4vermittelten aktivierung von b-lymphozyten und antigenspezifischen zytotoxischen cd8 + -lymphozyten (ganschow 2003) . seit den 1990er jahren wird fast ausschließlich die galenisch verbesserte mikroemulsion des ciclosporins (sandimmun optoral) verwendet, welche den vorteil einer geringeren inter-und intraindividuellen variabilität in der absorptionsund abbaukinetik sowie eine bessere korrelation der talspiegel mit der ciclosporin-a-exposition in der darstellung mittels "area under the curve" bietet (kovarik et al. 1994 (kelly 2001) . bei einigen patienten werden aufgrund der notwendigkeit einer intensivierung der immunsuppressiven therapie purinantagonisten eingesetzt. das thioguaninderivat des 6-mercaptopurins, azathioprin, zählt zu den ältesten dieser substanzen. weitere substanzen sind mycophenolatmofetil (cellcept) und dessen neue, verkapselte form (myfortic). dieses halbsynthetische pilzderivat blockiert als reversibler, nichtkompetitiver inhibitor der inosinmonophosphatdehydrogenase 2 enzyme der purinsynthese und führt somit zur verminderung der bildung der guanosinphosphate gmp, gdp und gtp. daraus resultieren eine selektive hemmung der lymphozytären reaktion sowie eine inhibierung der b-zell-proliferation und -funktion (antikörperbildung). eine selektive hemmung der t-zell-aktivierung und -proliferation ermöglicht der monoklonale interleukin-2-rezeptor-antagonist basiliximab (simulect), welcher die α-kette des interleukin-2-rezeptors aktivierter t-zellen blockiert. dieser antikörper wird als induktionstherapie in der frühen postoperativen phase eingesetzt und gilt praktisch als nebenwirkungsfrei. erfahrungen mit basiliximab zeigen, dass die rate akuter abstoßungsreaktionen bei kindern nach lebertransplantation mit einer 2-maligen gabe am ersten und vierten postoperativen tag signifikant gesenkt werden kann (ganschow et al. 2001b) . die induktionstherapie mit polyklonalen antikörpern (okt3) sowie anti-thymozyten-und anti-lymphozyten-globulinen sollte im bereich der pädiatrischen lebertransplantation aufgrund der unerwünschten wirkungen nicht mehr zum einsatz kommen. weitere immunsuppressiva sind sirolimus (rapamycin) und everolimus. sirolimus ist ein makrolidantibiotikum mit ausgeprägter immunsuppressiver wirkung auf t-zellen, everolimus ein synthetisches derivat dieser substanz. obwohl sirolimus strukturell dem tacrolimus ähnelt und dasselbe intrazelluläre bindungsprotein nutzt, sind die wirkweisen der beiden substanzen dennoch verschieden. der rapamycin-fk-bindungsprotein-komplex bindet an 2 zielproteine, auch "target of rapamycin" genannt, und hemmt hierdurch die intrazelluläre signalübertragung von interleukin 2 und weiteren t-zell-wachstumsfaktoren. die zellen verbleiben in der g1-phase des zellzyklus, vermutlich über die blockade einer proteinkinase, welche für die replikation der zelle erforderlich ist. dieser antiproliferative effekt kann gewisse vorteile bei der therapie von transplantationspatienten mit malignen erkrankungen (z. b. hepatoblastom) mit sich bringen. diese m-tor-inhibitoren sind bislang jedoch nicht für den einsatz bei kindern nach lebertransplantation zugelassen, erste erfahrungen mit diesen substanzen liegen in den transplantationszentren jedoch schon vor. im vergleich zur transplantation anderer solider organe ist nach lebertransplantation eine z. t. deutlich geringere immunsuppression ausreichend. darüber hinaus liegen klinische und inzwischen auch immunologische hinweise dafür vor, dass säuglinge aufgrund einiger unreifer immunologischer funktionen eine weniger ausgeprägte immunsuppression benötigen als ältere kinder (ganschow et al. 2001a ). bei der mehrzahl der kinder kann die steroiddosis nach der lebertransplantation zügig reduziert werden, und spätestens nach einem jahr kann man diese medikation ganz absetzen. die dann noch verbleibende monotherapie mit ciclosporin a oder tacrolimus ist selbst in niedriger dosis für eine vielzahl der patienten ausreichend. bislang wurden keine verlässlichen immunologischen parameter identifiziert, die ein komplettes absetzen der immunsuppression nach einigen therapiejahren bei selektiven patienten rechtfertigen würden, so dass derzeit von einer lebenslangen immunsuppressiven therapie ausgegangen werden muss. der impfstatus sollte vor transplantation vervollständigt werden. totimpfungen nach transplantation sind jederzeit möglich, zeigen aber in abhängigkeit von der immunsuppression nicht immer eine impfantwort. von lebendimpfungen wird generell abgeraten, einige zentren impfen aber im verlauf gegen masern und windpocken. z komplikationen wenn auch die lebertransplantation im kindesalter derzeit in großen zentren mit einem sehr hohen maß an erfahrung und routine durchgeführt wird, so sind doch verschiedene chirurgische und medizinische komplikationen möglich. das ausmaß der postoperativen probleme ist in nicht unerheblichen umfang von der transplantatqualität, der medizinischen ausgangssituation des organempfängers sowie der guten interdisziplinären zusammenarbeit des betreuenden teams abhängig. k chirurgische komplikationen auch wenn die ursächlichen gründe nicht näher bekannt sind, so kann es in seltenen fällen in der frühen postoperativen phase zu einem primären transplantatversagen kommen, welches eine zeitnahe retransplantation notwendig macht. insbesondere bei kleinen säuglingen erfolgt häufiger bei vergleichsweise großem transplantat nach vorübergehender anlage eines goretex-patches ein sekundärer bauchdeckenverschluss. klassische chirurgische komplikationen stellen primäre nahtinsuffizienzen im bereich der gefäße und des gallengangsystems dar, die oftmals sekundär chirurgisch revidiert werden müssen. besonderen stellenwert für die morbidität und mortalität haben thrombotische komplikationen, die vornehmlich die pfortader und die a. hepatica betreffen. nur bei rascher diagnostik kann hier eine chirurgische intervention abhilfe schaffen. thrombotische komplikationen sind jedoch nicht nur von der chirurgischen technik, sondern auch vom intensivmedizinischen management und einer adäquaten antikoagulativen therapie abhängig (ganschow et al. 2000) . entscheidend für die frühzeitige diagnosestellung einer solchen komplikation ist die sowohl intra-als auch postoperativ mit großer sorgfalt und erfahrung durchzuführende dopplersonographische untersuchung des transplantats. k infektionen in mehr als 50 % der fälle treten in der frühen postoperativen phase trotz antibiotischer prophylaxe unter der anfangs noch recht ausgeprägten immunsuppression und mit den in den ersten tagen noch vorhandenen eintrittspforten (zentraler venenzugang, wunddrainagen) infektionen auf. es handelt sich hierbei überwiegend um bakterielle infektionen (etwa 75 %), wohingegen virale (etwa 20 %) und pilzinfektionen (etwa 5 %) seltener beobachtet werden. als fokus findet sich häufig eine abdominale oder pulmonale infektion bzw. eine direkte assoziation mit in situ befindlichem fremdmaterial. wenn auch die mehrzahl der infektionen erfolgreich behandelbar ist, so ist dennoch eine sepsis mit konsekutivem multiorganversagen möglich und gefürchtet, vornehmlich bei hochrisikopatienten. von besonderer bedeutung nach pädiatrischer lebertransplantation ist das zytomegalievirus (cmv), da insbesondere säuglinge und kleinkinder zum zeitpunkt der transplantation einen cmv-negativen status aufweisen, dann jedoch mit hoher frequenz cmv-positive transplantate erhalten. je nach status der mütterlich übertragenen cmvspezifischen antikörper beim säugling mit risikokonstellation sowie zentrumspezifisch wird eine cmv-prophylaxe mit ganciclovir oder immunglobulinen durchgeführt. eine akute cmv-infektion oder -reaktivierung nach der transplantation kann sich klinisch insbesondere in form einer hepatitis sowie einer intestinalen oder pulmonalen beteiligung äußern. darüber hinaus liegen hinweise auf einen zusammenhang zwischen cmv-infektion und gallengangkomplikationen vor (halme et al. 2003) . eine frühzeitige diagnostik (polymerasekettenreaktion, "early antigen pp65") und eine spezifische therapie sind für den erhalt der transplantatfunktion von besonderer bedeutung (meyer-koenig et al. 2004) . weniger häufig und relevant sind infektionen mit dem epstein-barr-virus (ebv) nach lebertransplantation. die primäre ebv-infektion oder -reaktivierung ist klinisch oftmals durch eher milde und unspezifische symptome geprägt. eine spezifische therapie ist nicht möglich. bei klinischer indikation ist jedoch ein therapieversuch mit aciclovir oder auch eine immunglobulintherapie zu diskutieren. die hauptproblematik einer aktiven ebv-infektion nach lebertransplantation besteht in dem zusammenhang mit dem auftreten maligner lymphome ("posttransplant lymphoproliferative disease", ptld; . abb. 19.3). das ptld-risiko scheint nicht nur in hohem ausmaß mit der ebv-infektion, sondern auch mit der intensität der immunsuppression in verbindung zu stehen. so konnte gezeigt werden, dass sich das ptld-risiko durch therapieprotokolle mit geringer immunsuppression drastisch senken lässt -trotz primärer ebv-infektionen nach lebertransplantation im kindesalter (ganschow et al. 2004 ). therapeutisch stehen neben der reduktion der immunsuppression bei ptld auch anti-cd20-antikörper zur verfügung, mit denen gute erfolge erzielt werden können, sofern es sich um ein cd20 + -b-zell-lymphom handelt. z ergebnisse wie eingangs bereits erwähnt, sind mit der lebertransplantation im kindesalter hervorragende ergebnisse zu erzielen (spada et al. 2009 ). grundsätzlich ist der postoperative erfolg nach pädiatrischer lebertransplantation von verschiedenen prä-, peri-und postoperativen faktoren abhängig. zu den präoperativ relevanten zählen die grunderkrankung, voroperationen, der ernährungszustand des kindes, begleiterkrankungen oder fehlbildungen sowie das stadium des leberversagens (unos-kriterien oder peld-score). während der operation sind die dauer der kalten ischämie, die transplantatqualität, die chirurgische technik und das anästhesiologische management von besonderer bedeutung. auch ist der transplantationsmodus relevant, mit gewissen vorteilen der leberlebendspende (roberts et al. 2004) . nach erfolgter transplantation ist einerseits das intensivmedizinische vorgehen entscheidend, andererseits eine suffiziente diagnostik und eine adäquate therapie chirurgischer und internistischer komplikationen. für europa liegen nunmehr daten über pädiatrische lebertransplantationen seit anfang der 1990er jahre vor. es besteht kein zweifel daran, dass die patienten-und transplantatüberlebensraten seitdem kontinuierlich ansteigen, was vornehmlich auf den zugewinn an erfahrung zurückzuführen ist (burdelski et al. 1999) . auch die daten us-amerikanischer und kanadischer zentren weisen kontinuierlich verbesserte zahlen aus (martin et al. 2004) . ein vergleich der europäischen mit den amerikanischen daten zeigt, dass in europa aufgrund der häufiger angewandten lebendspende-und split-technik sowie der signifikant geringeren immunsuppression die gesamtrate an schweren komplikationen niedriger ist. daten zum langzeitverlauf müssen auf der anderen seite jedoch noch zeigen, ob z. b. die vergleichsweise geringe immunsuppression evtl. mit einer erhöhten rate an chronischer transplantatdysfunktion einhergeht, was allerdings bislang noch nicht aus der datenlage ersichtlich ist. bei der beurteilung des gesamtlangzeiterfolgs nach lebertransplantation im kindesalter sind neben den daten des patienten-und transplantüberlebens auch psychosoziale und sozioökonomische aspekte zu berücksichtigen (bucuvalas u. ryckman 2002) . es ist heutzutage -ein größenkompatibles transplantat vorausgesetzt -unproblematisch, auch bei sehr kleinen kindern in den ersten lebenstagen eine transplantation vorzunehmen (. abb. 19.4). die analyse der ergebnisse von 43 kindern, welche im alter von bis zu 6 monaten lebertransplantiert wurden, erbrachte exzellente kurz-und langzeitergebnisse mit einer überlebensrate von 90,7 % nach einem jahr (grabhorn et al. 2004) . neben der somatischen entwicklung nach lebertransplantation sind auch die kognitive entwicklung sowie die lebensqualität der kinder zur beurteilung des gesamterfolgs wichtig. entsprechende studien konnten zeigen, dass die kognitive entwicklung der kinder insgesamt sehr gut ist, wobei im säuglingsalter operierte kinder bei den psychologischen tests noch etwas besser abschnitten als später operierte patienten (schulz et al. 2003; kaller et al. 2010) . bei einigen kindern wird durch den einsatz der calcineurininhibitoren ciclosporin und tacrolimus eine einschränkung der glomerulären filtrationsrate (gfr) beobachtet. dies ist einerseits in den ersten monaten nach der transplantation der fall, wobei sich die nephropathie mittelfristig bei den meisten kindern signifikant bessert. auch bezüglich dieses aspekts scheint die transplantation im säuglingsalter vorteile zu haben (aroragupta et al. 2004) . auf der anderen seite ist die cni-bedingte nephrotoxizität im langzeitverlauf definitiv unterschätzt worden, was neuere daten zeigen (brinkert et al. 2011) . es konnte gezeigt werden, dass die allgemein übliche bestimmung der gfr mittels schwartz-formel bei lebertransplantierten kindern keine ausreichend validen daten liefert. dieser aspekt stellt eine große aktuelle herausforderung dar, denn es gilt, protokolle zur immunsuppression zu entwickeln, mit denen eine reduktion der cni-toxizität erreicht werden kann. da einige kinder und jugendliche vor der lebertransplantation eine signifikante wachstumsverzögerung aufweisen und sich immunsuppressive medikamente (steroide) nach der transplantation ungünstig auf die somatische entwicklung auswirken können, ist die analyse des wachstums nach lebertransplantation von besonderer bedeutung. es konnte gezeigt werden, dass bei der überwiegenden mehrzahl der patienten nach der transplantation ein aufholwachstum einsetzt und sich eine normale endgröße erreichen lässt (fine 2002) . z ausblick trotz der inzwischen sehr guten ergebnisse, die mit der lebertransplantation im kindesalter erzielt werden können, sind weitere verbesserungen zur reduktion der morbidität und zur verbesserung der lebensqualität möglich. vonseiten der leberchirurgie wird es in den kommenden jahren möglich sein, an weiteren zentren die leberlebendspende und die split-technik anzuwenden, was den vorteil mit sich bringen wird, die kinder wenn möglich in einem heimatnahen transplantationszentrum betreuen zu können. durch eine frühzeitige vorstellung von kindern mit akutem oder chronischem leberversagen in einem geeigneten transplantationszentrum und unter ausnutzung sämtlicher möglicher transplantationstechniken sollte es möglich sein, die mortalität auf der lebertransplantationswarteliste für kinder auf null zu senken. auch wenn derzeit schon hervorragende medikamente für die immunsuppression für kinder zur verfügung stehen, so werden weitere medikamente für spezielle einsatzgebiete anwendung finden. kombinationstherapien mit verschiedenen wirkansätzen werden nicht nur bei erwachsenen, sondern auch bei kindern zu einer optimierung der therapie führen. bei der induktionstherapie ist zu erwarten, dass der einsatz von monoklonalen interleukin-2-rezeptor-antagonisten eine signifikante reduktion der steroiddosis ermöglichen wird, wobei kontrollierte studien hierzu noch nicht publiziert sind. die von einigen zentren angestrebte steroidfreie lebertransplantation bei kindern wird hingegen sehr kontrovers diskutiert. inwieweit sich weitere substanzen für pädiatrische patienten eignen, wird sich in den kommenden jahren zeigen: fty720 ist ein neuer immunmodulator, welcher die einwanderung von lymphozyten in das transplantat erschwert und so zu einer hemmung von antigenkontakt, aktivierung und proliferation führt. ctla4-igg-fc und anti-cd40 l sind fusionsmoleküle, welche die kostimulation blockieren und die apoptose induzieren. alemtuzumab (campath 1h) ist ein humanisierter anti-cd52-antikörper, der im rahmen von studien zur toleranzinduktion eingesetzt wird. es besteht in jedem fall bedarf, durch eine geeignete auswahl immunsuppressiver medikamente, den nephrotoxischen effekt der calcineurininhibitoren im langzeitverlauf zu reduzieren. wesentlich bedeutender als die anwendung neuer substanzen zur immunsuppression wird künftig die individualisierung der protokolle zur immunsuppression nach pädiatrischer lebertransplantation werden. es liegen inzwischen ausreichende daten vor, welche dazu führen werden, dass eine risikoadaptierte therapie anwendbar ist. säuglinge wird man mit einer geringeren immunsuppression, kinder und jugendliche mit autoimmunhepatitis oder primär sklerosierender cholangitis eher mit einer intensivierten immunsuppression behandeln. ziel muss es darüber hinaus sein, immunologische parameter zu identifizieren, welche alters-und diagnoseunabhängig eine individuelle immunsuppression begründen können. neben parametern der individuellen t-zell-immunität (ganschow et al. 2001a ) könnten hierzu auch zytokingenpolymorphismen herangezogen werden (mazariegos et al. 2002) . derzeit noch ungelöst ist das problem, welche immunologischen parameter als surrogatmarker es erlauben werden, bei selektiven patienten im langzeitverlauf nach lebertransplantation die immunsuppression ganz abzusetzen. ebenso bleibt es spekulation, ob dies nach verwandtenspende aufgrund der zugrunde liegenden haploidentität besser gelingen wird. von besonderer relevanz ist auch die differenziertere pathogenetische charakterisierung der chronischen transplantatdysfunktion. zugrunde liegende pathomechanismen müssen ebenso wie evtl. vorhandene prädisponierende oder risikofaktoren erfasst werden. die chronische transplantatdysfunktion bis hin zum transplantatverlust gewinnt derzeit bereits zunehmend an bedeutung, da nunmehr viele lebertransplantierte kinder in das jugend-und erwachsenenalter gelangt sind. problematisch hierbei ist nicht nur die retransplantation per se, sondern auch die lange wartezeit auf der warteliste erwachsener. somit ist es auch schon jetzt im interesse der transplantierten kinder, dass durch eine höhere bereitschaft zur organspende und vielleicht auch durch einen weiteren ausbau der leberlebendspende zwischen erwachsenen dem aktuell noch bestehenden ausgeprägten mangel an spenderorganen entgegengewirkt wird. letztendlich gilt es für die weitere optimierung der ergebnisse zu untersuchen, welchen einfluss die transplantatqualität und das -alter auf die langzeitergebnisse nach lebertransplantation im kindesalter haben. das akute leberversagen (alv) wird im kindesalter unabhängig vom vorliegen einer enzephalopathie definiert, wenn bei patienten ohne vorher bekannte lebererkrankung innerhalb von 8 wochen eine schwere beeinträchtigung der leberfunktion mit einer konjugierten hyperbilirubinämie, einer lebersynthesestörung (quick-test <40 %; cholinesteraseaktivität <2,5 ku/l) und ggf. einer transaminasenerhöhung eintritt (melter et al. 1996) . es ist selten und war unter rein supportiven maßnahmen bis anfang der 90er jahre des letzten jahrhunderts mit einer verheerenden prognose (letalität 70 % bis >95 %) assoziiert. bei den überlebenden kommt es meist zu einer spontanen und kompletten "restitution". unter moderner pädiatrischer intensivmedizinischen maßnahmen gelang es in hierauf hochspezialisierten pädiatrischen zentren, die spontane restitution auf deutlich über 50 % zu erhöhen (lee et al. 2008) . die pädiatrische lebertransplantation ermöglicht bei alv heute überlebensraten von 70-87 %; in großen zentren macht diese indikation ca. 10-15 % der bei kindern durchgeführten lebertransplantation aus (lee et al. 2008; melter et al. 2002; baliga et al. 2004 ). z inzidenz epidemiologische untersuchungen zur inzidenz des pädiatrischen alv fehlen. der "erhebungseinheit für seltene pädiatrische erkrankungen in deutschland" (esped) wurden über 2 jahre aus 82 kinderkliniken 127 kinder gemeldet, von 19.2 • akutes leberversagen denen 70 die oben genannten kriterien erfüllten (brockstedt et al. 1997) . als eines der größten pädiatrisch-hepatologischen zentren behandelten wir an der medizinischen hochschule hannover in der zeit von 1986 bis 1996 50 kinder mit alv (einschließlich 21 % der esped-fälle). z ätiologie ursachen für das pädiatrische alv sind infektiöse, medikamentös/toxische, metabolische, autoimmunologische, ischämische und/oder maligne erkrankungen (. tab. 19.2). die ätiologie bleibt allerdings bei bis zu 50 % der fälle ungeklärt (lee et al. 2008) . in anbetracht der verbesserten diagnostischen möglichkeiten betrug der diesbezügliche anteil mitte der 1990er jahre in der esped-studie bundesweit 16 %, in unserem zentrum 7 % (. tab. 19.2) (brockstedt et al. 1997) . der anteil an den verschiedenen kategorien des pädiatrischen alv steht in direktem verhältnis zum alter der patienten. so stehen bei säuglingen hereditäre und metabolische erkrankungen, bei älteren kindern virushepatitiden im vordergrund (melter et al. 1996; brockstedt et al. 1997) . während prinzipiell jeder erreger ein alv auslösen kann, machen die klassischen hepatitiden den größten anteil aus, vor allem in entwicklungs-und schwellenländern. in regionen mit endemischer hepatitis e ist diese inzwischen der wesentliche ätiologische faktor des pädiatrischen alv (khuroo u. kamili 2003) . es gibt aber auch berichte über bakterielle erreger oder deren toxine, wie z. b. das toxin von bacillus cereus, die ein (pädiatrisches) alv verursachen können (posfaybarbe et al. 2008; mahler et al. 1997) . medikamentös-toxische ursachen werden in den meisten studien als zweithäufigste ursache gefunden (melter et al. 1996) . im prinzip kann jede substanz leberschädigend und ursächlich für ein alv sein. dabei wirken die substanzen entweder direkt oder über eine idiosynkrasie hepatotoxisch. häufigere auslöser sind paracetamol (ca. 20 % und damit die häufigste nachweisbare ursache des pädiatrischen alv in den usa), halothan, valproat, amanita phalloides (knollenblätterpilztoxin) und seltener kupfer (trinkwasser aus eigenem brunnen) (nguyen u. vierling 2011) . in den letzten jahren nehmen nichtparacetamolinduzierte idiosynkratische medikamentenverursachte alv zu (nguyen u. vierling 2011; kneiseler et al. 2010) . innerhalb dieser gruppe nehmen auch die komplementären, alternativen therapien (z. b. naturheilkundlich, pflanzlich), nahrungsergänzungsmittel etc. als auslöser eines alv deutlich zu (nguyen u. vierling 2011; stickel et al. 2005) . und selbst natürliche substanzen wie brokkolisaft (hoffentlich erfahren unsere kinder nichts davon, dass gemüse oder obst gefährlich sein kann) können im einzelfall für ein alv ursächlich sein (ekiz et al. 2010) . die metabolischen ursachen eines alv weisen eine deutliche altersabhängigkeit auf: autoimmunhepatitiden werden überwiegend bei älteren kindern diagnostiziert, wobei sehr selten auch systemische autoimmunerkrankungen (z. b. systemischer lupus erythematodes) beteiligt sein können. die riesenzellhepatitis wird zumindest teilweise durch paramyxoartige viren verursacht und deshalb auch "hepatitis g" ("giant cell hepatitis") genannt. sie findet sich sehr selten auch nach der säuglingszeit und dann z. t. in kombination mit immunhämolytischer anämie und/ oder autoimmunthrombozytopenie (grothues et al. 2003) . beim budd-chiari-syndrom, venenverschluss-syndrom ("venoocclusive disease"; vod), akutem kreislaufversagen und septischem schock ist die leberzellnekrose überwiegend auf die ischämie, bei malignen erkrankungen auf eine direkte hepatische infiltration zurückzuführen. beide erkrankungsgruppen sind nur ausnahmsweise ursache eines pädiatrischen alv. beim reye-/reye-artigen syndrom ist die transaminasen-und ammoniakerhöhung sowie enzephalopathie obligat, ein ikterus aber die ausnahme. es ist histologisch durch eine mikrovesikuläre steatose bei vorangegangenem akutem virusinfekt charakterisiert. salicylate, aber auch paracetamol wurden als auslösende kofaktoren postuliert. eine sog. non-a-bis non-e-hepatitis ist als ausschlussdiagnose, vor allem bei älteren kindern und erwachsenen, eine der häufigsten ursachen eines alv. einzigartig ist hierbei eine sonst bisher nur bei parvovirus-b19-oder hhv-6-infektionen beobachtete assoziation mit einer aplastischen anämie (panmyelopathie), die vor allem in der pädiatrischen klientel beobachtet wird. interessanterweise wird eine aplastische anämie bei dieser ätiologie auch nach erfolgreicher pädiatrischer lebertransplantation sowie im intervall nach restitution nach pädiatrischem alv beobachtet. z pathophysiologie und klinisches bild der hepatischen enzephalopathie (he) bestandteil der ursprünglichen definition eines fulminanten leberversagen war die hepatische enzephalopathie (he; melter et al. 1996) , die klinisch äußerst variabel verläuft und mit dem ausmaß, der geschwindigkeit und ursache des leberversagens bei erwachsenen korreliert. bei kindern und jugendlichen ist die he, wenn es nicht zu strukturellen zerebralen veränderungen oder intrakraniellen blutungen gekommen ist, oft mit einer guten prognose assoziiert. die pathophysiologie der he ist ungeklärt. außer einem zerebralen ödem lassen sich strukturell-anatomische veränderungen nicht nachweisen. das akute auftreten sowie die möglichkeit der restitutio ad integrum sprechen gegen eine strukturelle störung und für eine funktionelle (biochemische) grundlage. hierfür werden im wesentlichen 4 hypothesen diskutiert (melter et al. 1996): die ammoniak-hypothese, die hypothese synergistisch wirkender neurotoxine, die hypothese falscher neurotransmitter und die hypothese γ-aminobuttersäure-(gaba-)artig inhibierender neurotransmitter. keine dieser hypothesen allein vermag die gesamte pathophysiologie der he zu erklären, so dass man auch wegen der zentralen rolle der leber im intermediären stoffwechsel von einer multifaktoriellen genese ausgehen muss. zur beurteilung ist neben der klinischen einteilung, ein eeg obligat, für das ebenfalls eine einheitliche stadieneinteilung fehlt. z diagnostik die diagnostik dient zunächst der feststellung des alv und des ausmaßes der hepatozellulären schädigung. hierzu werden parameter der zellnekrose, der lebersyntheseleistung und des neurologischen status erhoben. darüber hinaus erfolgt eine evaluation des metabolischen und elektrolytstatus, der funktion anderer organe und der ätiologie des palv. (melter et al. 1996; ngyuen u. vierling 2011) . historisch betrug die überlebensrate unter rein supportiver therapie 6-29 % (melter et al. 1996) . eine verbesserung der prognose unter rein supportiven maßnahmen (lee et al. 2008; melter et al. 1995) . auch für das überleben nach pädiatrischer lebertransplantation hat sich der zeitpunkt des eingriffes als entscheidender parameter erwiesen. der rechtzeitige transfer eines kindes in ein spezialisiertes zentrum mit allen therapeutischen optionen (einschließlich der pädiatrischen lebertransplantation) hat sich daher auch als wesentlicher prädiktiver faktor für das überleben herausgestellt (vickers et al. 1988; arya et al. 2010) . die (lebenslange) immunsuppression bei einer lebertransplantation sowie die tatsache, dass es bei einem relevanten anteil an patienten mit pädiatrischem alv auf grund der hohen regenerationsfähigkeit der leber auch unter reiner supportivtherapie zu einer "spontanen" restitution kommt, zwingt zu einer möglichst präzisen individuellen prognostischen einschätzung. das größte dilemma bei pädiatrischem alv besteht daher darin, festzulegen, ob und wann eine lebertransplantation als einzige option verbleibt. wenngleich zahlreiche studien prädiktive faktoren für das transplantationsfreie überleben bei alv zu bestimmen versuchten, sind diese bisher -insbesondere für das pädiatrische alv -unzureichend definiert. klinisch hat sich bei erwachsenen gezeigt, dass eine volumenreduktion der leber ("schrumpfende leber") mit oder ohne sinkende transaminasen/gldh bei gleichzeitig unverändert persistierender oder gar eskalierender hyperbilirubinämie und plasmatischer gerinnungsstörung mit einer schlechten prognose assoziiert ist. darüber hinaus wurden, ebenfalls bei erwachsenen, folgende parameter als prognoserelevant evaluiert: alter, ätiologie, enzephalopathiegrad und die höhe verschiedener gerinnungsparameter (melter et al. 1996) . zusätzlich zeigte sich, dass die prognose invers mit einer früh einsetzenden enzephalopathie bzw. foudroyant verlaufenden hepatitis (je kürzer das intervall, desto besser die prognose) korreliert ist (o'grady et al. 1989) . beim toxischen leberversagen korreliert die überlebensrate nicht mit dem blutspiegel oder der eingenommenen dosis des jeweiligen toxins (o'grady et al. 1989) . für das alv bei paracetamolintoxikation konnte gezeigt werden, dass die paracetamoldosis weder mit den klinischen prognoseindikatoren noch mit der prognose assoziiert ist, dass diese aber sehr eng mit einer metabolischen acidose (ph-wert <7,3) korreliert (gegory et al. 2010) . insbesondere bei pädiatrischem alv sind aufgrund der begrenzten patientenzahlen, der großen ursachenvarianz und des großen altersspektrums (0-18 jahre) aber prädiktoren eines spontanen überlebens nur begrenzt zu definieren. in einer eigenen und in zwei weiteren studien in großen pädiatrischen zentren erwiesen sich in dem kollektiv der patienten mit pädiatrischem alv neben einer ungeklärten ätiologie, der schwere der he und der nierenersatztherapiepflichtigen niereninsuffizienz nur die höhe des bilirubins und verschiedener plasmatischer gerinnungsparameter (faktor v <20 %, ptt-verlängerung, "international normalized ratio" (inr) ≥4) als unabhängige prädiktoren des versterbens bei pädiatrischem alv (bhaduri u. mieli-vergani 1996; devictor et al. 1992) . unter zugrundelegung der hypothese, dass auf grund der raschen teilung von leberzellen bei einer schnellen regeneration der leber eine erhöhte adenosintriphosphat-(atp-)synthese und konsekutiv hierzu ein erhöhter bedarf an anorganischem phosphat besteht, haben baquerizo und mitarbeiter den prädiktiven wert einer hypophosphatämie bezüglich des transplantationsfreien überlebens bei alv untersucht (baquerizo et al. 2003) . es hat sich hierbei gezeigt, dass eine hypophosphatämie und deren frühzeitige substitution mit phosphat mit einer guten prognose assoziiert ist, während eine hyperphosphatämie einen prädiktor für eine ausgeprägte leberzellnekrose und schlechte leberrestitution darstellt (ozturk et al. 2010) . im tiermodell wurden die serumspiegel von α-aminoadipinsäure und α-aminobuttersäure (aab)als valide prädiktoren einer guten hepatischen regeneration gefunden (rudnick et al. 2009 ). um den prognostischen wert dieser parameter bei pädiatrischem alv zu überprüfen, wurden diese retrospektiv im serum gut definierter patienten mit pädiatrischem alv gemessen. dabei fand sich bei patienten mit spontanem überleben im vergleich zu patienten, die verstarben oder transplantiert wurden, sowohl ein signifikant erhöhter aab-spiegel, als auch eine erhöhte aab-leucin-ratio (rudnick et al. 2009 ). neben diesen diagnoseunabhängigen faktoren ist die grunderkrankung selbst ein wesentlicher prädiktor des transplantationsfreien überlebens bei pädiatrischem alv. so ist eine autoimmunhepatitis, vor allem bei jüngeren kindern, mit einer schlechten prognose (selbst nach lebertransplantation) assoziiert, wenn diese nicht kurzfristig auf eine immunsuppressive therapie (steroid, azathioprin) anspricht (vogel et al. 2004) . demgegenüber ist unter suffizienter therapie die chance auf eine restitution bei m. wilson, wenn es nicht zu extrahepatischen komplikationen (vor allem he) kommt, selbst bei extremen gerinnungsstörungen relativ gut. allerdings scheint auch beim m. wilson z. b. eine ausgeprägte hyperbilirubinämie (≥300 µmol/l bzw. 17,5 mg/dl), evtl. auch eine hyperphosphatämie, zeichen für eine schlechte prognose zu sein. z therapie und klinisches management die therapie des pädiatrischen alv beruht auf 3 säulen: -supportivtherapie, spezifische therapie der zugrunde liegenden erkrankung und -leberersatztherapie. eine spezifische therapie der zugrunde liegenden erkrankung (. tab. 19 .3) ist nur selten möglich. beispiele hierfür sind die elimination der auslösenden substanz bei hämochromatose oder m. wilson. obwohl die wirksamkeit einer virostatischen therapie bei replikativer hepatitis noch umstritten bzw. deren effektivität (noch) nicht nachgewiesen ist, empfehlen wir generell auch hier einen therapieversuch. (. tab. 19 .3), einschließlich einer "aggressiven" pädiatrischintensivmedizinischen betreuung, dar. hierzu ist eine frühzeitige verlegung des patienten in ein pädiatrisches zentrum, das alle therapeutischen optionen (einschließlich der pädiatrischen lebertransplantation) vorhält, notwendig. bei entsprechendem mangel ist ein versuch mit einer intravenösen vitamin-k-substitution über mehrere tage indiziert. im gegensatz dazu ist eine prophylaktische verabreichung von gerinnungspräparaten ohne protektiven wert. (kamat et al. 2012) . dieses vorgehen ist allerdings umstritten und keinesfalls als standard zu verstehen. andererseits ist es unstrittig, dass bei he frühzeitig maßnahmen zur durchführung einer lebertransplantation zu ergreifen sind. eine therapie mit n-acetylcystein scheint sauerstofftransport und -aufnahme bei patienten mit pädiatrischem alv unabhängig von der ätiologie zu verbessern (harrison et al. 1991) . darüber hinaus erscheint uns auch eine erweiterte antioxidanzientherapie (vitamin e, selen, prostaglandin e 1 / e 2 ) sinnvoll. bei einer assoziierten aplastischen anämie kann die verabreichung von "granulocyte colony-stimulating factor" (g-csf) therapeutisch hilfreich sein. auf der hypothese, dass multiplen toxinen eine wesentliche rolle in der entstehung und unterhaltung der symptomatik bei alv zuzuschreiben ist, beruht der therapeutische ansatz der "systemischen detoxifikation". hierzu wurden zahlreiche therapeutische verfahren eingesetzt, zellfreie systeme (z. b. hämodiafiltration, plasmapherese, hämoperfusion [kohle-, bindungsharzfilter], dialyse, austauschtransfusion) und sog. bioartifizielle leberstützende systeme, die vitale hepatozyten beinhalten (ringe et al. 2011; carpentier et al. 2009; schaefer et al. 2011) . dieser verschiedenen leberunterstützenden bzw. -ersetzenden maßnahmen und die hepatozytentransplantation sind in der entwicklung befindliche, z. t. vielversprechende ansätze. mittels dieser therapien könnte zukünftig eine überbrückung bis zur restitution der eigenleberfunktion oder bis zu einer lebertransplantation sicherer oder länger gelingen (carpentier et al. 2009 ). bisher konnte allerdings für keines dieser verfahren -einschließlich des "molecular absorbent recyc-ling system" (mars; dialyse über eine albuminbeschichtete membran) -ein signifikanter einfluss auf das transplantationsfreie patientenüberleben nachgewiesen werden. dennoch werden diese verfahren heute zunehmend eingesetzt. indikationen hierfür sind vor allem die he, hepatorenales-und/ oder hepatopulmonales syndrom. die wenigen vorliegenden vergleichenden studien zu verschiedenen extrakorporalen methoden bei pädiatrischem alv können dabei zumindest keinen vorteil für das mars-verfahren gegenüber z. b. einer kombination von plasmapherese und hämodialyse zeigen (schaefer et al. 2011) . der einsatz anderer therapien, wie die applikation von leberwachstumsfaktoren, passagere "ex-vivo-leber", passagere ex-vivo-oder orthotope xenografttransplantationen, sind heute rein experimentell oder erfolglos. die lebertransplantation stellt somit derzeit die einzig kurative therapie bei "nichtregenerativer" lebernekrose dar. sie kann aufgrund ihrer entwicklung in den letzten 15 jahren als etablierte standardtherapie auch bei pädiatrischem alv bezeichnet werden und sollte prinzipiell bei allen hiervon betroffenen patienten rechtzeitig erwogen werden. hierbei ist auch zu bedenken, dass nach den derzeit geltenden regeln von euro-transplant eine höchst dringliche ("high urgency") meldung für eine lebertransplantation nur innerhalb der ersten wochen eines alv möglich ist. eine lebertransplantation ist dann indiziert, wenn die chance auf eine spontanremission deutlich verringert ist (s. unten). daher stellen wir die indikation zur lebertransplantation z. b. bei autoimmunhepatitis, wenn es unter einer suffizienten immunsuppressiven therapie nicht innerhalb von wenigen wochen zu einer merklichen verbesserung kommt. in einer großen kanadischen single-center-studie bezüglich des langzeitüberlebens bei erwachsenen mit lebertransplantation aufgrund eines alv wurde gezeigt, dass ein sehr gutes langzeitüberleben nur dann erzielt werden kann, wenn auf der einen seite entsprechend "gute" transplantate (ab0-kompatibel, keine extended-criteria-organe) verwendet werden und es auf der anderen seite beim empfänger vor der lebertransplantation nicht zu einer irreversiblen zerebralen störung gekommen ist (chan et al. 2009 ). in abhängigkeit von der grundkrankheit, den anatomischen und histologischen gegebenheiten sollte dabei heute auch eine sog. temporäre auxiliäre (unterstützende) partielle orthotope lebertransplantation (apolt) erwogen werden. diese technik, bei der ein teil der eigenleber durch einen teil einer spenderleber ersetzt wird, ist schon früh vereinzelt auch bei kindern erfolgreich eingesetzt worden (melter et al. 1995) . ein wesentliches problem dieser technik stellt die (portale) perfusion dar, um die beide leberanteile konkurrieren. allerdings scheinen moderne chirurgische verfahren dieses problem überwinden zu können (faraj et al. 2010; knoppke et al. 2011) . eine apolt könnte dabei, ebenso wie bei anderen formen einer leberresektion, selbst trigger für eine frühzeitige und vermehrte regeneration hepatischer zellen via wachstumsfaktoren wie "hepatocyte growth factor" (hgf) und andere sein. der vorteil dieser therapie besteht darin, dass im falle der restitution der eigenleber die immunsuppression beendet werden kann, wonach die transplantatleber eigenständig atrophiert (knoppke et al. 2011) . wir sehen daher, ebenso wie z. b. die kollegen des king's college, london, die apolt -wenn sie möglich ist -als ideale option insbesondere dann an, wenn die ätiologie des pädiatrischen alv unklar ist und die möglichkeit einer regeneration der nativen leber besteht (shanmugam u. dhawan 2011) . allgemein akzeptierte kontraindikation für eine lebertransplantation stellen eine unkontrollierte sepsis, schwerwiegende, irreversible neurologische störungen und "globale" mitochondriale hepatopathien dar (thomson et al. 1998) . wenn solche nicht vorliegen, stellen wir die indikation zur lebertransplantation entsprechend folgender parameter: hepatische enzephalopathie > grad 2 und/oder -faktor v <20 % ohne signifikanten anstieg nach (2-maliger) suffizienter, gewichtsadäquater ffp-verabreichung (10-20 ml/kg kg) und/oder -gesamtbilirubin ≥300 µmol/l (17,5 mg/dl) und/oder -serumphosphat oberhalb der oberen normgrenze 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toxins key: cord-342808-yonbowkb authors: francque, sven title: innovative liver research continues during the current pandemic date: 2020-05-24 journal: jhep rep doi: 10.1016/j.jhepr.2020.100121 sha: doc_id: 342808 cord_uid: yonbowkb nan whilst the covid-19 pandemic runs its devastating course, heavily impacting on the health and quality of life (qol) of many, the scientific community continues to work hard to reconcile the need for new knowledge to fight this unprecedented threat to individual and global health, the high clinical needs, as well as the continuous need to invest in research for the non-covid-19related diseases that continue to impact on our populations. the editorial team of jhep reports is therefore pleased to present the next issue of the journal on schedule. first, we tackle the covid-19 issue by providing a key position paper that comprehensively summarises our current knowledge on how covid-19 impacts on the liver, how preexisting liver disease might impact on covid-19 infections and how to continue to ensure care for patients with acute and chronic liver disease in the current context. 1 this document is a joint effort of the european association for the study of the liver and the european society of clinical microbiology and infectious diseases and gives guidance regarding the burning issues. patients with pre-existing medical conditions are regarded as populations at risk of a severe disease course, but it is currently unclear to what extent chronic liver diseases should be considered as risk factors. patients with advanced liver disease as well as liver transplant patients likely represent vulnerable patient cohorts with an increased risk of infection and/or a severe course of covid-19. furthermore, an unusual allocation of healthcare resources is currently required in virtually all countries around the world. this may negatively impact the care of patients with chronic liver disease who continue to require medical attention. this position paper offers guidance on how to balance the need to avoid nosocomial dissemination of the virus to patients and healthcare providers, whilst at the same time maintaining a high standard of care for patients who require immediate and continued medical attention. the question of whether nucleos(t)ide analogues (nucs) can be safely discontinued in certain patients with chronic hepatitis b remains a matter of debate. one of the factors that could potentially allow for the safe withdrawal of nucs is the depletion of cccdna that might occur with long-term treatment. in the current issue of jhep reports, lai et al. report that viral rebound occurred in most cases following nuc cessation in a series of 19 patients with undetectable intrahepatic cccdna (covalently closed circular dna). 2 to what extent the determination of cccdna on liver biopsy can reliably reflect liver cccdna depletion is obviously questionable, but this study adds to the growing knowledge on how to handle patients following successful nuc treatment. van hees et al. 3 also reported a >50% rebound rate after treatment cessation in caucasian patients with hbeag seroconversion, with potentially fatal outcomes. the study of lai et al. adds a note of caution regarding nuc cessation, but unfortunately it does not fill the unmet need of identifying reliable predictors for the safe discontinuation of nucs. whilst hepatitis c has been successfully tackled in the last decade, hepatitis b and hepatitis b-d coinfection continue to challenge the hepatology community, as many knowledge gaps persist and viral eradication is rarely achieved in hepatitis b, whilst the treatment of hepatitis d is even more challenging. the complex interaction between the virus and host immune system is incompletely understood, which hampers the development of efficacious treatments. 4 the development of animal models that reliably mimic the infection in patients is particularly challenging. usai et al. present their results on a large and comprehensive study on the immunological mechanisms driving disease progression in hepatitis b-d coinfection. 5 in an adeno-associated vector-mediated mouse model of hepatitis b-d the authors surprisingly found a limited role of different immune cell populations (e.g. t cells or natural killer cells) in the induction of liver damage. by contrast, interferon-and tumour necrosis factor alpha (tnf-a)-mediated pathways appeared to be of crucial importance. inhibition of tnf-a by etanercept ameliorated hepatitis-d-induced liver damage. although the conclusion that pharmacological inhibition of tnf-a represents an attractive strategy to control hepatitis-d-induced acute liver damage is premature, the findings are innovative and substantially contribute to our understanding of the immunology of hepatitis-d-related disease, which should allow further exploration of the manipulation of these pathways in the quest for an effective treatment. although distinct diseases, alcoholic and non-alcoholic fatty liver disease (ald and nafld) share multiple features. not only the histological picture, but also several pathophysiological mechanisms overlap, e.g. the impact of alcohol on metabolic pathways including sterol regulatory element-binding protein 1c or peroxisome proliferator-activated receptor-a that are key metabolic pathways in the pathophysiology of nafld, 6 or the role of genetic factors. furthermore, in many patients both the drivers of nafld (such as overweight and diabetes) and the use of alcohol are concomitantly present. this can impact on the management of ald and nafld, elegantly summarised in the review by louvet et al. 7 one of the aspects that is poorly touched upon in the (medical) management of patients, is their perceived qol. several tools have been developed over time to capture the impact of the disease on this important aspect of disease burden, which is particularly challenging in the context of nafld, as the aforementioned comorbidities intrinsically affect qol. sweeney et al. extensively reviewed the literature on qol impairment in the specific population of patients with nafld-related compensated cirrhosis, concluding that those patients suffer significantly from lower qol and poorer physical health. 8 they also reviewed the available patient-reported outcome measures (proms) that are increasingly important in the design of clinical trials and patient management policies. they highlight the current limitations of the proms that have been used so far and conclude that there is a need for a standardised and structured approach to these measures in clinical trials of patients with nafld-related cirrhosis, as well as in daily clinical practice. rare liver diseases ask for collaborative efforts to provide high quality data that can solve clinical research questions. the european union has actively promoted this by recognising european reference networks (ern) for rare diseases, including rare liver for rare liver diseases. a group of centres, some from this ern, report on their pooled series of patients with autoimmune hepatitis with normal igg levels, which corresponds to 10% of their patient population. 9 although these studies inevitably struggle with their retrospective nature and with selection bias (reinforced in this study by the fact that igg levels are part of the diagnostic criteria for autoimmune hepatitis), the findings of hartl et al. are insightful and substantially contribute to our understanding of this clinical entity. it appears that these patients differ little in their characteristics at diagnosis compared to patients with a typical elevation of igg, but have a higher, albeit with 24% still rather low, chance of remaining in stable remission after cessation of all treatment. the issue of whether to stop any immunosuppressive treatment and the selection of patients who might qualify for this, is still ill-defined and hence the study by hartl et al. is an important contribution to help settle this clinical challenge. wilson's disease is an even rarer condition, and acute liver failure in this disease is usually confined to childhood or young adulthood. in this issue of jhep reports, shribman et al. not only report on a very rare case of acute liver failure as a primary manifestation of wilson's disease in a patient over 60 years of age, but also report the experience of the transplant community in the united kingdom with liver transplantation in this specific setting over the past 2 decades. 10 only 8 such cases of acute liver failure as the first presentation of wilson's disease in patients older than 40 years, or 1 in 1,250 listed for transplantation, could be identified. although rare, this implies that this cause of acute liver failure should be part of the differential diagnosis of acute liver failure at any age. budd-chiari syndrome is another rare disease with multiple challenges in clinical management. one of these is the frequent occurrence of benign regenerative lesions that need to be discriminated from malignant lesions, especially in case of mild elevations of alpha-foetoprotein that frequently accompany these benign lesions. as the contrast dynamics in budd-chiari syndrome are altered compared to a classical cirrhotic condition (especially impacting on the venous wash-out that is an important criterion in imaging-based diagnosis of hepatocellular carcinoma), and as the regenerative lesions in budd-chiari syndrome have features of focal nodular hyperplasia and hence show hyperenhancement in the arterial phase of contrast-enhanced imaging, the classical imaging criteria do not always suffice to reliably rule out malignancy in focal liver lesions of patients with budd-chiari syndrome. in a retrospective analysis of 26 patients with suspected liver lesions (a large single centre series for this condition), van wettere et al. report on the accuracy of mri with hepatobiliary excretion contrast agent in differentiating hepatocellular carcinoma lesions, which were all homogenously hypointense in the hepatobiliary excretion phase, from the benign lesions, which were almost all hyperintense, at least in the periphery of the lesion, or also homogenously. 11 diagnostic accuracy was very high, especially in combination with an alphafoetoprotein level >15 ng/ml. this important observation is of great relevance in the management of budd-chiari syndrome. from the same group, the current issue of jhep reports offers a comprehensive state-of-the-art overview of the role of imaging in the diagnosis of liver tumours, focusing mainly but not exclusively, on hepatocellular carcinoma. besides a detailed and critical appraisal of what has become standard of care, gregory et al. shed light on the future role of imaging in the clinical management of patients with a large variety of liver tumours. 12 of particular interest is the assessment of tumour response to various therapies. this goes beyond the assessment of viable tumour tissue, for some years the cornerstone of treatment response assessment in hepatocellular carcinoma. the response to immunotherapy is particularly challenging due to specific radiological images that appear in line with dynamics of treatment-induced alterations. 3d volumetry and radiomics offer new perspectives, the latter working on voxel level, mathematically analysing and manipulating their binary signatures in complex models, allowing to go beyond size or human-eye based semantic descriptors. also, quantitative and functional imaging provide insight into microscopic tumour changes that may be used as early predictors of response. this elegant review will guide clinicians through the fascinating innovations that imaging is offering in diagnosing these various liver tumours and in assessing treatment responses (to therapies with different modes of action, which require different corresponding imaging modalities). we hope you enjoy the issue! care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper rebound of hbv dna after cessation of nucleos/tide analogues in chronic hepatitis b patients with undetectable covalently closed circular dna stopping nucleos(t)ide analogue treatment in caucasian hepatitis b patients after hbeag seroconversion is associated with high relapse rates and fatal outcomes treating chronic hepatitis delta: the need for surrogate markers of treatment efficacy tnf-alpha inhibition ameliorates hdv-induced liver damage in a mouse model of acute severe infection ppara gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis combined alcoholic and non-alcoholic steatohepatitis health-related quality of life and patient-reported outcome measures in nash-related cirrhosis features and outcome of aih patients without elevation of igg liver transplantation for late-onset presentations of acute liver failure in wilson's disease: the uk experience over 2 decades hepatobiliary mr contrast agents are useful to diagnose hepatocellular carcinoma in patients with budd-chiari syndrome evaluation of liver tumour response by imaging key: cord-033821-i14dmmps authors: guo, yue-cheng; lu, lun-gen title: antihepatic fibrosis drugs in clinical trials date: 2020-08-24 journal: j clin transl hepatol doi: 10.14218/jcth.2020.00023 sha: doc_id: 33821 cord_uid: i14dmmps liver fibrosis is not an independent disease. it refers to the abnormal proliferation of connective tissues in the liver caused by various pathogenic factors. thus far, liver fibrosis has been considered to be associated with a set of factors, such as viral infection, alcohol abuse, non-alcoholic fatty liver disease, and autoimmune hepatitis, as well as genetic diseases. to date, clinical therapeutics for liver fibrosis still face challenges, as elimination of potential causes and conventional antifibrotic drugs cannot alleviate fibrosis in most patients. recently, potential therapeutic targets of liver fibrosis, such as metabolism, inflammation, cell death and the extracellular matrix, have been explored through basic and clinical research. therefore, it is extremely urgent to review the antihepatic fibrosis therapeutics for treatment of liver fibrosis in current clinical trials. hepatic fibrosis, a reversible response to various chronic liver injuries, may progress to cirrhosis, hepatocellular carcinoma and liver failure. cirrhosis is the common end-stage of a series of chronic liver diseases and can be divided according to its compensated and decompensated states. a series of complications of cirrhosis (such as portal hypertension, infection, ascites, and esophageal bleeding) are associated with significant morbidity and mortality. the mechanism involved in the progression and reversal of liver fibrosis is still not clear. up to now, alcohol, hepatocyte lipid deposition, and insulin resistance (ir) are recognized to be the major risk factors in patients with chronic hepatitis. besides, intrahepatic oxidative stress, viral and schistosomiasis infection, hepatic sinus microcirculation disturbance, and microbiota dysbiosis also participate in the occurrence and development of liver fibrosis. 1 current therapies for liver fibrosis encompass two aspects: etiology treatments and antifibrotic therapeutics. removal of underlying etiology of liver injury makes liver fibrosis reversible. however, up to now, there is lack of cause-specific treatment for certain liver diseases, such as non-alcoholic fatty liver disease (nafld), cholestatic liver diseases (clds), and some genetic liver diseases. therefore, there remains a need to develop direct antifibrotic therapies for liver fibrosis. over the past several decades, many researchers have proposed potential targets and alternative therapies for liver cirrhosis. unfortunately, there is no effective antifibrotic drug approved for human use, up to now. this review will focus on the representative drugs for liver fibrosis in clinical trials. fibrosis in epithelial organs is produced by the reticular deposition of extracellular matrix (ecm) and chronic inflammation, accompanied by compromised immune systems and pathological angiogenesis. liver fibrosis is an abnormal perpetuation of fibrogenesis due to various constant chronic liver injuries. apoptosis of liver parenchymal cells and continuous accumulation of ecm gradually replace the liver parenchyma with scar tissue, eventually forming liver architectural distortion, cirrhosis, portal hypertension, liver cancer, and liver failure. 2 as illustrated in fig. 1 , activation of hepatic stellate cells (hscs) is well established as the central driver of hepatic fibrosis. hscs are derived from embryonic mesothelial cells. quiescent hscs mainly store vitamin a and produce type iv collagen, while activated hscs produce collagen type i, iii and other proteins (fibronectin, elastin, laminin) after liver injuries. in addition, activated hscs exhibit a developed proliferative phenotype and contractile function. abundant ecm deposition and collagen production result in the increase of liver stiffness, thereby stimulating the activation of hscs and forming a positive feedback loop to develop cirrhosis. 3, 4 besides hscs, other liver cells also contribute to matrix protein production. hepatocytes, sinusoidal endothelial cells, and lymphocytes are involved in the development of liver fibrosis through releasing cell contents and cytokines. 1 macrophages in the liver comprise kupffer cells and monocytederived macrophages, and the latter stimulates hscs to become collagen-producing myofibroblasts. 5 compelling evidence from animal models indicates that liver fibrosis is reversible. firstly, modifications in ecm composition may regulate cellular functions partly through cell adhesion molecules and participate in the regression of fibrosis. 1, 6, 7 secondly, cell death not only induces inflammation and promotes fibrogenesis but may also contribute to fibrosis resolution through influencing the apoptosis and senescence of activated hscs. 6 lastly, infiltrating macrophages play a detrimental role in liver fibrosis. infiltrating macrophages have shown profibrogenic and proinflammatory features in the progression of fibrosis. however, in a murine model of hepatic fibrosis, the increased restorative macrophages are associated with the accelerated resolution of fibrosis. 8 overall, the resolution of liver fibrosis is also a complex process. currently, the most important treatment is controlling the underlying cause of the liver diseases. these include effective suppression or elimination of hepatitis virus replication (hepatitis b virus and hepatitis c virus), drug eradication of schistosomiasis, relieving cholestasis, reduction of body weight in nafld, improvement of associated metabolic disorders, cessation of alcohol use in patients with alcoholic liver disease (ald), phlebotomy in hemochromatosis, and use of corticosteroids/immunosuppressants for autoimmune liver disease. all these therapies can reduce persistent hepatic injury, thereby inhibiting liver fibrogenesis and/or promoting fibrolysis. many researchers demonstrated that effective inhibition of hepatitis b virus or hepatitis c virus replication significantly improved the fibrosis stage in patients with hepatitis b or c, and that liver fibrosis can be reversed in some participants. [9] [10] [11] however, there are still some patients with virological and biochemical attenuation, whose liver fibrosis still exists and even progresses, eventually into liver cancer. 12 additionally, it takes a prolonged period of time to promote the resolution of liver fibrosis after viral elimination. antiviral benefits may be offset by the increased rates of drug resistance over time. 11 treatment based on the etiology may not completely attenuate all fibrosis patients, as there are currently no effective managements for eliminating the cause of certain liver diseases, such as autoimmune hepatitis. 12 thus, direct antifibrotic therapies targeting ecm metabolism and hsc activation are indispensable. liver fibrosis plays an important role in liver tissue repair in the early stage of injury. however, the management of liver fibrosis is required in significant or advanced fibrosis and cirrhosis. at present, there is no recognized and effective antifibrotic agent. chronic inflammatory response is the premise of fibrogenesis and the driving force of fibrotic progression. antiinflammation, hepatocyte protection and antioxidation are important methods for hepatic fibrosis. in the following sections, we mainly review representative drugs based on therapeutic targets. table 1 displays phase ii/iii/iv clinical trials for current therapies. clds represent a series of hepatobiliary diseases accompanied by disorders of bile formation, secretion, and excretion. the clds mainly include primary biliary cholangitis (pbc) and primary sclerosing cholangitis (psc), both having a risk of progression to liver fibrosis. ursodeoxycholic acid (udca), a physiologic hydrophilic dihydroxy bile acid, is the primary drug for treating cld. its antifibrotic property may be associated with the stimulation of bile secretion and the suppression of inflammation. 13 parés et al. 14 pointed out that udca (15 mg/kg daily, 1.5-14 years) improved survival and delayed the histologic stage progression of pbc patients. udca use can significantly reduce alkaline phosphatase (alp; an essential indicator of cld activity) levels in patients. the most frequent adverse event in clinical trials is diarrhea, which rarely leads to discontinuation of therapy. however, only 20-30% of pbc patients had a complete response to udca, and udca discontinuation deteriorates liver biochemical indicators and is associated with pruritus. 15 udca has an optimal dose of 13 to 15 mg/kg/d. lindor et al. 16 found that high-dose udca (28-30 mg/kg daily, 5 years) increased the risk of advanced cirrhosis, esophageal varices, and bile duct cancer. a new phase iv clinical trial (nct03345589) has been proposed by west china hospital, chengdu, china. this trial aims to define whether an alternative dose (18-22 mg/kg daily) of udca is effective in refractory pbc. for psc, the american association for the study of liver diseases recommends not to use udca as a treatment, as psc patients did not exhibit significant improvements after a long-term udca therapy. 17 farnesoid x receptor agonist farnesoid x nuclear receptor (fxr), also known as bile acid receptor, is involved in the synthesis, secretion and reabsorption of bile acids. fxr activation effectively improves lipid metabolism through improving insulin sensitivity, decreasing hepatic gluconeogenesis, and reducing circulating triglycerides. 18 thus, fxr is a potential target for non-alcoholic steatohepatitis (nash) and cld-related liver fibrosis. obeticholic acid (oca), an agonist of fxr, decreases bile acids synthesis and exerts antiinflammatory and antifibrotic effects. in a multicenter, randomized, placebo-controlled trial, 19 patients with nash exhibited improvements of liver histological features after treatment with oca (25 mg, 72 weeks). however, oca therapy was accompanied by a deterioration involving dyslipidemia and ir, indicating an increased risk of atherogenesis. given these adverse events, oca treatment for nash should be further evaluated in long-term prospective trials. oca has also been widely used in clinical trials for pbc. nevens et al. 20 conducted a phase iii clinical trial in pbc patients with inadequate response to conventional drugs. after 12 months of therapy, the oca had significantly reduced alp, high-density lipoprotein, and total bilirubin levels. these effects were sustained for 2 years. although improvements were observed at the lower dose (5 mg) in some patients, incremental benefits occurred with adjustment to the higher dosage (10 mg). another double-blind phase ii trial, 21 including 165 patients with inadequate response to udca, showed that oca resulted in a clinically significant reduction in alp at all doses (10, 25, and 50 mg). there were also significant improvements in gamma-glutamyltransferase, alanine aminotransferase (alt), aspartate aminotransferase (ast), and conjugated bilirubin levels. in 2016, oca was approved by the food and drug administration for patients intolerant or with a null response to udca. dose-dependent pruritus was the only clinical adverse event that differed between oca treatment and placebo. 21, 22 pruritus can be controlled by antipruritic agents or temporary oca interruption, but high oca doses may result in the discontinuation due to pruritus. the mechanism of cholestatic pruritus may be related to the activation of tgr5, 23 as oca is a weak tgr5 agonist. non-bile acid fxr agonists have also been developed and are expected to cause less pruritus than oca. 24 tropifexor (also known as ljn-452), a well-tolerated fxr agonist, is suitable for once-daily dosing 25 and has been used in phase ii clinical trials for pbc and nash (nct02516605, nct04065841, and nct03517540). similarly, cilofexor (otherwise known as gs-9674) is also currently undergoing phase ii trials for nash and cld. cilofexor therapy (30 mg or 100 mg daily, 12 weeks) reduced alp levels in a dose-dependent manner in patients with psc, while adverse events (pruritus) were similar among groups. 26 in another trial, 27 cilofexor (30 mg or 100 mg daily, 24 weeks) significantly improved hepatic steatosis and liver biochemistry in patients with nash. however, no significant improvements in liver stiffness were observed with magnetic resonance imaging-proton density fat fraction and magnetic resonance elastography. pruritus was more common in patients receiving cilofexor at 100 mg than those receiving cilofexor at 30 mg or placebo. additional studies of cilofexor in nash with a longer duration are warranted. ppar is a key regulator of lipid metabolism in the liver and has been approved by the food and drug administration as a molecular target for dyslipidemia. as distinct ppar isoforms, ppara regulates cholesterol and bile acid homeostasis, 28 while pparg contributes to inhibiting the activation of hscs and reducing collagen production. 29 hence, ppar is a potential target for hepatic fibrosis. bezafibrate, an agonist of ppar, is a widely used antihyperlipidemic agent. in a phase iii clinical trial, 30 bezafibrate treatment (400 mg, 24 months) in addition to udca contributed to a significant biochemical attenuation in pbc patients with an inadequate response to udca. the bezafibrate group also exhibited decreased liver stiffness and improved fibrosis scores compared with placebo. another prospective study 31 has compared the long-term efficacy between combination therapy (udca + bezafibrate) and udca monotherapy for pbc patients. the combination therapy significantly decreased alp levels and mayo risk scores but did not improve the survival rate. bezafibrate may result in dose reduction or discontinuation due to the renal dysfunction or muscle pain. other ppar agonists such as fenofibrate and seladelpar have also been used in phase ii/iii clinical trials (table 1) . according to a meta-analysis, 32 combination therapy of udca and fenofibrate had a superior efficacy to udca monotherapy in reducing alp levels, whereas no improvements were observed in clinical symptoms. no significant differences were observed in the incidence of adverse events. however, all trials included in this meta-analysis had a small sample size and no adequate histological results were reported. in prior studies, several serious adverse events were found after fibrates therapy, including hepatotoxicity, elevated creatinine, and increased creatinine kinase. 33 hence, larger controlled multicenter studies are required to confirm the long-term efficacy and safety of ppar agonists. journal of clinical and translational hepatology 2020 vol. 8 | 304-312 cenicriviroc approaches directly targeting the inflammatory recruitment are also important. ccr2 and its ligand are significantly increased in liver fibrosis, and fibrosis is significantly ameliorated in ccr2-deficient mice, 34 suggesting that ccr2 is involved in liver fibrosis. ccr5 is also associated with the accumulation of collagen and ecm. 35 therefore, inhibition of ccrs may be a novel approach for liver fibrosis. cenicriviroc (cvc), an oral antagonist of the dual ccr2/ ccr5 receptor, is safe, well-tolerated and has been used in phase ii clinical trials in nash patients with liver fibrosis. cvc (150 mg daily, 2 years) can significantly promote nash regression and decrease the collagen area (as detected by morphometry on liver biopsy). treatment benefits were more significant in patients with higher disease activity and fibrosis stage at baseline. safety and tolerability of cvc were comparable to placebo, and adverse events (fatigue, diarrhea) had a mild or moderate severity. 36 another phase iii trial (nct03028740) has been posted which investigates the long-term efficacy and safety of cvc in advanced cirrhosis patients. this trial mainly aims to evaluate liver histological improvements based on a larger sample. overall, cvc has a favorable prospect in the application for liver fibrosis. galectins are secreted proteins that bind to terminal galactose residues in glycoproteins on components of the ecm. galectin-3 is highly expressed on kupffer cells and plays a vital role in cell adhesion, inflammation, and fibrogenesis. 37 in animal models, glectin-3 inhibitors significantly reduced fibrosis stages and portal pressure. 38, 39 gr-md-02, a galectin-3 inhibitor, was safe and well-tolerated in subjects who had a definite histological diagnosis of nash with advanced fibrosis. 40 fibroscan test showed the potential benefits of gr-md-02 therapy (8 mg/kg daily) on liver stiffness. 40 recently, chalasani et al. 41 conducted a phase ii clinical trial of gr-md-02 in 162 patients with nash, cirrhosis and portal hypertension. though improvements of fibrosis or nafld activity scores did not differ significantly among groups, a subgroup analysis showed that gr-md-02 therapy (2 mg/kg biweekly, 52 weeks) did reduce hepatic venous pressure gradient and development of varices in patients without esophageal varices. spasmodic cough was the only adverse event related to the study drug. a phase iii trial has been initiated to evaluate to safety and efficacy of gr-md-02 in nash cirrhosis patients without esophageal varices (nct04365868). the fibroblast growth factor (fgf) family of hormones regulate metabolic functions and participate in tissue repair and regeneration. 42 among them, fgf19 and fgf21 are considered to be closely related to nafld. ngm282, an engineered fgf19 analogue, has been used in phase ii clinical trial for nash. 43 ngm282 therapy (3 or 6 mg daily, 12 weeks) can significantly reduce liver fat content and decrease alt and ast levels without severe adverse event. similarly, pegbelfermin (bms-986036), a pegylated human fgf21 analogue, was well-tolerated and significantly reduced hepatic fat fraction in patients with nash. 44 no severe adverse events or drug-related discontinuations were reported. however, whether fgf analogs contribute to improving liver fibrosis histology is still unknown. it's worthy of further discussion in the follow-up clinical trials. currently, two active phase ii clinical trials (nct03486912, nct03486899) are investigating the efficacy and safety of pegbelfermin in patients with nash-related fibrosis as determined by liver biopsy. apoptosis signal-regulating kinase 1 inhibitor continuous cell death of hepatocytes is a critical trigger for liver fibrosis. the apoptosis signal-regulating kinase 1 (ask1) regulates intracellular pathways of cell death, and its activation results in a deterioration in hepatic inflammation, apoptosis, and fibrosis. ask1 inhibitor treatment protects against liver injury through decreasing oxidative stress reaction and the expression of pro-inflammatory cytokines. 45 as an ask1 inhibitor, selonsertib (6 or 18 mg daily, 24 weeks) decreased collagen content and reduced lobular inflammation on liver biopsy in patients with nash and stage 2-3 fibrosis, accompanied by improvements in liver stiffness on magnetic resonance elastography. 46 however, the majority of patients receiving selonsertib experienced at least one adverse event. side effects, such as numbness of upper extremities and elevated liver enzymes, resulted in the discontinuation in several patients. a recent phase iii clinical trial (nct03053063) investigated the long-term (240 weeks) efficacy and safety of selonsertib in liver cirrhosis due to nash. however, this trial was terminated early due to lack of efficacy based on the results of the week 48 analysis. apoptosis-mediated inflammation and immune response also play a crucial role in the process of fibrosis. caspases are intracellular cysteine proteases that participate in the process of apoptosis and inflammation through proinflammatory cytokines. emricasan is a well-tolerated pan-caspase inhibitor. emricasan (25 mg twice daily, 28 days) can effectively decrease alt levels, reduce hepatic venous pressure gradient, and improve intrahepatic inflammation in patients with compensated cirrhosis. 47 most reported adverse events were not serious and the frequent side effect was fatigue. despite improvements in liver enzymes and hepatic inflammation, it is unlikely that emricasan exerted an antifibrotic effect in 28 days. 47 although improvements of hepatitis may occur relatively rapidly, attenuation of fibrosis will take longer. a recent phase ii trial 48 demonstrated that emricasan (25 mg twice daily, 3 months) improved liver function and reduced child-pugh scores in patients with advanced cirrhosis. incidence of adverse events was similar between emricasan and placebo groups. however, it is worth noting that the multiple etiologies of cirrhosis may obscure the actual efficacy when setting clinical scores as endpoints. optimal doses and long-term efficacy of emricasan will be explored in an active phase ii trial (nct03205345) in nash patients with cirrhosis. journal of clinical and translational hepatology 2020 vol. 8 | 304-312 309 transforming growth factor-b inhibitor transforming growth factor-b (tgf-b) is considered to be the central factor participating in liver fibrosis. various strategies have been developed to inhibit the tgf-b pathway. pirfenidone, an inhibitor of tgf-b, can ameliorate fibrogenesis through inhibiting the activation of hscs and reducing collagen synthesis in vitro. 49 borunda et al. 50 found that pirfenidone was well-tolerated and attenuated histological injuries in patients with established advanced cirrhosis after 12 months of treatment. only 15% of patients developed adverse events, and these included photosensitivity, rash, itching, and nausea. a phase ii clinical trial 51 showed that pirfenidone (1200 mg daily, 24 months) improved inflammation, fibrosis, and steatosis in patients with hepatitis c virusrelated cirrhosis. all patients on pirfenidone displayed improvements of life quality and child-pugh scores. side effects such as gastritis and nausea were observed. as viral clearance is indispensable to resolve liver damage, a combination therapy of pirfenidone and antiviral agents may be a new approach for viral fibrosis. similarly, based on structural modification of pirfenidone, hydronidone is a novel antifibrotic agent for hepatic fibrosis. 52 an open-label and randomized clinical trial showed that hydronidone was well tolerated and rapidly absorbed in young, healthy volunteers. 52 a phase ii clinical trial (nct02499562) has explored the effective dose and safety of hydronidone capsules in patients with liver fibrosis induced by hepatitis b virus infection in shanghai general hospital, shanghai, china. however, related results have not been posted. the lysyl oxidase (lox) family is crosslinking enzymes overexpressed in liver cirrhosis and contributes to fibrogenesis by catalyzing cross-linkage of collagen and increases the stability of fibrosis in chronic hepatitis settings. 53 among the five isoforms, loxl2 is overexpressed by activated hscs in liver fibrosis. selective inhibition of loxl2 suppresses hepatic fibrosis progression and accelerates its reversal in animal fibrosis models. 54 however, simtuzumab, a humanized monoclonal antibody directed against loxl2, did not show a promising benefit in clinical trials. in two phase iib trials, simtuzumab alone (subcutaneous injections: 75 or 125 mg; intravenous infusions: 200 or 700 mg, 96 weeks) was ineffective in decreasing hepatic collagen content or fibrosis stage in patients with bridging fibrosis and hepatitis c virus infection. 55 in another 6-month open-label safety trial, though treatment was welltolerated, no significant improvements were observed in hepatic venous pressure gradient or liver biopsy after simtuzumab therapy (700 mg, intravenous infusion every 2 weeks for 22 weeks). 56 also, in patients with psc, simtuzumab therapy (75 or 125 mg daily, 96 weeks) did not reduce liver collagen content or ishak fibrosis stage. 57 the renin-angiotensin system is a crucial regulator of liver fibrosis and portal hypertension. 58 activated hscs can secrete angiotensin ii that promotes liver fibrosis through angiotensin receptors. yoshiji et al. 59 demonstrated that angiotensin-converting enzyme inhibitor (acei) combination (perindopril: 4 mg daily, 12 months) with interferon decreased serum fibrosis markers (hyaluronic acid, 7-s-collagen, p-iii-p, tgf-b) in patients with chronic refractory hepatitis c. another clinical trial 60 demonstrated that acei (captopril: 25-75 mg daily, 3 months) effectively reduced the portal pressure and prevented variceal bleeding in portal hypertensive patients. the adverse effects of captopril (orthostatic hypotension and dry cough) were not severe enough to stop medication. however, these studies are limited by their lack of histological and immunohistochemical examinations. similarly, angiotensin receptor blockades may also attenuate liver fibrosis. losartan (50 mg daily, 48weeks) can decrease serum aminotransferase levels and promote histological improvements in nash with no adverse events. 61 in a randomized open-label controlled study, a combination therapy of candesartan (8 mg daily) and udca (600 mg daily) for 6 months also suppressed the expression of fibrosis biomarkers and decreased arterial blood pressure in alcoholic liver fibrosis with no significant complications or side effects. 62 recently, a phase iii trial (nct03770936) has been posted to compare the efficacy of candesartan and ramipril in hepatitis c virus-related liver fibrosis. at present, due to the complex mechanisms involved in hepatic fibrosis, antihepatic fibrosis therapies still face many problems, as outlined here: 12,63 1) hepatic fibrosis has a long disease course. the most reliable method for evaluating the efficacy of treatments is to observe the histopathological changes. in contrast, it is not appropriate to use the clinical serum or radiologic measurements directly as the primary endpoints. 2) currently, the mechanism involved in liver fibrosis has not been elucidated thoroughly. murine models may not accurately mimic human liver diseases. 3) up to now, many antifibrotic trials still assess the efficacy by liver biopsy, which is costly and can be affected by sampling. the invasive inspection may result in complications. 4) though many clinical trials confirm the efficacy and safety of combination therapy, the role of each component is not defined. 5) many scholars consider stem cells as a potential option. however, the cellular and molecular basis of liver regeneration in liver fibrosis have not been elucidated totally. cirrhosis is a severe form of chronic liver disease and is the typical outcome of various etiologies-induced liver injuries. in this article, we reviewed current therapies in clinical trials. safe and effective therapies may delay the development of decompensated cirrhosis and even reverse fibrosis stage. apart from conventional cause-specific therapies, the most promising treatments for liver fibrosis are those that target cellular and molecular mechanisms involved in the reversal of fibrosis. as we continue to explore the mechanisms and targets of hepatic fibrosis, effective antifibrotic therapies will be a reality in the future. journal of clinical and translational hepatology 2020 vol. 8 | 304-312 hepatic fibrosis: concept to treatment liver cirrhosis hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver mechanical stiffness of liver tissues in relation to integrin b1 expression may influence the development of hepatic cirrhosis and hepatocellular carcinoma therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis pathobiology of liver fibrosis: a translational success story regression of liver fibrosis differential ly-6c expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis b sofosbuvir and velpatasvir for hcv in patients with decompensated cirrhosis regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis b: a 5-year open-label follow-up study chinese society of hepatology chinese medical association; chinese society of gastroenterology chinese medical association; chinese society of infectious diseases, chinese medical association. consensus on the diagnosis and therapy of hepatic fibrosis in natural history of primary biliary cholangitis in the ursodeoxycholic acid era: role of scoring systems excellent long-term survival in patients with primary biliary cirrhosis and biochemical response to ursodeoxycholic acid prospective evaluation of ursodeoxycholic acid withdrawal in patients with primary sclerosing cholangitis high-dose ursodeoxycholic acid for the treatment of primary sclerosing cholangitis diagnosis and management of primary sclerosing cholangitis bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease farnesoid x nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (flint): a multicentre, randomised, placebo-controlled trial a placebo-controlled trial of obeticholic acid in primary biliary cholangitis efficacy of obeticholic acid in patients with primary biliary cirrhosis and inadequate response to ursodeoxycholic acid a randomized trial of obeticholic acid monotherapy in patients with primary biliary cholangitis bile acids reach out to the spinal cord: new insights to the pathogenesis of itch and analgesia in cholestatic liver disease investigational drugs in phase ii clinical trials for primary biliary cholangitis safety, tolerability, pharmacokinetics, and pharmacodynamics of the novel non-bile acid fxr agonist tropifexor (ljn452) in healthy volunteers the nonsteroidal farnesoid x receptor agonist cilofexor (gs-9674) improves markers of cholestasis and liver injury in patients with primary sclerosing cholangitis cilofexor, a nonsteroidal fxr agonist, in patients with noncirrhotic nash: a phase 2 randomized controlled trial fibrates and cholestasis peroxisome proliferator-activated receptors and hepatic stellate cell activation a placebo-controlled trial of bezafibrate in primary biliary cholangitis a prospective randomized controlled study of long-term combination therapy using ursodeoxycholic acid and bezafibrate in patients with primary biliary cirrhosis and dyslipidemia combination therapy of fenofibrate and ursodeoxycholic acid in patients with primary biliary cirrhosis who respond incompletely to udca monotherapy: a meta-analysis safety considerations with fibrate therapy dual role of ccr2 in the constitution and the resolution of liver fibrosis in mice effects of treatment with maraviroc a ccr5 inhibitor on a human hepatic stellate cell line a randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis 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inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment selective targeting of lysyl oxidase-like 2 (loxl2) suppresses hepatic fibrosis progression and accelerates its reversal simtuzumab is ineffective for patients with bridging fibrosis or compensated cirrhosis caused by nonalcoholic steatohepatitis simtuzumab treatment of advanced liver fibrosis in hiv and hcv-infected adults: results of a 6-month open-label safety trial simtuzumab for primary sclerosing cholangitis: phase 2 study results with insights on the natural history of the disease role of the renin-angiotensin system in hepatic fibrosis and portal hypertension interferon augments the anti-fibrotic activity of an angiotensin-converting enzyme inhibitor in patients with refractory chronic hepatitis c captopril reduces portal pressure effectively in portal hypertensive patients with low portal venous velocity therapeutic efficacy of an angiotensin ii receptor antagonist in patients with nonalcoholic steatohepatitis beneficial effects of candesartan, an angiotensin-blocking agent, on compensated alcoholic liver fibrosis -a randomized open-label controlled study antifibrotics in liver disease: are we getting closer to clinical use? the authors would like to acknowledge everyone who contributed to the completion of this project. none to declare. the authors have no conflict of interests related to this publication. drafting of the manuscript and creation of figure and table (ycg), critically revising the document for important intellectual content (lgl), approval of the final version of this manuscript to be published (ycg, lgl). key: cord-284693-mgpxnnk0 authors: jothimani, dinesh; venugopal, radhika; vij, mukul; rela, mohamed title: post liver transplant recurrent and de novo viral infections date: 2020-09-26 journal: best pract res clin gastroenterol doi: 10.1016/j.bpg.2020.101689 sha: doc_id: 284693 cord_uid: mgpxnnk0 survival following liver transplantation has changed dramatically owing to improvement in surgical techniques, peri-operative care and optimal immunosuppressive therapy. post-liver transplant (lt) de novo or recurrent viral infection continues to cause major allograft dysfunction, leading to poor graft and patient survival in untreated patients. availability of highly effective antiviral drugs has significantly improved post-lt survival. patients transplanted for chronic hepatitis b infection should receive life-long nucleos(t)ide analogues, with or without hbig for effective viral control. patients with chronic hepatitis c should be commenced on directly acting antiviral (daa) drugs prior to transplantation. daa therapy for post-lt recurrent hepatitis c infection is associated with close to 100% sustained virological response (svr), irrespective of genotype. de novo chronic hepatitis e infection is an increasingly recognised cause of allograft dysfunction in lt recipients. untreated chronic hev infection of the graft may lead to liver fibrosis and allograft failure. similarly, cmv and ebv can reactivate leading to systemic illness following liver transplantation. with covid-19 pandemic, post-transplant patients are at risk of sars-co-v2 infection. majority of the lt recipients require hospitalisation, and the mortality in this population is around 20%. early recognition of allograft dysfunction and identification of viral aetiology is essential in the management of post-lt de novo or recurrent infections. optimising immunosuppression is an important step in reducing the severity of allograft damage in the treatment of post-transplant viral infections. viral clearance or control can be achieved by early initiation of high potency antiviral therapy. liver transplantation (lt) is the only curative therapy for patients with decompensated cirrhosis, with an one year and 5-year survival of around 90% and 70%, respectively 1 survival following olt has improved over the years from a 1 and 5 year survival of 70% and 50% to 90% and 70%, respectively. significant improvement in surgical techniques, peri-operative care and immunosuppression therapy has translated in to improved survival. however, in this review, we focus on the prevalence, natural history and clinical outcomes of hepatitis c (rhcv), hepatitis b (rhbv), hev and other viral infections such as cmv, hsv and ebv in lt recipients. in addition, given the current covid-19 pandemic, we discuss impact of sars-cov-2 in the liver allograft and their outcomes. recurrent hepatitis c (rhcv) infection of the liver allograft is universal in untreated patients transplanted for this indication. allograft colonisation by viral particles occurs at the time of portal reperfusion inducing liver damage as early as 72 hours post-liver transplantation. a study on hcv viral kinetics demonstrated an initial sharp decline in the rna levels within 24 hours of reperfusion followed by a rapid increase to nearly 20 fold higher than the pretransplant values during the first week 6 . untreated rhcv may progress with necroinflammation, leading to allograft injury and fibrosis. in a study to evaluate the natural course of 183 hcv liver transplanted patients using protocol liver biopsies, fibrosis score progressed from 1.2 to 2.2 over 10 year period. in addition, patients with severe fibrosis at 1 j o u r n a l p r e -p r o o f year due to rhcv was associated with poor survival. furthermore, donor age >33 years and hcv genotype 1 or 4 developed rapid fibrosis 7 . post-transplant immunosuppression accelerates rhcv related liver damage with fibrosis progression at the rate of 0.3%-0.8% per year leading to cirrhosis and graft loss in 20-40% of patients within 5 years 8, 9 . up to 40% patients with rhcv develop hepatic decompensation within a year of cirrhosis 10 . these patients follow an accelerated course with a three year survival of only 10% following hepatic decompensation unlike 60% in those with native cirrhotic liver. over all patients transplanted for hcv have a lower 3 and 5 year survival of 73% and 67%, respectively 11 . rarely (<10%), rhcv can present in a severe form, fibrosing cholestatic hepatitis (fch), characterized by bilirubin >6 mg/dl, alp and ggt >5x upper limit normal, high hcv rna typically occurring in the first 6 months post liver transplantation. liver histology characterised by severe cholestasis, hepatocyte ballooning spotty necrosis and kupffer cell hypertrophy. progressive fch is associated with severe allograft dysfunction leading to graft failure leading to death within 12 months. in summary, hcv lt recipients had worst long term survival until 5 years ago. hcv viral clearance described as sustained virological response (svr) defined as undetectable hcv rna at 24 weeks following antiviral therapy, but recently reduced to 12 weeks (svr12). svr has a major implication in disease progression in rhcv. similar to chronic hepatitis c in pre-transplant patients, achieving svr in post-transplant patients results in stability of the disease or even regression of fibrosis in 75% of patients 12 . whereas, failure to achieve svr results in worsening of fibrosis. unlike, in patients with chronic liver disease, fibrosis in rhcv progresses at faster pace. in patients untreated rhcv, fibrosis score ≥2 progress rapidly to cirrhosis and graft failure than patients with absent or mild fibrosis 13 . in a 10 year follow up italian study of 358 lt patients for hcv, patients who did not achieve svr had the worst 10-year survival 39.8% compared to 84.7% in those with svr 14 . a number of studies evaluated recipient, donor and virus related risk factors associated with rhcv. advanced recipient age, diabetes mellitus, severe liver disease (child pugh >10), il-28b polymorphism, high hcv rna >10 7 iu/ml, ischemic/reperfusion injury, cmv, donor age >65 years, cold ischaemic time over 8 hours and warm ischemia over 90 minutes, marginal graft, dcd donor, higher immunosuppression in particular high dose corticosteroids for acute cellular rejection, use of anti-thymocyte globulin were significantly associated with rhcv in the liver allograft 15, 16 . calcineurin inhibitors (cni) such as tacrolimus and cyclosporine are the most commonly used immunosuppressive drugs in lt recipients. ideal immunosuppressive regimen in hcv lt recipients to avoid rejection and at the same time to reduce the risk of rhcv is not established. corticosteroid was considered to be the main drug associated with increased risk of rhcv. in particular, use of steroid boluses and rapid tapering has been shown to reactivate hepatitis c in the graft 17 . various immunosuppressive modifications were adopted in the past to reduce rhcv of the allograft. low dose slow steroid tapering has shown to reduce rhcv in the graft. a study by berenguer et al., showed 29% rhcv in patients with cni and tapering steroids over 9-12 months compared to 48% in the historical controls 18 . studies attempted to eliminate corticosteroids using il-2receptor antagonist induction regimens to reduce rhcv. in a prospective randomised multi-centre trial evaluation dacluzimab induction followed by tacrolimus and mmf (n=153) vs standard treatment rhcv occurred in 33% vs 40% (p not significant) 19 with increasing demand for cadaveric organs, dcd organ harvest has been increasingly utilized. studies showed increased rhcv in patients undergoing lt from a dcd compared to donation after brain death (dbd) liver, probably related to ischaemic reperfusion injury 22 . interestingly, a large case control study on hcv patients who received dcd liver showed no significant differences in the hcv rna titres (p=0.7), severe rhcv with fibrosis 8% vs 15%, p=0.38) and graft loss (5% vs 9%, p=0.6) between the two groups at 12 months posttransplant 23 . previously, interpretation of abnormal lfts in patients transplanted for hcv infection was difficult to distinguish between a rejection episode and rhcv infection, in the absence of liver biopsy. therefore, management of graft dysfunction in hcv transplant recipients was a challenge. this led to the development of non-invasive markers of liver fibrosis. fibroscan using transient elastography technique plays a major role in the assessment of disease severity in these patients 24 . liver histology is definitive in the diagnosis and management of rhcv. acute hepatitis c in the liver allograft shows mild lobular activity with mononuclear inflammatory infiltrate, necroinflammatory foci and apoptotic bodies. nodular lymphoid aggregates may also be present. fch is characterised histologically by irregular portal expansion, portal fibrosis with immature pericellular/sinusoidal fibrous bands, extensive hepatocyte ballooning and degeneration, bile ductular reaction, marked canalicular and intracellular bilirubinostasis, j o u r n a l p r e -p r o o f and mild to moderate mononuclear inflammation. confluent or bridging necrosis may be present. pre-lt hcv rna level strongly predicts post-transplant rhcv severity. therefore, treatment should be aimed at making the patient virus free prior to liver transplantation.. svr has consistently shown to improve several aspects of hcv related complications. achieving svr in patients awaiting liver transplantation has shown to decrease the rate of disease progression, symptomatic improvement, improvement in meld score and importantly improves post-transplant survival 25, 26 treatment of hcv in the previous era: a decade ago, the treatment of hcv in patients with decompensated cirrhosis was nearly impossible. in the absence of advanced liver disease, the use of peg ifn and ribavirin achieved 50-70% svr depending on the genotype. unfortunately, both these drugs are contraindicated in patients with decompensated cirrhosis due to higher adverse events, like anaemia and sepsis. subsequent introduction of first generation directly acting antiviral (daa) therapy, protease inhibitors telaprevir and boceprevir achieved svr around 70% given with peg ifn and ribavirin. however, protease inhibitors were effective only against hcv genotype 1 and similarly these were contraindicated in advanced liver disease. both these drugs were discontinued in 2015. treatment of hcv in patients with decompensated cirrhosis changed significantly following the introduction of sofosbuvir, the second generation daa with high efficacy, shorter treatment course, better safety profile and importantly interferon free regime. sofosbuvir with ribavirin for up to 48 weeks in 61 hcv hcc patients awaiting liver transplantation, showed an undetectable hcv rna at the time of lt was associated with j o u r n a l p r e -p r o o f 70% svr12 in the post-transplant period. moreover, patients with undetectable hcv rna at least 4 weeks prior to lt never developed rhcv. adverse events leading to drug discontinuation was noted in 2 patients (sepsis and kidney injury) which was unrelated to sofosbuvir 27 . the success of this therapy changed the perspective of hcv management in patients undergoing liver transplantation. subsequent studies were conducted in hcv patients with decompensated cirrhosis with second generation daa. solar-1 was a phase 2 open label multi-centre study on 12 or 24 weeks of ledipasvir (ns5a polymerase inhibitor), sofosbuvir and ribavirin in patients with advanced cirrhosis (child pugh b or c, n=108). svr12 for child pugh b was 87% and 89%, and for child pugh c was 86% and 87%, with 12 and 24 weeks treatment irrespective of previous therapy. adverse events occurred in 23% of patients but only 4% discontinued treatment 28 . similarly, solar-2 study was conducted across european and new zealand sites on hcv child pugh b (n=56) and child pugh c (n=51) with svr12 of 87% and 96%, and 85% and 78% for 12 and 24 weeks, respectively. adverse events occurred in 11-50% of patients, predominantly in child pugh c, including 3 (12%) patients death. these two studies clearly demonstrated high svr rates in patients with 12 weeks of this combination therapy with effective hcv clearance in patients with decompensated cirrhosis. with the higher viral eradication rates, meld score improved in these patients over a period of time; however, in many of the study centres patients have undergone liver transplantation in view of shorter waiting times in different parts of the study sites 29 .data from our liver unit showed a weeks daclatasvir 60 mg daily in combination with sofosbuvir 400 mg twice a day and weight based ribavirin in patients with decompensated cirrhosis (n=60) with an svr12 of 83% 30 . patients with child pugh a and b had a higher svr12 than c, 94%, 94% and 56%, respectively. likewise, genotype 1b had a better svr12 than genotype 1a (100% vs 76%). in patients with decompensated cirrhosis, meld score improved by -0. higher rates of svr with daa has shown to reduce hcv related death by 74% 31 . achieving svr has shown to reduce the chance of liver disease progression with improvement in portal pressure 32 . in several cases there were significant improvement in meld score leading to delisting of patients waitlisted for lt. in a study involving 409 patients with child b and c cirrhosis, the mean meld declined by 0.85 within 6 months compared to untreated patients (p<0.0001) and they encountered reduced episodes of hepatic decompensation (3.7% vs 10%, p=0.009) in patients with svr 33 . importantly, a cohort study from srtr database showed 32% reduction in the hcv lt waitlist after they cleared hcv with daa therapy 34 . in the previous decade, management of post-transplant rhcv has been posed several difficulties. introduction of peg-ifn and ribavirin in the treatment of rhcv slightly improved clinical outcome. unfortunately, these drugs were associated with significant side effects including graft rejection. therefore, treatment was recommended only in patients with moderate and severe rhcv. fatigue, diarrhoea and anaemia occurred in 30%, 28% and 20% of patients 37 . initial studies with sofosbuvir and ribavirin combination therapy for post-transplant rhcv showed poor drug tolerance, however, the main adverse event was anaemia related to ribavirin in 62% of patients, and subsequent hepatic decompensation related to the low haemoglobin 38 . solar 1 and solar 2 also evaluated the efficacy of the combination of sofosbuvir and ledipasvir also included post liver transplantation rhcv patients. solar-1 study involving 12 or 24 weeks of ledipasvir, sofosbuvir and ribavirin in post-lt rhcv (n=229), the svr12 was 96%, 96%, 86%, 60%, 100% in patients with no cirrhosis, child a, child b, child c and fch patients with 12 weeks and, 98%, 96%, 88%, 75% and 100% for 24 weeks therapy. adverse events such as mild hyperbilirubinemia and anaemia however, mostly managed by reducing median dose of ribavirin to 600 mg daily 37 . solar-2 trial evaluated 226 post-transplant rhcv patients with ledipasvir sofosbuvir combination however, with child pugh score above 13 were excluded. svr12 was 93%, 100% and 95% in patients with no cirrhosis, child a and child b cirrhosis with 12 weeks j o u r n a l p r e -p r o o f therapy and 100%, 96%, 100% with 24 weeks therapy. these drugs were well tolerated with 5% adverse events, 2% discontinuation rates, and 2% viral relapse rates 39 .the success of solar trial with all oral daa in this challenging population opened the avenue for patients post lt rhcv. these two studies clearly demonstrated high svr rates in patients with 12 weeks of this combination therapy with effective hcv clearance post-transplant rhcv, than the previous generation drugs. ally-1 study also recruited patients with post-transplant rhcv infection (n=53) to assess the efficacy of 12 weeks daclatasvir 60 mg daily in combination with sofosbuvir 400 mg twice a day and weight based ribavirin. svr12 was 94% in lt recipients. viral relapsed in 3 posttransplant patients and were subsequently treated with 24 weeks of daclatasvir, sofosbuvir and ribavirin. there was no significant drug interactions with cni requiring dose adjustments 30 . interestingly, a real world analysis of daclatasvir and sofosbuvir showed 87-100% svr12 in patients with child b and c cirrhosis, and 58% of decompensated cirrhotic patients showed improvement in meld score 40 . a large multicentre study on daclatasvir in combination with sofosbuvir or simeprevir with or without ribavirin for 24 weeks was carried out on 97 lt recipients with severe rhcv including 37% patients with fch and 31% with cirrhosis. svr12 with dac + sof was 91% and dac + smv was 72%. there was a significant improvement in child pugh score from 6.8 to5.7 (p<0.001) and meld score from 12.1 to 9.7 (p<0.001) following svr. however, these scores worsened in 13% patients despite antiviral therapy 41. introduction of pangenotypic velpatasvir-sofosbuvir combination further simplified hcv therapy with maximal efficacy. a phase 3 study on 624 patients with hcv increased svr to 99% including in cirrhotic patients 42 . higher alt was shown to accelerate disease progression and cirrhosis. alt>100 u/l was shown to predict cirrhosis at 5 years due to rhcv (35% vs 6%). similarly, serum bilirubin >3.5 mg/dl was strongly associated with development of cirrhosis following rhcv (prieto 1999 hepatology, rosen 1999 transplantation) 43, 44 . practice of protocol liver biopsies at regular intervals post transplantation were carried out in the past. presence of moderate to severe lobular inflammation was associated with disease progression (30% vs 0.10%) compared to no or minimal inflammation over 5 years post transplantation. hcv rna titres has a direct correlation with the severity of post-transplant rhcv infection. in a study by shakel et al., 45 .peak hcv rna in the first year of untreated patients was associated with poor patient survival. a level of less than 10 7 , 10 7 -10 8 and >10 8 49 .in a recent innovative approach non-liver recipients of hcv positive donors were prescribed a short 7 day course of newer daa glecaprevir/pibrentasvir along with ezetimibe (hcv entry inhibitor) showed 100% viral clearance at week 12 50 . in summary, long term survival of hcv lt recipients has improved dramatically following second generation daa. with current svr close 100%, excellent safety profile daa therapy are increasingly used in decompensated cirrhosis and for post-transplant rhcv treatment. most centres treat hcv decompensated cirrhosis because it is associated with significant improvement in meld and may help reducing transplant waitlist. there was a debate j o u r n a l p r e -p r o o f whether to treat these patients while waiting for transplant or to treat in the posttransplant period, because some studies observed an increased risk of hcc following svr. in the management of rhcv, the timing of antiviral therapy is not well established. most experts recommend starting daa after first 3 months with stable immunosuppression onboard. a study by pellicelli et al., showed significant adverse events including hepatic decompensation and 25% mortality in those with advanced disease following treatment with daclatasvir and sofosbuvir for post-transplant rhcv 51 . similarly, forns et al, provided compassionate access sofosbuvir and ribavirin to 104 patients with severe rhcv with life expectancy less than 12 months. svr was 59% and much higher (73%) in patients with severe rhcv. however, severe adverse event occurred in 47% of patients including 13% mortality 52 . therefore, it is advisible to commence antiviral therapy prior to hepatic decompensation. post-transplant rhcv management previously included allograft biopsy to assess the severity of rhcv before commencing anti-viral therapy. however, the necessity of biopsy may be arguable with the currently available daa. abnormal liver enzymes in the presence of high hcv rna level may suffice to commence daa. retransplantation for graft failure secondary to rhcv had much worse outcomes. retransplantation for rhcv related graft failure is associated with prolonged hospitalization, increased cost and reduced survival. however, outcome and patients selection differed amongst various studies. in a multi-centred study from the us showed 1 and 3 year survival of hcv retransplantation was 69% and 49% with no difference in survival compared to other indications 53 .however, the currently available daa, the need for retransplantation likely to have reduced. hepatitis b is the major cause of chronic liver disease across the world affecting 350 million population, with higher prevalence in asian and african countries despite universal vaccination. hepatitis b was a considered an absolute contraindication in the initial years of liver transplantation due to early graft reinfection and poor survival benefit (less than 50%, j o u r n a l p r e -p r o o f therefore, most liver transplant centres adopted lifelong entecavir or tenofovir to reduce viral relapse following liver transplantation, particularly in low risk patients such as those with decompensated cirrhosis and low viral load (<100 copies/ml), alf presentation. low dose and short course hbig with antivirals may be useful in patients with high hbv dna, hbeag positive patients. tenofovir alafenamide (taf) is the most recently introduced formulation of tenofovir, to overcome renal and bone related adverse events related to tenofovir disoproxil fumarate. the efficacy is similar to its prodrug tenofovir disoproxil fumarate with a good safety profile. it is recommended in patients with chronic kidney disease and osteoporosis. data in posttransplant patients with taf is scarce. small case series found switching from tdf to taf hepatic involvement occurs in 14-53% of patients with covid-19, particularly in severe cases 73 . ace2 receptor is highly expressed in cholangiocytes (59.7%), vascular endothelial cells. interestingly, only 2% hepatocytes express ace2 and no expression observed in sinusoidal endothelial cells 72 . in a recent multicentre study on covid-19 patients with underlying cirrhosis (n=50), 97% required hospitalization and 71% required respiratory support. the number of patients with meld>15 increased from 13% to 26% (p=0.037) and aclf occurred in 28% of patients. in that study the 30-day mortality was 34% 74 . sars-cov-2 in decompensation in patients is associated with higher mortality 75 . analysis of apasl covid-19 liver injury spectrum study (apcolis) on 228 patients (43 cirrhotics), hepatic decompensation occurred in 9% and aclf in 11.6% of patients. child pugh score >9 predicted higher mortality (roc 0.94, hr 19.2). in patients with decompensated cirrhosis, mortality was 33% compared to 16.3% in compensated cirrhosis 76 to the non-transplant setting, however, chronic presentation defined as detectable hev rna or hev igm in the serum for 6 months, is exclusively observed in the post-transplant patients 86 . chronic hev following in transplant recipients was first recognised by kamar et al, including in lt patients. these patients continued to have allograft dysfunction following an acute infection and persistent positive hev rna in serum or stool for 10 to 24 months 87 . exact prevalence of hev seroprevalence in lt recipients is unknown. tests based on hev rna or hev igg level, a retrospective analysis of frozen sera showed 1% to 16% seroprevalence of hev in lt recipients 88 . in a french study, hev seroprevalence (hev igg) was observed in 12.9% of patients during pre-lt evaluation, interestingly one third of these patients became hev negative in the post-transplant period 89 late onset cmv disease occurs after completion of prophylaxis. especially in (r-/d+) recipients and its incidence is 25% which when compared to 8.3% in preemptive treatment 3-6 months reduces the advantage of universal prophylaxis. some studies report mitigation of this by extending the prophylaxis to 200 days, but data are insufficient 118 varicella zoster, that causes chicken pox remains latent in the dorsal root ganglion. this can reactivate following immunosuppression leading to herpes zoster, characterized by painful vesicular rash along the nerve distribution, usually restricted to one side. the 1-year incidence is 3% whereas the 5-and 10-year incidences are 14% and 18% respectively 146 human herpes virus 6 (hhv-6) belongs to beta herpesviridae subfamily under roseolovirus genus. this is a common name for 2 similar viruses hhv-6a and hhv-6b, the latter accounting in most cases 149 . asymptomatic exposure to hhv-6 occur in childhood such that 90-95% adult population are seropositive 150 . the circular dna of the virus integrates with the host genome and may remain latent for several years in the mononuclear cells 151, 152 . hhv-6 infection occurs as a result of reactivation in the post-transplant state. occasionally, donor derived infections or rarely through blood products have been reported 153 . incidence of hhv6 varies between 14% to 82% 154 , commonly occurring in the first 2-8 weeks after liver transplantation. sporadic cases were reported as early as 10 days and rarely after 5 years following liver transplantation 155 . in a prospective study of 51 lt patients, 11 (21.5%) developed hhv-6b reactivation, of which 4 had fever and abdominal pain 156 . reactivation rates are less when patients are covered up for cmv prophylaxis with ganciclovir. most hhv-6 reactivation are asymptomatic with low viral load 156 and they usually do not require treatment. clinically, hhv-6 reactivation presents with fever, and skin rash and raised liver enzymes 158, 159 . histologically, hhv-6 hepatitis may mimic acute cellular rejection with elevated liver enzymes and portal lymphocytic infiltration and confluent periportal necrosis on liver 153, 158 . in 170 lt patients with graft hepatitis, high levels of intra-hepatic hhv-6 dna and hhvantigenemia was significantly associated with decreased graft survival 160 . hhv-6 j o u r n a l p r e -p r o o f encephalitis though rare in lt recipients occurs usually within 4-6 weeks of transplantation. hhv-6a is neurotrophic and is detected in plasma as well as csf. brain imaging shows characteristic signal intensity in medial temporal lobes involving amygdala and hippocampi. hhv-6 infection may also present as post-transplant colitis in some 161 . it can also occur as co infection with cmv where hhv-6 antigenemia precedes cmv antigenemia 162 to summarise, hhv-6 infection after liver transplantation is rare, but its reactivation is associate with significant increase in graft failure, mortality, hepatitis c progression and cmv disease 169 . hhv infections in lt patients can be treated successfully with cmv antivirals ganciclovir, cidofovir and foscarnet. post lt viral infections can cause significant allograft dysfunction. early recognition, diagnosis and systematic approach can ameliorate the infective process and preserve allograft function. newly evolving less familiar viral infections such as covid-19 should also be considered in the differential diagnosis of post-transplant viral infections. management j o u r n a l p r e -p r o o f of post-transplant viral infection has improved over the last decade due to significant changes in immunosuppression protocols with optimal usage, and introduction of effective anti-viral drugs with high genetic barrier for resistance. these high potency anti-viral drugs have translated in to better long-term allograft and patient survival. • patients with decompensated cirrhosis due to chronic hepatitis b or hepatitis c infection should be commenced on high efficacy antiviral therapy • pathogenesis of covid-19 in post-liver transplant patients requires larger studies. • ideal immunosuppressive regimen to reduce the chance of recurrent or de novo viral infection needs to be studied. j o u r n a l p r e -p r o o f liver transplantation around the world. curr opin organ transplant hepatitis b virus epidemiology, disease burden, 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cytomegalovirus dna in liver donor biopsies and their correlation with hla matches and acute cellular rejection acute hepatitis with periportal confluent necrosis associated with human herpesvirus 6 infection in liver transplant patients encephalitis caused by human herpesvirus-6 in a liver transplant recipient treating hhv-6 infections diagnosis and clinical management hhv-6 in liver transplantation: a literature review key: cord-288721-3bv3aak6 authors: schneider, annika; kurz, sandra; manske, katrin; janas, marianne; heikenwälder, mathias; misgeld, thomas; aichler, michaela; weissmann, sebastian felix; zischka, hans; knolle, percy; wohlleber, dirk title: single organelle analysis to characterize mitochondrial function and crosstalk during viral infection date: 2019-06-11 journal: sci rep doi: 10.1038/s41598-019-44922-9 sha: doc_id: 288721 cord_uid: 3bv3aak6 mitochondria are key for cellular metabolism and signalling processes during viral infection. we report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies. mitochondria are crucial for cellular energy metabolism, critically involved in the coordination of signalling processes within cells and orchestrate induction of apoptotic cell death 1, 2 . besides this, cell-autonomous defence mechanisms during viral infection link innate immune sensing of infection and inflammation at the level of mitochondria 3, 4 . the research in the recent years has expanded our knowledge about the different roles of mitochondria. for the different functions mitochondrial shape and motility, but also size, are important and are highly dynamic processes 5 . mitochondrial shape and size are continuously changed during the dynamics of mitochondrial fusion and fission and mitochondrial turnover is controlled by mitophagy 5 . viruses modify the host cell to create an ideal ambience, which includes metabolic support for viral gene expression and replication. such modifications of cellular metabolism and structure of viruses can also affect mitochondria. there are more and more reports about viruses known to influence mitochondrial dynamics. viruses known to enhance mitochondrial fission are hepatitis b virus (hbv), hepatitis c virus (hcv) and epstein-barr virus [6] [7] [8] [9] . viruses, which interfere with or enhance mitophagy are hbv, hcv and measles virus [6] [7] [8] 10 . sars coronavirus is reported to enhance the fusion of mitochondria 11 . but the influence of viral infection on mitochondrial membrane potential and stress response has not been addressed in detail because of methodological constraints. so far, analysis of mitochondria and their functions relied mostly on bulk analysis of mitochondrial populations analysed ex vivo. in infected tissues where both, infected and non-infected cells are simultaneously present, it is very difficult to discriminate between mitochondria from infected versus healthy non-infected cells. this may be achieved by serial tissue sections analysed by electron microscopy, where viral particles could be visualized. however, this is a very time demanding process yielding results with little statistical power. we therefore aimed to develop a technology, where high numbers of single mitochondria and their function can be analysed in the context of viral infection in order to characterize changes induced by viral infection. we chose the liver, and more specifically hepatocytes, as viral infection increases size and mitochondrial fragility of liver mitochondria. we first aimed to determine the influence of viral infection of the liver on the size of liver mitochondria by flow cytometry. to that end, we established a reference curve using polystyrene microparticles with defined sizes (0.88 µm, 1,34 µm and 3 µm). forward scatter analysis of these polystyrene microparticles revealed clear demarcation of the differently sized microparticles and a direct linear correlation of forward scatter results with microparticle size (r 2 = 0.99) ( fig. 2a) , consistent with earlier reports that forward scatter measurements directly correlate with microparticle size down to 0.5 µm 20, 21 . the flow cytometric analysis revealed that mitochondria isolated from healthy non-infected liver ranged in size from 0.8 µm up to 1.4 µm (fig. 2b ) assuming that isolated mitochondria are spherical in morphology, which is indicated by electron microscopy (see fig. 1b ). since mitochondria from hepatocytes are much larger than those from non-parenchymal liver cells or immune cells, we assume that mitochondria ≥0.8 µm in size are derived from hepatocytes. mitochondria purified from virus-infected livers had a slightly higher mean size compared to healthy liver (1.04 ± 0.06 µm compared to 0.97 ± 0.04 µm, respectively) and ranged in size from 0.8 µm up to 3 µm (fig. 2b) . infection with recombinant replication-deficient adenoviruses is a well-established preclinical model system to study hepatotropic infections [22] [23] [24] . however, to confirm the results we repeated the experiments by infection with wildtype replication-competent lymphocytic choriomeningitis virus (lcmv). also after lcmv-infection, we detected an increase of mitochondrial size confirming the results obtained after adenoviral infection (supp. fig. 1a ). in order to investigate whether innate immunity generated during viral infection, was responsible for this increase in mitochondrial size, we induced a type i interferon response by application of poly i:c 25 . flow cytometric analysis of mitochondria isolated after poly i:c application did not reveal any differences in their size compared to the control groups suggesting other mechanisms (supp. fig. 1b) . the exact determination of the size of single mitochondria now opened the possibility to use this information for further analysis. next, we evaluated mitochondrial functionality by determining the mitochondrial membrane potential using the potentiometric dilc 1 (5) fluorescence dye. dose titration experiments of the dilc 1 (5) dye demonstrated a dose-dependent increase in fluorescence intensity in purified mitochondria (supp. fig. 1c ). upon addition of the electron chain uncoupling agent cccp, we found a profound reduction in dilc 1 (5) fluorescence (fig. 2c ) demonstrating that flow cytometric determination of changes in dilc 1 (5) fluorescence reflected mitochondrial membrane potential. by flow cytometric analysis we observed a significant decrease in the mitochondrial membrane potential of mitochondria isolated from virus-infected vs. healthy livers after either adenoviral or lcmv infection compared to healthy controls ( fig. 2d and supp. fig. 1d ). in contrast, we did not detect changes in the membrane potential after innate immune stimulation by poly i:c (supp. fig. 1e ). yet, the size of mitochondria may influence dilc 1 (5) signal intensity. indeed, we found a direct correlation between mitochondrial size and dilc 1 (5) staining ( fig. 2e and supp. fig. 1f ) suggesting that larger mitochondria purified from virus-infected livers should show higher dilc 1 (5) fluorescence intensity. we therefore compared mitochondria with the same size isolated from healthy or virus-infected livers. such direct comparison demonstrated that mitochondria of the same size from healthy vs. virus-infected livers showed a remarkable decrease in the membrane potential of mitochondria from infected livers (fig. 2f ) and suggested that viral infection caused changes in mitochondrial functionality. mitochondria also function to take up calcium from the cytosol and thereby coordinate cellular function 26 , which can also serve as a stress test. when challenged with high concentrations of calcium (100 µm), mitochondria isolated from virus-infected livers are much more fragile shown by time-dependent loss of membrane potential and change of their morphology indicated by decrease in side-scatter (fig. 2f ). this accurately detects mitochondrial swelling after loss of membrane potential following ca 2+ challenge which is also detected by bulk analysis with a classical stress test by adding ca 2+ and detection of loss of membrane potential by rh123-fluorescence and swelling by measuring optical density at 540 nm (supp. fig. 1g ) 18 . consistent with the loss of membrane potential and changes in side-scatter signals, we detected loss of mitochondrial integrity after www.nature.com/scientificreports www.nature.com/scientificreports/ calcium challenge. number of viable mitochondria detected per second by flow-cytometry declined after calcium challenge, consistent with loss of mitochondrial integrity, and did so much faster in samples from virus-infected livers (fig. 2f ). comparing mitochondria with different sizes, it became evident that larger mitochondria are more fragile and disappeared more rapidly after ca 2+ -challenge (fig. 2f ). taken together, here we detected an increase in size and a decrease in membrane potential as well as mitochondrial fragility of liver mitochondria after viral infection. however, since both, non-infected as well as infected hepatocytes are present in livers after adenoviral infection (see fig. 1b ), current protocols for isolation and analysis yield a mixture of mitochondria derived from healthy as well as infected hepatocytes. this makes it necessary to develop a methodology, by which mitochondria from healthy and virus-infected hepatocytes can be separated in order to characterize changes in mitochondrial function specifically in virus-infected cells. we generated a recombinant adenovirus expressing the fluorescent protein dsred fused to a mitochondrial localization sequence (ad-cmv-mitorl) that accumulates and selectively labels mitochondria within infected cells (supp. fig. 2 ). we combined this mitochondrial labelling in infected cells with a multispectral flow cytometric single organelle measurement of isolated mitochondria. upon infection of hepatocytes with ad-cmv-mitorl in vitro we detected mito-dsred-fluorescence in mitochondria using confocal microscopy (fig. 3a) . since ad-cmv-mitorl also codes for luciferase, we detected in vivo bioluminescence of the liver after infection, thus www.nature.com/scientificreports www.nature.com/scientificreports/ confirming successful infection of hepatocytes in vivo (fig. 3b ). this allowed us to test whether mitochondria from ad-cmv-mitorl-infected hepatocytes (mito-dsred + mitochondria) could be distinguished from those of non-infected hepatocytes (mito-dsred − mitochondria) within the same liver. after density-gradient purification, mitochondria isolated from virus-infected livers were counterstained with mitotracker green and analysed by flow cytometry allowing discrimination of mito-dsred + mitochondria from mito-dsred − mitochondria from the same liver (fig. 3c ). mito-dsred + mitochondria from virus-infected hepatocytes had a mean size of 1.19 ± 0.06 µm as compared to mito-dsred − mitochondria from healthy hepatocytes with a mean size of 0.96 ± 0.01 µm (fig. 3d ). this confirmed the results obtained from mitochondria isolated from non-infected livers and further demonstrated a more pronounced size difference when mitochondria from virus-infected could be distinguished at the single organelle level from those of healthy hepatocytes. this was most likely related to a relative underestimation of size for mitochondria from virus-infected livers due to contamination with mitochondria from non-infected cells that are smaller than hepatocyte mitochondria. yet, we cannot formally exclude that mito-dsred localizing to mitochondria after infection with ad-cmv-mitorl may have contributed to the size difference. the almost identical forward scatter results and size of mito-dsred − mitochondria compared to mitochondria isolated from non-infected livers (fig. 3d) indicated that there was no influence of viral infection in neighbouring hepatocytes on mitochondrial size after isolation. differences in size of mitochondria may have also an influence on other parameters detected by flow cytometry and we therefore systematically measured mitochondrial autofluorescence from 450 to 800 nm using a spectral flow cytometer. as expected, we detected increased fluorescence at 590 nm in mito-dsred + mitochondria, where the maximum of dsred fluorescence emission (590-650 nm) is expected 27 . interestingly, we detected higher autofluorescence signals between 500 and 550 nm as well as above 650 nm in mito-dsred + mitochondria (fig. 3e) . as the strength of autofluorescence may be influenced by the size of mitochondria, we analysed autofluorescence signals against the size of isolated mitochondria (fig. 3f ). we found that autofluorescence intensity between 430 and 550 nm directly correlated with mitochondrial size, which may explain the higher autofluorescence observed in larger mito-dsred + mitochondria. together, these data demonstrate the usefulness of single-organelle analysis by flow cytometry in combination with in vivo mitochondrial labelling in virus-infected hepatocytes to exactly determine physical parameters such as size or autofluorescence. mitochondrial crosstalk enables changes in membrane potential. since discrimination of mitochondria isolated from virus-infected compared to non-infected hepatocytes was reliably achieved using flow cytometry, we proceeded to test for changes in mitochondrial functionality upon infection. we assumed that the difference in membrane potential detected between mitochondria isolated from virus-infected livers compared to non-infected livers (see fig. 2 ) has previously been underestimated, and that our method would allow to more specifically discriminate mitochondria from virus-infected hepatocytes compared to non-infected hepatocytes. we determined whether mito-dsred + differed from mito-dsred − mitochondria with respect to dilc 1 (5) fluorescence intensity. to our surprise, we found that the dilc 1 (5) signal was similar for all sizes of mito-dsred + compared to mito-dsred − mitochondria (fig. 4a ). since dilc 1 (5) fluorescence was homogenous in all mitochondria isolated from ad-cmv-gol infected livers (see fig. 1 ), although they consisted of a mixture of mitochondria from infected and non-infected hepatocytes, we wondered whether there was an exchange of molecules between mitochondria. therefore, we mixed dilc 1 (5)-labelled mitochondria with non-labelled mitochondria and by time-dependent flow cytometric analysis found that dilc 1 (5) fluorescence decreased in pre-labelled and increased in un-labelled mitochondria reaching an equilibrium of intermediate fluorescence intensity within 30 seconds (fig. 4b) . however, mito-dsred was not exchanged between mitochondria, because we found clearly distinct dsred staining of mitochondria isolated from ad-cmv-mitorl-infected livers, and mito-dsred − mitochondria showed the same absent dsred fluorescence intensity as mitochondria isolated from non-infected livers. in order to further evaluate mitochondrial functionality, we challenged mitochondria with ca 2+ as stress test and performed time kinetic measurements of dilc 1 (5) fluorescence and side-scatter of mito-dsred + and mito-dsred − mitochondria isolated from ad-cmv-mitorl infected livers. remarkably, the differences in mitochondrial characteristics observed when comparing mitochondria isolated from infected livers to mitochondria from non-infected livers (see fig. 2f ) where not present any more when comparing mito-dsred + to mito-dsred − mitochondria originating from the same liver. in fact, loss of dilc 1 (5) fluorescence, decrease in side scatter and mitochondrial events were the same for mito-dsred + mitochondria as compared to mito-dsred − mitochondria (fig. 4c) . when in direct physical contact with mito-dsred + mitochondria, also mito-dsred − mitochondria showed the same fragility as mitochondria from virus-infected hepatocytes. there, was still a small difference in the large mitochondrial group after calcium stimulation and flow cytometric analysis of the ssc and dilc 1 (5) which could be explained by the fact that 5 to 10 minutes after calcium stimulation the number of events was drastically reduced. only approximately 10% from the initial number of events are still detectable (shown by number of events/s). because of the statistical variation the conclusions at later time points has to be taken with caution. interestingly, also mixing of dilc 1 (5) labelled mitochondria isolated from either ad-cmv-golor lcmv-infected with those from healthy uninfected livers yielded in rapid loss of mitochondrial membrane potential to that measured in mitochondria from infected livers (fig. 4d and supp. fig. 2 ) taken together these data demonstrate that mitochondria which are in close physical proximity exchange information leading to changes in mitochondrial membrane potential but not in mitochondrial size. here, we describe the influence of viral infection on the phenotype and function of mitochondria employing a new methodology combining spectral flow cytometry with virus-encoded markers to simultaneously evaluate multiple mitochondrial parameters at the level of single organelles. most studies involve confocal microscopy to detect mitochondria, which is also available in an automated manner to quantify large datasets of mitochondria 28 . www.nature.com/scientificreports www.nature.com/scientificreports/ while most of these microscopic studies are performed in cell cultures to explore mitochondrial dynamics at the level of single cells, there are only few reports specifically detecting tagged mitochondria in tissues for ex vivo or in vivo analysis 29, 30 . since in vivo microscopic analysis of mitochondria requires a complex experimental setup, is rather time consuming and does not allow for analysis of large numbers of mitochondria, we aimed to establish a methodology to evaluate mitochondria directly ex vivo following isolation from virus-infected tissue. so far, most of available methods analyse properties of mitochondria ex vivo at the level of mitochondrial populations, www.nature.com/scientificreports www.nature.com/scientificreports/ such as extracellular flux analysis, western blot analysis, calcium uptake or swelling assays. beyond visualization by microscopy, flow cytometry has emerged as technology to characterize mitochondria 18, 31, 32 . however, mitochondria isolated from virus-infected tissues can be derived from both, virus-infected cells as well as healthy cells, which may skew the experimental results. we therefore generated recombinant adenoviruses containing a mito-dsred expression cassette to selectively label mitochondria of infected cells. fusion of a fluorescent marker to mitochondrial target sequences has previously been reported to reliably and specifically label mitochondria as shown by confocal microscopy 33, 34 . we combined virus-encoded mito-dsred labelling of mitochondria to separate mitochondria of virus-infected cells from those originating from healthy cells, with the power of multi-parameter analysis by spectral flow cytometry. using this methodology, we provide evidence that mitochondria can be reliably separated from virus-infected cells and that viral infection led to an increase in size as well as a decrease of mitochondrial membrane potential. such changes in biophysical and functional properties of mitochondria were not triggered by innate immunity following recognition of infection through microbe-associated pattern recognition receptors indicating other reasons for these changes, which still have to be defined. time kinetic measurements of single mitochondria by flow cytometry further allowed us to detect a previously unknown mitochondrial cross-talk that involves rapid exchange of small molecules like the potentiometric dye dilc 1 (5) . such exchange of molecules among mitochondria required physical contact, occurred within seconds and did not include mitochondrial matrix-embedded proteins. this indicates a dynamic regulation of mitochondrial properties by cell autonomous mechanisms that require further investigation. taken together, the combination of mitochondrial labelling through mito-dsred together with single organelle analysis using spectral flow cytometry is ideally suited to further unravel biophysical and functional properties of mitochondria as well as mechanisms and consequences of mitochondrial interconnectivity in virus-infected cells. given the important role of mitochondria in cellular metabolism, anti-viral defence, cell signalling and cell death, the multiparametric analysis of single mitochondria opens new avenues to explore these complex mitochondrial functions in more detail in virus-infected cells. mice. c57bl/6 j mice were purchased from charles river (sulzfeld, germany). mice were maintained under specific pathogen-free (spf) conditions in the central animal facility of the klinikum rechts der isar, in accordance with the guidelines of the federation of laboratory animal science association. animal experiments were approved by the animal care commission of bavaria. male mice between the ages of 6-10 weeks were used. the expression cassette for cloning into recombinant adenovirus consists of the genes for the fluorescent protein dsred linked to a mitochondrial targeting sequence and cbg99-luciferase separated by p2a linker sites from the porcine teschovirus 1 followed by a bgh poly(a) signal. gene expression was driven by the ubiquitous minimal cmv-promoter (ad-cmv-mitorl). ad-cmv-gol generation has been reported before 23 . recombinant second generation serotype 5 adenoviruses were generated using the gateway ® technology from thermofisher as described before 23 . briefly, expression cassettes with cmv promotor, dsred linked to the mitochondrial targeting site, cbg99-luciferase and the bgh poly(a) signal were synthesized (eurofins genomics, germany) and cloned into gateway ® pentr ™ 11 dual selection vector (thermofisher scientific, germany). recombination of pentr ™ with expression cassette into pad/pl-dest ™ gateway ® vector (thermofisher scientific, germany) was performed in vitro via the lr clonase ® enzyme mix (thermofisher scientific, germany). the obtained pad/pl-dest ™ with expression cassette was linearized using the paci restriction enzyme and the resulting adenoviral dna was transfected with lipofectamine 2000 (thermofisher scientific, germany) into hek293 cells (crl-1573 ™ ; atcc, usa). cell debris and supernatant were harvested when complete detachment of the cells occurred. this suspension was freeze/thawed, centrifuged and used for further infection of hek293 cells. cells from several cell culture dishes were harvested and resuspended in tris-buffer and freeze/thawed three times. cell debris was removed by centrifugation and supernatant purified by a two-step cscl gradient ultracentrifugation. the band containing adenovirus was harvested and dialyzed. virus titer was determined via adenovirus hexon titration. hek293 cells were infected with serial dilutions of purified adenovirus. after 35 to 40 hours, cells were fixed with methanol, and virus infected cells were stained with anti-hexon antibody (anti-hexon 2297hrp, acris, germany) and detected via dab (dako, usa). the infected cells were counted and the titer was calculated. bioluminescence imaging. imaging of luciferase expression in infected mice was monitored by ivis lumina lt-series iii instrument (perkinelmer las, germany). five minutes before measurement mice have been anesthetized with 2.5% isofluran and treated intraperitoneally with 100 mg/kg bodyweight d-luciferin-k-salt (pjk gmbh, germany). isolation of mitochondria from murine liver tissue. heparin/nacl (300 u/150 µl) was injected i.p. into the mouse 5 minutes prior to preparation. mice were sacrificed and livers were perfused via portal vein for 1 minute with pbs to remove blood. liver was removed and weighed, and the liver was rinsed with isolation buffer www.nature.com/scientificreports www.nature.com/scientificreports/ (220 mm mannitol, 80 mm sucrose, 10 mm hepes, 1 mm edta, ph 7.4). the whole isolation procedure was performed on ice and in ice-cold isolation buffer. the tissue was rinsed with 1 ml isolation buffer and cut with a blunt end scissor into small pieces. the liver fragments were resuspended in 1 ml isolation buffer supplemented with 0.5% bsa and protease inhibitor (protease inhibitor cocktail, edta-free, roche, switzerland) per 0.1 gram of weighted organ and homogenized in a potter-elvehjem with 3 strokes at 800 rpm. the homogenate was transferred to cooled 50 ml falcon and centrifuged at 600 x g for 10 minutes to remove nuclei, intact cells and cellular debris. the supernatant was transferred to a glass tube and centrifuged at 4000 x g for 10 minutes to sediment mitochondria. the received crude pellet was gently dislodged with a glass pestle from the side of the glass tube. mitochondrial purification by density gradient centrifugation. mitochondria were purified as previously described 35, 36 . in brief, a discontinuous percoll density gradient was used for mitochondrial purification. crude mitochondria were resuspended in ip-buffer (300 mm sucrose, 5 mm tes, 0.2 mm egta, ph 6.9), loaded on a percoll density gradient (60%, 30% and 18% diluted in ipp buffer: 300 mm sucrose, 10 mm tes, 0.2 mm egta, 0.1% w/v bsa, ph 7.2) and separated at 9000 × g for 10 minutes. the phase containing mitochondria between 60% and 30% percoll-layer was recovered with a glass pipette and transferred to a 30 ml glass tube, resuspended in 15 ml ip-buffer and centrifuged for further 10 minutes at 9000 × g. the pellet was washed again in 10 ml ip-buffer and centrifuged at 9000 × g for 10 minutes to get rid of remaining percoll. the supernatant was removed and mitochondrial pellet was dislodged from the side of the glass tube. the received mitochondria were resuspended in 100 µl ip-buffer and kept on ice. determination of protein concentration. the protein content in the mitochondrial preparations was determined using the dc tm protein assay kit (bio rad laboratories, germany). the assay was performed according to the manufacturer´s protocol. four different bsa-dilutions reaching from 0.25 mg/ml to 1.5 mg/ml in ip-buffer were used as standards. the optical density was measured at 750 nm with a multiplate reader (infinitem100 pro, tecan, germany). determining mitochondria by flow cytometry. mitochondria were diluted to 10 µg protein per µl in ice-cold mitochondrial staining buffer msb (0.2 m saccharose, 10 mm mops-tris, 5 mm succinate, 1 mm phosphoric acid, 10 µm egta). the different mitochondrial probes were diluted in msb, mixed with the mitochondrial dilution in a 1:1 ratio and incubated at room temperature for 20 minutes. the cell permeable carbocyanine-based mitotracker green probe (mtg, 200 nm), which contains a mildly thiol-reactive chloromethyl moiety, was used to selectively stain all undamaged mitochondria regardless of the membrane potential. dilc1(5) (100 nm), a cationic carbocyanine dye, was used to measure the membrane potential of isolated mitochondria. mitochondria were pelleted at 9000 x g for 2 minutes and washed once in ice cold pbs. mitochondrial pellet was resuspended in msb to a final concentration of 10 µg/µl and stored on ice for analysis. immediately before analysis, samples were diluted in ice-cold and filtered pbs to the final analysis concentration of 0.05 µg/µl. samples were analysed using the spectral cell analyzer sp6800 (sony biotechnology inc, japan). the sample flow rate was set to record about 1500 events per second. as mitochondrial uncoupling by the protonophore cccp is well known to dissipate mitochondrial membrane potential (mmp), 5 µm cccp (sigma-aldrich, st. louis, missouri, usa) was used as a positive control for membrane potential dependence of diic 1 (5) (biotium, hayward, usa). the mitochondrial permeability transition (mpt), a process characterized by a large increase of permeability of the inner mitochondrial membrane (imm), leading to an influx of solutes with a molecular weight less than 1.5 kda and water into the mitochondrion, is a ca 2+ -induced process. the influx of solutes and water leads to swelling of mitochondria. in mpt-measurements 100 µm ca 2+ in msb was added to induce swelling and samples were analyzed immediately after administration and every following 5 minutes for 45 minutes in total. cyclosporina (sigma-aldrich, st. louis, missouri, usa) inhibiting mpt and thereby reversing the effect of ca 2+ , was added at a concentration of 5 µm. mitochondrial size was determined using polystyrene particle size standard beads (flow cytometry grade, spherotech) in three sizes: 0.88 μm, 1.34 μm and 3 μm. beads of each size were separated via ultrasound, vortexed and 20000 beads/size were added per ml filtered pbs. immediately before analysis, mitochondria were diluted in bead mixture to the final analysis concentration of 0,05 µg/ml. data were analysed using flowjo software (version 10, flowjo, oregon, usa). western-blot. 30 µg of protein per sample was loaded onto 4-20% mini-protean ® tgx stain-free ™ precast gels (bio rad laboratories, münchen) and separation was performed within a gel chamber filled with 1x sds electrophoresis buffer at 100 v for 1 to 2 hours. after separation, proteins were blotted using the trans-blot ® turbo ™ mini pvdf transfer packs (bio rad laboratories, germany). proteins were transferred onto membranes at 2.5 a for 30 minutes using the trans-blot turbo ™ (bio rad laboratories, germany). membranes were blocked with 10% milk in tbs-t (tbs + 0.1% tween-20) for 1 hour at room temperature, washed three times with tbs-t and incubated with primary antibodies in 5% bsa in tbs-t overnight at 4 °c. the membranes were washed three times with tbs-t and incubated for 4 hours at room temperature with hrp-coupled secondary antibodies in 10% milk powder in tbs-t. blots were washed three times and developed using cheluminate-hrp picodetect (applichem gmbh, germany), which was evenly distributed on the membrane. the luminescence was detected for up to 20 minutes using the imaging-system chemidoc tm xrs (bio rad laboratories, germany). to visualize www.nature.com/scientificreports www.nature.com/scientificreports/ several proteins on the same blot, primary and secondary antibodies were removed by incubating membranes for 45 minutes at 50 °c in stripping buffer containing ß-mercaptoethanol. subsequently membranes were washed three times with tbs-t and incubated as previously described with primary and secondary antibodies. following primary antibodies were used: adenine nucleotide translocator (ant) (santa cruz biotechnology usa), cytochrome-c-oxidase (cox iv), cytochrome-c (cyt-c), glyceraldehyde 3-phosphate dehydrogenase (gapdh), glucose-regulated-protein 78 (grp78), histon 2b (h2b), voltage dependent anion channel (vdac) (all cell signaling technology, usa), lysosome-associated membrane protein 2 (lamp2) (thermo fisher scientific, usa), peroxisomal membrane protein 70 (pmp70) (origene technologies, usa), following secondary antibodies were used: rabbit anti-goat hrp (santa cruz biotechnology, usa), mouse anti-rabbit hrp, goat anti-mouse hrp (jackson immunoresearch, uk). histology. mouse livers were fixed for 48 hours in 4% paraformaldehyde. dehydrated livers (leica asp300s, germany) were embedded in paraffin. serial 2 µm-thin sections were prepared with a rotary microtome (hm355s, thermofisher scientific, usa) and subjected to histological and immune-histochemical analysis. hematoxylin-eosin (he) staining was performed on deparaffinized sections with eosin and mayer's haemalaun according to standard protocol. immunohistochemistry was performed using a bondmax rxm system (leica, wetzlar, germany, all reagents from leica) with primary antibody against egfp (a-11122, diluted 1:500 in antibody diluent, invitrogen, thermofisher scientific, usa). slides were deparaffinized, pre-treated with epitope retrieval solution 1 for 30 minutes. bound antibody was detected with a polymer refine detection kit without post primary reagent and visualized with dab as a dark brown precipitate. counterstaining was done with hematoxyline. electron microscopy. tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 m sodium cacodylate buffer ph 7.4 (science services, munich, germany), postfixed in 2% aqueous osmium tetraoxide 37 , dehydrated in gradual ethanol (30-100%) and propylene oxide, embedded in epon (merck, darmstadt, germany) and cured for 48 hours at 60 °c. semithin sections were cut and stained with toluidine blue. ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (zeiss libra 120 plus, carl zeiss nts gmbh, oberkochen, germany). pictures were acquired using a slow scan ccd-camera and item software (olympus soft imaging solutions, münster, germany). statistics. student's t tests were calculated using graphpad prism software. significance was set at p < 0.05 and denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001. all results are expressed as the mean ± standard deviation (sd). the data within this manuscript are available from the corresponding author upon reasonable request. mitochondrial control of cellular life, stress, and death mitochondrial signaling pathways: a receiver/integrator organelle mechanisms of mavs regulation at the mitochondrial membrane identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 mitochondria: dynamic organelles in disease, aging, and development hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis hepatitis c virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy epstein-barr virus latent membrane protein-2a alters mitochondrial dynamics promoting cellular migration mediated by notch signaling pathway mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-i-like receptor signaling sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/ traf3/traf6 signalosome living in the liver: hepatic infections correlated morphometric and biochemical studies on the liver cell. i. morphometric model, stereologic methods, and normal morphometric data for rat liver bioluminescence imaging allows measuring cd8 t cell function in the liver isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses analysis of mitochondria by flow cytometry real-time flow cytometry analysis of permeability transition in isolated mitochondria flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death measurement of mitochondrial mass by flow cytometry during oxidative cell sizing: a light scattering photometer for rapid volume determination overcoming limitations of microparticle measurement by flow cytometry tnf-induced target cell killing by ctl activated through cross-presentation outcome of anti-viral immunity in the liver is shaped by the level of antigen expressed in infected hepatocytes perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis reduced type i interferon production by dendritic cells and weakened antiviral immunity in patients with wiskott-aldrich syndrome protein deficiency calcium uptake mechanisms of mitochondria biochemistry, mutagenesis, and oligomerization of dsred, a red fluorescent protein from coral deep analysis of mitochondria and cell health using machine learning multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo in vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system flow cytometry of isolated mitochondria during development and under some pathological conditions why to compare absolute numbers of mitochondria analysis of mitochondrial dynamics and functions using imaging approaches strategies for imaging mitophagy in high-resolution and high-throughput a semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies progressive stages of mitochondrial destruction caused by cell toxic bile salts a chrome-osmium fixative for electron microscopy this work was funded by the deutsche forschungsgemeinschaft (dfg, german research foundation) -projektnummer 272983813 -trr 179 to d.w and p.k. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44922-9.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-342930-f7cw2ca6 authors: portincasa, piero; krawczyk, marcin; machill, antonia; lammert, frank; ciaula, agostino di title: hepatic consequences of covid-19 infection. lapping or biting? date: 2020-06-01 journal: eur j intern med doi: 10.1016/j.ejim.2020.05.035 sha: doc_id: 342930 cord_uid: f7cw2ca6 the outbreak of coronavirus disease 2019 (covid-19) starting last december in china placed emphasis on liver involvement during infection. this review discusses the underlying mechanisms linking covid-19 to liver dysfunction, according to recent available information, while waiting further studies. the manifestations of liver damage are usually mild (moderately elevated serum aspartate aminotransferase activities), and generally asymptomatic. few patients can still develop severe liver problems, and therapeutic options can be limited. liver dysfunction may affect about one-third of the patients, with prevalence greater in men than women, and in elderly. mechanisms of damage are complex and include direct cholangiocyte damage and other coexisting conditions such as the use of antiviral drugs, systemic inflammatory response, respiratory distress syndrome-induced hypoxia, sepsis, and multiple organ dysfunction. during new covid-19 infections, liver injury may be observed. if liver involvement appears during covid-19 infection, however, attention is required. this is particularly true if patients are older or have a pre-existing history of liver diseases. during covid-19 infection, the onset of liver damage impairs the prognosis, and hospital stay is longer. a novel coronavirus was reported to world health organization on dec 30, 2019, as the cause of a 2 cluster of pneumonia cases in china, city of wuhan, hubei province. the first name of 2019-3 ncov(human) was adopted on jan 7, 2020, lately changed to severe acute respiratory syndrome 4 coronavirus 2 (sars-cov-2). covid-19 infection became an outbreak throughout china on feb 5 11, 2020 and subsequently was identified as a global pandemic on march 11, 2020, spreading to 6 more than 120 countries, as a major threat to public health [1] [2] [3] . the covid-19 pandemic suddenly 7 represented an enormous burden of care [4] , and raised issues related to medical ethics [5] , since 8 specific therapies and/or vaccines are missing, to date. covid-19 may manifest in different ways. 9 many subjects may remain asymptomatic [6] , but the exact number is still unknown. specific 10 settings might facilitate the spread of infection e.g., in skilled nursing facility where more than half 11 of residents with positive test results were asymptomatic at the time of testing and most likely 12 contributed to transmission [7, 8] . the proposed 3-stage classification system of potential increasing 13 severity for covid-19 infection encompasses stage i (early infection), stage ii (pulmonary phase), 14 and stage iii (hyperinflammation phase) [9] . although the most frequent and critical clinical 15 presentation is secondary to the involvement of the lung (fever, cough), the infection by sars16 cov-2 virus may lead to a systemic and multi-organ disease [10] , also involving the gastrointestinal 17 tract (nausea/vomiting, or diarrhea) [11, 12] . the liver appears to be the second organ involved, 18 after the lung [13] [14] [15] . 19 the present paper explores the available evidences on liver involvement in patients with covid-19 20 infection, to provide a comprehensive understanding of the phenomenon, and to anticipate effective 21 follow-up. during covid-19 infection, patients can be asymptomatic or present clinical symptoms ranging 25 from fever, dry cough, headache to dyspnea and fatigue, to acute respiratory distress syndrome 26 6 (ards), shock, and cardiac failure [9, 16] . a nasopharyngeal swab is the collection method used to 1 obtain a specimen for testing. because the likelihood of the sars-cov-2 being present in the 2 nasopharynx increases over time, repeated testing is often used [17] . multi-organ involvement 3 secondary to covid-19 infection occurs in a subgroup of patients [10] . covid-19 infection can be 4 associated with myocardial injury [18] [19] [20] , heart failure [18] , vascular inflammation, myocarditis, 5 cardiac arrhythmias [19] , and hypoxic encephalopathy [21] . the progression and prognosis of 6 covid-19 infection is worse in the presence of diabetes mellitus [22, 23] . the case-fatality rate 7 increases with age (from 8% to 15% in the age range 70-79 years, and ≥80 years, respectively) and 8 with associated diseases, i.e., 11%. 7%, 6%, 6%, and 6% in patients with cardiovascular disease, 9 diabetes mellitus, chronic respiratory disease, hypertension, and cancer, respectively [ another study in zhejiang province [26] . gastrointestinal involvement could be the consequence of 15 covid-19-angiotensin-converting enzyme 2 (ace2) receptors at the enterocyte level (i.e. 16 glandular cells of gastric, duodenal and distal enterocytes), resulting in malabsorption, unbalanced 17 intestinal secretion and activated enteric nervous system, therefore diarrhoea) [28, 29] . in human 18 small intestinal organoids, sars-cov-2 rapidly infects the enterocytes and strongly induces a 19 generic viral response program, pointing to a marked viral replication in the intestinal epithelium 20 [30] . 21 notably, continuous viral rna shedding occurs into feces up to 11 days negativity of respiratory a study reported that the virus can be detected but not cultivated from stool (despite high rna 1 concentration), consistent with the lack of transmission [33] . in a case-control study from usa 2 (enrolling 278 covid-19 positive patients and 238 covid-19 negative patients), the presence of 3 gastrointestinal symptoms was predictive of covid-19 positivity, and symptoms were associated 4 with slower and less severe disease course [34] . when the virus binds to ace2 receptors [35] [36] [37] to enter the target cell [38] . receptors are well 10 expressed in epithelia of the lung, gastrointestinal tract, and vascular endothelium, also in the liver 11 [39]. this early period of covid-19 infection can evolve to the second stage of viral pneumonia.; (ii) extra-pulmonary systemic hyperinflammation syndrome occurs in the minority of infected 13 patients, and is characterized by the so-called "cytokine storm". at this moment, several cytokine 14 levels increase, namely interleukin (il)-2, il-6, il-7, il-10, and tumor necrosis factor (tnf)α. 15 additional inflammatory biomarkers include granulocyte-colony stimulating factor, interferon 16 (ifn)-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory patients with severe and fatal disease had significantly increased white blood cell count, and 24 decreased lymphocyte and platelet counts compared to non-severe disease and survivors. biomarkers of inflammation, muscle and cardiac injury, as well as liver and kidney function and year in 43% of the 99 covid-19 cases from wuhan [48] . this aspect deserves further attention. although the level of serum transaminases could be already elevated before the onset of covid-14 19, results from clinical reports and autopsy studies [26, 49, 50] suggest that liver dysfunction can 15 be an expression of a worse disease evolution, and that an isolated elevation of transaminases alone 16 is likely to be the indirect expression of a systemic inflammation. previous data from covid-19 outbreak in china found that 2-11% of patients had liver 18 comorbidities, 14-53% of patients presented with abnormal serum aminotransferases levels during 19 the disease, and that the rates of liver dysfunction were more present in subjects with the most 20 severe clinical presentation [26, 49] . in another large series of 417 chinese covid-19 patients, 21 abnormal liver tests (ast, alt, total bilirubin, ggt) were present in 76.3% of patients and 21.5% 16 china, in 6% to 22% and 21% to 28% of patients, respectively. in studies from wuhan, ast levels 17 were increased in 24% to 37% of patients, a proportion higher than in other chinese regions 18 (zhejiang), reporting a proportion of 16%. a gender difference might exist in this respect [62] , since 19 the prevalence of ast increase is higher in men than women, as documented by six case series (i.e., 20 average 66% vs. 35%, respectively). case reports and case series also suggest that the probability of 21 developing liver dysfunction increases with older age [61] . notably, the elevation of additional elements possibly concurring to liver damage are drug-related injury and the progression 14 of underlying liver diseases. 15 it is still under debate if these alterations can really be an expression of a clinically relevant liver 16 injury requiring particular attention in the management of the disease [13, 68]. in one study, patients 17 developing abnormal liver tests had higher risks of progressing to severe disease [51] , and the 18 finding is associated with longer hospital stay [62] . in addition, the more severe form of covid-19 19 infection is a predisposing condition to a more evident liver damage [10, 49, 69] , and therefore also promoted by systemic sepsis [71] . viral inclusions seem to be absent in the liver [57] , but this 2 possibility deserves further investigations, because of potential viral rna translocation from 3 intestine though portal blood. 4 another possibility is the direct damage from covid-19. cholangiocytes express ace2 receptors 5 (more that 20-fold than in hepatocytes). although cell damage can also occur at the level of bile covid-19 infection can progress to the inflammatory cytokine storm [75] , which involve both the 13 innate (toll-like receptors, tlrs) and the cellular adaptive immunity (killer t lymphocytes) [76, increased neutrophil counts and neutrophil to lymphocyte ratios, as well as hyperferritinemia [10] . elderly patients go worse, in this respect [79] . this sudden and immense immune hyperactivation 24 may result in multiple organ failure lungs but also to the liver, heart, and kidneys [75] . serum levels liver disorder in western industrialized countries, (prevalence ranging from 10 to 46% in the united 1 states [89-91]) and a median of 20% worldwide with a documented rising trend with time [92] . this 2 trend in north america and europe is the consequence of the rising prevalence of major risk factors 3 for nafld, including obesity, sedentary lifestyles, type 2 diabetes mellitus, dyslipidaemia, and 4 metabolic syndrome [92] [93] [94] [95] . however, lean non-alcoholic steatohepatitis (nash) can develop as 5 well [94] and is frequent in asia [96] . overall, factors contributing to nafld include the 6 environment, the gut microbiome, disrupted gluco-lipid metabolic pathways, metabolic was more frequent among those receiving lopinavir/ritonavir after hospital admission [62] . 14 remdesivir, a nucleoside analog prodrug developed by gilead sciences (usa), is effective against 15 covid-19 replication in vitro [116] and in infected patients [117] . this drug produced similar 16 effects on liver enzymes [118] . hydroxy chloroquine sulphate is also effective in vitro [116] and, in 17 covid-19 patients for short periods, appears to safe. rare case of fulminant hepatic failure have 18 been described with hydroxy chloroquine [119, 120] . acute liver injury is also possible after 19 azithromycin treatment, with a clinically evident presentation following about two weeks after drug 20 cessation, and after an average duration of treatment of 4 days [121] . several patients with 21 concomitant diseases (i.e. diabetes type 1 or 2, or hypertensive), undergo antihypertensive therapies 22 with ace inhibitors and angiotensin ii type i receptor blockers. in this context, a possibility is the 23 onset of ace2 overexpression. whether this condition will facilitate covid-19 infection and 24 penetrance, deserves further attention [122, 123] . there is no evidence, however, that ace 25 inhibitors will worsen the consequence of infection [123] . many patients with fever use antipyretic 1 agents, namely acetaminophen [124] . this drug might mediate, at least in part, the liver damage [57] . 2 patients with underlying metabolic abnormalities and nafld might be more exposed to drug-3 induced liver damage (dili) [99, 108]. as mentioned earlier, the cytokine mcp-1 is often increased 4 in covid-19 patients [42] and act as a further hit for steatohepatitis [125] . in addition, patients with 5 nafld/nonalcoholic steatohepatitis (nash) covid-19 infection, might be more susceptible to 6 dili, as well as to therapy with steatogenic drugs (amiodarone, sodium valproate, tamoxifen and world health organization. coronavirus disease 2019 (covid 19) association of coronavirus disease prominent changes in blood coagulation of patients 12 with sars-cov-2 infection abnormal coagulation parameters are associated with poor 14 prognosis in patients with novel coronavirus pneumonia disseminated intravascular coagulation in patients with 2019-ncov pneumonia hematologic, biochemical and 18 immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 19 2019 (covid-19): a meta-analysis tissue-based map of the human proteome epidemiological and clinical characteristics of 23 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus 26 disease 2019 in china gross examination report of a covid-19 28 death autopsy characteristics of liver tests in covid-19 patients patients with coronavirus disease 2019 (covid-19) gastrointestinal and liver manifestations in patients with covid-19 liver impairment associated with disease progression in 36 covid-19 patients liver injury in covid-19: management and challenges radiological findings from 81 patients with 40 covid-19 pneumonia in wuhan, china: a descriptive study high expression of ace2 receptor of 2019-14 ncov on the epithelial cells of oral mucosa analysis of angiotensin-converting enzyme 2 (ace2) from different 16 species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov sars-associated viral hepatitis 19 caused by a novel coronavirus: report of three cases overexpression of 7a, a protein 21 specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a 22 caspase-dependent pathway liver impairment in covid-19 patients: a 27 retrospective analysis of 115 cases from a single center in wuhan city a descriptive study of the impact of diseases control and 29 prevention on the epidemics dynamics and clinical features of sars-cov-2 outbreak in shanghai, 30 lessons learned for metropolis epidemics prevention the mechanisms and strategies to protect from hepatic ischemia-32 reperfusion injury liver -guardian, modifier and target of sepsis specific ace2 expression in cholangiocytes may 39 cause liver damage after 2019-ncov infection ischaemia reperfusion injury in liver 41 transplantation: cellular and molecular mechanisms immune responses in covid-19 and potential vaccines: 1 lessons learned from sars and mers epidemic pathogen recognition and toll-like receptor targeted therapeutics in innate 3 immune cells infection of neonatal 5 mice with sindbis virus results in a systemic inflammatory response syndrome systemic viral infections and collateral damage in the liver evolution of the immune system in humans from infancy 10 to old age mcp-1 expression in an autocrine manner in hepatocytes during acute mouse liver injury signaling axis is a key mediator of hepatic ischemia-reperfusion injury mitochondrial 17 oxidative injury in rat fatty livers exposed to warm ischemia-reperfusion the reduced 20 tolerance of rat fatty liver to ischemia reperfusion is associated with mitochondrial oxidative injury people who are at higher risk for severe illness study of the relationship sars and hepatitis virus b care of patients 26 with liver disease during the covid-19 pandemic: easl-escmid position paper the global burden of liver disease: the major 29 impact of china prevention of sars-cov-2 infection in patients with 31 decompensated cirrhosis fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population 34 utilizing ultrasound and liver biopsy: a prospective study systematic review: the epidemiology and natural history of 36 non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults prevalence of 39 nonalcoholic fatty liver disease in the united states: the third national health and nutrition 40 examination survey changes in the prevalence 1 of the most common causes of chronic liver diseases in the united states from exercising the hepatobiliary-gut axis. the impact of physical activity performance liver disease in non-obese individuals: prevalence, pathogenesis and treatment epidemiological feature of nafld from 10 1999 to 2018 in china the global nafld epidemic nonalcoholic fatty liver disease the common adiponutrin 16 variant p. i148m, a common genetic risk factor for severe forms of nafld and ald, in gallstone 17 patients non-alcoholic fatty liver diseases in patients with 19 covid-19: a retrospective study potential implications of covid-19 in non-alcoholic fatty liver disease chronic liver injury in rats 23 and humans upregulates the novel enzyme angiotensin converting enzyme 2 associated with markers of inflammation and oxidative stress in analysis of data from the 26 framingham heart study clinical and 28 metabolic characterization of obese subjects without non-alcoholic fatty liver: a targeted 29 metabolomics approach adipokines and 31 cytokines in non-alcoholic fatty liver disease advice for hepatology and liver transplant providers during the covid-19 pandemic: aasld 34 expert panel consensus statement highlights for management of patients 36 with autoimmune liver disease during covid-19 pandemia the covid-19 pandemic will have a long-lasting impact on the quality of 38 cirrhosis care covid-19 and 40 drug-induced liver injury: a problem of plenty or a petty point establishment of a new animal model of 1 azithromycin-induced liver injury and study the molecular pathological change during the process acute hepatocellular injury associated with azithromycin lopinavir/ritonavir 6 induces the hepatic activity of cytochrome p450 enzymes cyp2c9, cyp2c19, and cyp1a2 but 7 inhibits the hepatic and intestinal activity of cyp3a as measured by a phenotyping drug cocktail in 8 healthy volunteers hydroxychloroquine-induced toxic hepatitis in a patient with systemic lupus 10 erythematosus: a case report association of polymorphisms 12 of cytochrome p450 2d6 with blood hydroxychloroquine levels in patients with systemic lupus 13 in vitro evaluation of hepatotoxic drugs in human hepatocytes from 15 multiple donors: identification of p450 activity as a potential risk factor for drug-induced liver injuries expression and activities of cytochrome p450 enzymes in an age-dependent manner in mouse 19 liver remdesivir and chloroquine effectively inhibit 21 the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel 23 coronavirus in the united states fulminant hepatic failure secondary to 26 hydroxychloroquine hydroxychloroquine, a less toxic derivative of 28 chloroquine, is effective in inhibiting sars-cov-2 infection in vitro clinical and 30 histologic features of azithromycin-induced liver injury are patients with hypertension and diabetes mellitus at 33 increased risk for covid-19 infection? the lancet misguided drug advice for covid-19 characteristics of and public health responses to the coronavirus disease 36 2019 outbreak in china inflammation in alcoholic and nonalcoholic fatty liver disease: friend or 38 foe? we confirm that there are no known conflicts of interest associated with this publication and there has been 3 no significant financial support for this work that could have influenced its outcome. 4 5 key: cord-025168-be7zube4 authors: saleh, mahshid; taher, mohammad; sohrabpour, amir ali; vaezi, amir abbas; nasiri toosi, mohsen; kavianpour, maria; ghazvinian, zeinab; abdolahi, shahrokh; verdi, javad title: perspective of placenta derived mesenchymal stem cells in acute liver failure date: 2020-05-24 journal: cell biosci doi: 10.1186/s13578-020-00433-z sha: doc_id: 25168 cord_uid: be7zube4 acute liver failure (alf) is a life-threatening disease and is determined by coagulopathy (with inr ≥ 1.5) and hepatic encephalopathy as a result of severe liver injury in patients without preexisting liver disease. since there are problems with liver transplantation including lack of donors, use of immunosuppressive drugs, and high costs of this process, new therapeutic approaches alongside current treatments are needed. the placenta is a tissue that is normally discarded after childbirth. on the other hand, human placenta is a rich source of mesenchymal stem cells (mscs), which is easily available, without moral problems, and its derived cells are less affected by age and environmental factors. therefore, placenta-derived mesenchymal stem cells (pd-mscs) can be considered as an allogeneic source for liver disease. considering the studies on mscs and their effects on various diseases, it can be stated that mscs are among the most important agents to be used for novel future therapies of liver diseases. in this paper, we will investigate the effects of mesenchymal stem cells through migration and immigration to the site of injury, cell-to-cell contact, immunomodulatory effects, and secretory factors in alf. liver is one of the largest vital organs in human body that controls various biological processes, including the production of multiple hormones, storage of glycogen, neutralization of toxins and drugs, control of metabolism, metabolism of urea, and synthesis of plasma protein. typically, most physiological features of liver function are controlled by liver cells or hepatocytes; therefore, the loss of hepatocytes is the main cause of liver failure. several diseases related to malfunction of the liver are caused by damage to or loss of hepatocytes, including viral hepatitis, fatty liver disease, drug and toxin-induced liver injury, hepatocellular carcinoma, and hepatic abnormalities associated with autoimmunity and cirrhosis [1] . (aspartate aminotransferase), alt(alanine transaminase), tbil (total bilirubin indirect level), alb (albumin)). liver failure is divided into three forms as follows. alf within 48 h to several days with jaundice, coagulopathy and encephalopathy; acute-on-chronic liver failure (aclf) with a background of chronic liver disease leading to rapid progression of liver injury and associated with jaundice and ascites; and clf occurring within months to years [7] . alf is an unpredictable and potentially catastrophic condition often encountered in intensive care units, with more than 2500 cases reported each year in the united states. the progression potential of acute hepatic dysfunction toward multi-organ failure demands rapid diagnosis and management of the disease. due to a set of hepatic and non-hepatic complications, alf indirectly leads to immediate follow-up for liver transplantation [8] . alf, formerly known as fulminant hepatic failure, means the development of hepatocellular disorders such as coagulopathy and encephalopathy with inr ≥ 1.5 in patients without a history of liver disease within 26 weeks. more than half of the cases of alf progression require liver transplantation and significant improvements have been reported in the last decade after liver transplantation. alf mortality is usually due to intracranial hypertension (ich) and infection [9] [10] [11] ). however, patients with varying degrees of hemodynamic disorders and renal failure have also been reported [12, 13] . clinically, the patients show coagulopathy, jaundice and hepatic encephalopathy. the period between the onset of the first clinical symptoms and hepatic encephalopathy is crucial in determining the prognosis of these patients [14, 15] . there are obvious differences in the development mechanisms of early alf. the three main factors determining the prognosis of this disease include metabolic problems leading to the loss of liver cells, secretion of toxins and mediators from the liver tissue, and capacity of the remaining hepatocytes to repair the liver [15, 16] . common treatments are therapies that are often meant to improve the complications of acute liver failure (alf). multiple organ failure (mof) and severe infection are the most prevalent factors of mortality in these patients. therefore, management of treatment for alf patients should focus on the handling and prevention of infection [17] . alf patients with severe hepatic encephalopathy, those with renal failure and patients who have any of sirs criteria use broad-spectrum antibiotics [18] . application of vasoconstrictors and dialysis reduce the incidence of cerebral edema [19] . in case of hepatic encephalopathy, the patient is transferred to icu and ventilator devices are used to regulate the level of blood gases patients with alf have qualitative and quantitative coagulation abnormalities. in control of bleeding and during invasive procedures, there is indication for ffp and platelet administration [20] . to prevent gastrointestinal bleeding in alf, patients admitted to icu are treated with h2 blockers or proton pump inhibitors (ppi) [21] . patients with alf are at risk of hypovolemia for a number of reasons, including poor oral fluid intake, vomiting, and vasodilation, in which case bolus fluids are used and level of fluids is frequently maintained if necessary to keep serum sodium levels and prevent fluid overload [22] . in addition to the mentioned treatments, 10-20% glucose is administered when glycemic target is 140 mg/dl and na level is 135-145 mmol/l, as well as n-acetylcysteine and stress ulcer prophylaxis agents [17] . a wide variety of factors cause alf (table 1) [23] [24] [25] . the most common causes of this disease are viral infections and drug-induced liver inflammation. in asia and parts of europe, mainly viral hepatitis agents are involved and acetaminophen is the predominant factor in countries such as usa and australia [26] . impaired function of both humoral and innate immunity is implicated in the pathophysiology of alf [27] . the mechanism of alf begins with necrosis of hepatocytes [28] . oxidative stress is triggered when liver injury is caused by factors such as viral infections, alcohol consumption, drug intoxication, autoimmune diseases, herbal remedies and many other factors [29] [30] [31] . oxidative stress results in the production of reactive oxygen species, which in turn activates the janus kinase (jnk) signaling pathway [32] and generates damageassociated molecular patterns (damps), followed by liver inflammation. liver inflammation is a major factor in immunopathology of several hepatic diseases [33, 34] . damps activate hepatic macrophages (kupffer cells (kcs)) and induce the formation of inflammasome [32, 35] that eventually leads to the secretion of il-1, il-18, and caspase 1 [32] . damps are detected by kupffer cells [33, 36] that express a large number of damp receptors, including tlr4, tlr9, and rage [36] . kcs are activated in this process and release inflammatory cytokines such as tnfα, oxygen radicals, and chemokines such as ccl2 under the effect of inflammatory signals. the presence of inflammatory factors mobilizes inflammatory cells such as neutrophils and monocytes and thereby increases inflammation [33, 34] . hepatic encephalopathy (he) is a function of neurotoxins that reach the brain through the bloodstream [37] . various factors such as blood ammonia levels, infection, necrotic liver, toxins, and systemic inflammatory response syndrome (sirs) can lead to he [38] . in normal conditions, the ammonia produced in the body is efficiently excreted by the liver through the urea cycle and glutamine synthesis and thus a small amount of ammonia remains in hepatic vessels. in alf, ammonia levels rise in the hepatic vein, and the liver loses the ability to release ammonia from the hepatic veins. the muscles and brain begin ammonia detoxification through glutamine synthesis. therefore, both of these tissues are considered as an ammonia scavenging and glutamine releasing organs [39] . tissue damage is the first factor triggering sirs reaction. as explained above, the injury leads to the release of inflammatory mediators such as damps, tnfα, il-6, and il-18. inflammatory cells such as lymphocytes and monocytes reach the damage site and enhance the inflammatory response. coagulation factors as well as primary and secondary homeostasis also become involved and result in sirs reaction [38] . these reactions are associated with the development of he [40, 41] , bacteremia [42] and, in some cases, infection [41, 43] . compensatory anti-inflammatory response syndrome (cars) occurs in reaction to sirs, leading to the secretion of anti-inflammatory factors (including il-10 and spli) from hepatic macrophages during the early stages. this reaction is meant to alleviate the inflammatory status [44, 45] . both of these reactions eventually lead to dysregulation of the immune system and defective immune responses to microbial agents [46, 47] . mscs are fusiform non-hematopoietic cells capable of adhering to plastic surfaces, which can be isolated from various tissues, including placenta, umbilical cord, bone marrow, adipose, and other tissues [48] . despite their morphological and phenotypical similarities, mscs have different regeneration potentials [49] , which is due to the microenvironment and cellular niches affecting their fate [50] . the number of stem cells in many adult tissues is small and isolation of them is associated with several risks; for example, the cells exhibit a limited capacity for differentiation and proliferation after removal from the body, making it difficult to produce large numbers of stem cells [51] . in comparison to adipose tissue and bm, in which mscs are affected by donor's age, placenta is a rich source of stem cells [52] and high differentiation capacity and pluripotentiality of placental cells are related to their origin [53] . pd-mscs have a higher proliferative potential than bm-mscs [54] which reduces the number of passages to reach a large number of cells as well as the risk of cell aging [55, 56] . among mcss, pd-mscs have a higher potential for in vitro proliferation and differentiation of hepatocytes [57] . human bm-msc cells are involved in neovasculogenesis and synergize with endothelial colony forming cells (ecfcs) to create microvessels in vivo [58, 59] . bm-msc cells serve as the gold standard for bone and cartilage repair [60] . adipose tissue-derived mesenchymal stem cells (ad-mscs) are isolated from adipose tissue by liposuction, are capable of differentiation to hepatocytelike cells in the presence of hgf, fgf-1, and fgf-4 factors and participate in the regeneration of hepatocytes and vasculogenesis [61] . wharton's jelly mesenchymal stem cells (wj-mscs) exhibit stemness and pluripotential properties and have been shown to generate various types of neurons and connective tissue cells [62, 63] . umbilical cord-derived mesenchymal stem cells (uc-mscs) have been recognized as low-immunogenicity cells because of their immunomodulatory properties. uc-mscs are involved in neovascularization and differentiation into hepatocyte-like cells [64, 65] . umbilical cord blood has always been considered as a source of hematopoietic stem cells (hscs) [66] . the phenotypic characteristics of uc-mscs are consistent with bm-msc cells [67] . dental tissue-derived mesenchymal stem cells (dp-msc) have limited differentiation capacity relative to bm-mscs [68] . dental pulp stem cells (dpscs) are dental stem and progenitor cells that are capable of selfrenewal and differentiation, which differentiate into neurons and adipocytes in addition to odontogenic cells [69, 70] . the definition of mscs according to international society for cell therapy (isct) is as follows: mscs are (1) able to bind plastic surfaces, (2) able to differentiate into all three classes of chondrocytes, adipocytes and osteocytes in vitro, and (3) capable of expressing cd73, cd90, and cd105 markers but not hematopoietic markers like cd45, cd14, cd19, cd34, and hla-dr [71] . mscs release numerous factors such as vascular endothelial growth factor (vegf), insulin-like growth factor 1 (igf-1), basic fibroblast growth factor (bfgf), nerve growth factor (ngf), transforming growth factor beta-1 (tgf-b1), placental growth factor (pgf), stromal cell-derived factor 1 (sdf-1/cxcl12), monocyte chemoattractant protein-1 (mcp 1/ccl2), hepatocyte growth factor (hgf), interleukin-6 (il-6), il-8, il-10, il-13, g-csf and gm-csf [72] [73] [74] [75] . there are various tissue-specific factors in mscs depending on the tissues from which mscs are isolated. for example, factors such as hgf, bfgf, and il-6 are mainly secreted by mscs isolated from placental tissue or interferon-γ (ifn-γ), tumor necrosis factor α (tnfα), interleukin-1 alpha (il-1α), and interleukin-1 beta (il-1β) secreted by mscs from menstrual blood-derived stem cells (menscs) [76, 77] . hence, it can be said that the selection of mesenchymal stem cells extracted from tissues is an important consideration in the treatment of diseases with respect to the secretory factors they produce. embryonic stem cells are isolated from embryonic tissues, especially multiple extraembryonic tissues. tissues such as amniotic fluid, wharton's jelly, amnion, chorion, embryonic membrane and placenta have mscs. the placenta is one of the largest organs with an essential role in the development of the fetus, which plays a role in the secretion of nutrients for the fetus and immune protection (tolerance) of it. it has recently been observed that pd-msc are a new alternative source of mscs for regenerative therapies [78] . studies have shown that pd-mscs possess self-renewal capacity, have multilineage differentiation, lack ethical problems, are accessible, abundant, and show strong immunosuppressive effects [79] [80] [81] ). in addition, placental tissue derived from the fetus is voluminous and can be easily manipulated to increase the number of mscs, which exceeds the number of mscs present in bone marrow and adipose tissue [81, 82] . another advantage of these placental stem cells is that we do not require an invasive method to isolate them, whereas invasive methods are needed to isolate adult mscs [78] . typically, pd-mscs can maintain a high proliferative capacity in culture medium for at least 20 passages [83] . some studies have recently suggested the differentiation of pd-mscs into hepatocyte-like endodermal cells [57, 84] . investigations have shown that many perinatal resources of mscs such as amniotic membrane (am), chorionic plate (cp), parietal decidua [85] , and umbilical cord (uc) have advantages relative to adult sources, including bone marrow (bm) [86] [87] [88] . the mscs isolated from these tissues have their own characteristics as follows. vcam1 is a biomarker of chorionic plate with unique immunosuppressive activity that plays an important role in immune responses [86] . cp-derived mesenchymal cells copiously secrete hgf and vcam1. parietal decidua derived mesenchymal stem cells (dmscs) [85] show a high secretion of ang1 and vegf but the lowest secretion of tgfβ1. umbilical cord (uc) derived mscs have a high secretion level of igf1 and amniotic membrane (am) derived mscs highly release peg2 and tgfβ1 [89] . considering the above statements, we show in this research that amniotic membrane-derived mesenchymal stem cells may be effective in treatment of premature ovarian aging due to overexpression of peg2 and tgfβ1, cp-derived mscs could be used for angiogenic therapy because of pro-angiogenic activity, and parietal decidua derived mscs [85] might be useful for the treatment of vital organ ischemia, and uc-mscs may be used for other therapies because of secreting a large number of factors [90] . most animal and human studies on mscs have indicated therapeutic effects of these cells. however, there is evidence for low engraftment of mscs due to shortterm viability after injection [77, 91] . mscs are trapped in the lung after injection and a lower number of these cells may reach their destination [92] . therefore, the reduction of cell loss during migration is an advantage of topical over intravenous injection [93] . several studies have indicated that a single injection of mscs is safe for the patient and does not stimulate the immune system, but re-injection of mscs may lead to the generation of alloantibodies [94] . in addition, the fbs that is used to grow mscs could induce an immune response in the patient [95] . in general, mscs show a dual behavior when faced with tumors and pd-mscs are no exception in this regard. for example, some in vitro studies have indicated that uc-mscs increase the expression of proliferating cell nuclear antigen (pcna) [96] , induce the proliferation promoting genes like epgn/mzt2a, downregulate transcription factors associated with the suppression of tumor development such as tal1/fos/egr1/klf10, which stimulates different tumor populations [97] . pursuant to this dual role of pd-mscs, one study introduces the antitumor role of these cells in a particular type of tumor but suggests a promoter role in another type. wj-mscs have an antitumor role in the face of squamous cell carcinoma in vitro, but stimulate the growth of cancer in vivo [98] . clinicians have observed that a number of patients with alf may recover spontaneously and that the clinical outcome of these patients largely depends on the balance between loss and repair of hepatocytes [99] . the damaged hepatocytes are rapidly replaced by normal hepatocytes in moderate disease, but in case of severe injury and widespread death of hepatocytes, the repair capacity of remaining hepatocytes may not be complete and lead to the deployment of liver progenitor cells (lpc) that act as hepatocytes [100] . in most alf patients, these progenitor cells are insufficient to repair and replace hepatocytes, eventually leading to the adoption of limited therapeutic approaches by physicians [101] . today, liver transplantation is the only way to treat liver failure patients. however, liver transplantation has failed for a number of reasons such as lack of proper organs, high costs, and the administration of immunosuppressive agents for long periods of time. other treatment strategies include bioartificial liver with less hepatocytes and drug therapy [102] . hepatic failure is a disastrous consequence of liver loss, in which the repair of residual hepatocytes is not performed in a timely and appropriate manner, resulting in increased mortality [103] . massive hepatic necrosis in acute liver failure [97] is caused by sudden loss of hepatocytes due to a variety of acute injuries induced by hepatotoxic drugs, immune system attack, and viral infections [104] [105] [106] . while most hepatocytes are completely destroyed in alf, the circulating bone marrow-derived cells and endogenous hepatocyte progenitor cells can rapidly regenerate the liver [107] . cell-based therapies have been promising in regenerative medicine. mscs can be important sources of alternative therapy because of various properties such as self-renewal, proliferation and differentiation [108] . the precise mechanism of mscs in alf is not completely understood [109] . according to several studies, it can be stated that placenta-derived mesenchymal stem cells (pd-msc) are able to affect the liver damages in several ways: 1. pd-mscs are recruited to the damaged area by vcam-1 and vla-4 adhesion molecules [104, 110, 111] affecting the remaining hepatocytes through cell-cell contact and secretion of tgf-α, egf, hgf, and vegf tropic factors [112, 113] . increase treg cells, modulating the immune system as well as suppressing activated t-cells, nk cells, b-cells and il-10 production [113, 114] . 3. pd-mscs decrease the inflammation of hepatocytes and prevent their apoptosis by suppressing tnfα and ifnγ, which leads to the regeneration of hepatocytes by releasing hgf, il-6, paf and vegf [115, 116] . 4. mscs are capable of secreting various angiogenic factors, including vegf, sdf-1α, and mmp1, which promote angiogenesis [117] [118] [119] . 5. in addition to their immunomodulatory properties, mcss differentiate into vascular cells and pericytes in vivo [117] . they also have the potential to differentiate into hepatocyte-like cells both in vivo and in vitro, leading to improvement of liver damage ( fig. 1) [115, 120] . a majority of studies have used the intravenous route to inject mscs, after which most mscs are trapped in lungs in the early stages [121, 122] . after 24 h, mscs move toward other organs (especially the liver and spleen) and settle in them [123] . they also migrate to damaged tissues [123] . for instance, in a study on patients with cirrhosis, mscs labeled with 111 in-oxine were detected in the liver after 48 h (through radioactivity assay) where they remained for 10 days [124] . elimination of mscs may be related to the immune system, which does not rule out the functional effect of these cells. one study has reported that phagocytosis of dead mscs induces the production of regulatory macrophages modulating the immune response by producing il-10 factors [125, 126] . moreover, a small fraction of these cells that have been spared elimination could be responsible for the therapeutic effects [126] . mscs play a critical role in liver regeneration because of their ability to produce and regulate platelet-activating factor (paf), hepatocyte growth factor (hgf) and vascular endothelial growth factor (vegf) [104] . several studies have demonstrated the significance of mscs in liver diseases. mscs have been used in various investigations on alf in both animal models [127, 128] and clinical trials [129, 130] . nevertheless, the precise mechanism of the function of these cells remains unclear. since mscs are able to move to the site of injury and inflammation [131] as well as being capable of proliferating and differentiating into hepatocytes [132, 133] , they play an essential role in regenerative therapies. mscs show immunomodulatory feature because they do not express stimulatory molecules or hla ii [134] and are therefore a good source for allogeneic and autologous transplantation. several studies have shown that mscs secrete tropic factors and can be effective in reducing inflammation, fibrosis and apoptosis of liver cells as well as repairing damaged tissue by stimulating angiogenesis [74] . high migration ability is a major advantage of pd-mscs. migration involves the movement of mscs toward damaged and inflamed sites through interactions between mscs with cytokines and adhesion molecules secreted from the injured tissue environment [135] . migration of mscs has been investigated in both animal [136] and in human studies [137] . for example, various researches have revealed that mscs express adhesion molecules and integrins such as vcam-1 and vla-4, which are composed of cd29 and cd49d components. compared with bm-derived mesenchymal stem cells (bm-mscs), placental mscs express a higher level of vla-4 and animal studies have indicated mscs binding to endothelial cell surface markers such as p-selectin and vcam-1, which is indicative of the high implantation capacity of pd-mscs into damaged tissue [111, 138] . a clinical trial of cirrhotic patients showed that 111 in-oxinelabeled mscs were trapped in the lungs in the early hours after injection through peripheral blood and that they left there after 48 h and migrated to the liver and spleen, remaining in these tissues for 10 days [137] . there are various mechanisms in the creation of an immunologically safe environment by placenta for the fetus [139] . this feature is a strong advantage for pd-msc cell therapy in allogeneic transplantation, which prevents graft rejection, stabilizes the transplant and drives mscs, including bm-mscs and amniotic fluidderived mscs (af-mscs), toward the site of injury [140] . embryonic-derived mscs are also capable of migrating to the placenta and blood brain barrier (bbb) [141] . it can be argued that the beneficial effects of mscs in liver diseases (including alf) are not limited to hepatocyte repair, but rather the tropical factors released by fig. 1 mesenchymal stem cells and its effects on acute liver failure them modulate the deleterious effects of the immune response [142] . the immunosuppressive effects of mscs on the secretion of tnfα and ifn γ prevent from apoptosis of hepatocyte cells and reduce hepatic inflammation, and the suppression of these cytokines appears to be systemic [143] . mscs in mice with alf suppress activated t-cells, decreasing the inflammatory cytokines tnfα, γ ifn and il-4 and exerting their immunosuppressive effects by increasing il-10 levels [143, 144] . cells such as natural killer t (nkt) are of high importance in the pathogenesis of alf and are immunomodulatory targets mediated by mscs along with dendritic cells (dcs), macrophages and t-cells [143, 145] . hgf is one of the most important factors in the repair of hepatic tissue, which is secreted by mscs. hepatocyte growth factor is an effective mitogen for hepatic tissue repair that is dependent on c-met receptor during tissue damage [146] . the hgf/c-met signaling pathway is essential for liver repair and implantation of mscs in the affected area [147] . many studies have reported the protective effects of hgf/c-met signaling pathway on liver injury [148, 149] . hgf as well as other factors like tnfα and egf is considered a mitogenic factor associated with hepatocyte proliferation [112, 150] . on the other hand, hgf together with ngf factor secreted by mscs induces apoptosis of hepatocyte stellate cells (hsc), indicating the antifibrotic property of these cells [151] [152] [153] . many studies have shown that angiogenesis plays a crucial role in hepatic repair so that the injection of antiangiogenic factors such as anti-vegf inhibits hepatic repair [154, 155] but factors such as bfgf enhance it [156] . vegf boosts angiogenesis and contributes to the healing process [157] . angiogenesis is essential for wound healing, regeneration and organogenesis [158] . il-6 binds to gp80 and gp130 receptors, which activate the jak pathway and in turn phosphorylate tyrosines in the intracellular domain of gp130, subsequently activating the mapk pathway and stat 1 and 3 transcription factors that lead to hepatocyte proliferation [159] [160] [161] . recent experiments on animal models have shown that il-6 and tnf-2 are involved in regeneration of liver mass [162] . limited information is available on the repair mechanism of mscs in various diseases; therefore, further in vivo studies provide a broad perspective for mscs use in clinical practice. choosing the right cell, determining the proper dose, selecting the appropriate injection site and timely injection can help improve the function and implantation of mscs in the target tissue, and they can be highly important and applicable for further research in the future. in this review paper, we concluded that pd-mscs can be considered as a good allogeneic source for alf in future because of their safety, easy accessibility, lack of immune system stimulation, secretion of appropriate factors for liver tissue and healing properties. mscs: mesenchymal stem cells; pd-mscs: placenta-derived mesenchymal stem cells; alf: acute liver failure; aclf: acute-on-chronic liver failure; clf: choronic liver failure; ich: intracranial hypertension; damps: damage-associated molecular patterns; sirs: systemic inflammatory response syndrome; cars: compensatory anti-inflammatory response syndrome; isct: international society for cell therapy; vegf: vascular endothelial growth factor; igf-1: insulin-like growth factor 1; bfgf: basic fibroblast growth factor; ngf: nerve growth factor; tgf-b1: transforming growth beta-1; ifn-γ: interferon-γ; tnf-α: tumor necrosis factor α; il-1α: interleukin-1 alpha; il-1β: interleukin-1 beta; pgf: placental growth factor; sdf-1/cxcl12: stromal cell-derived factor 1; mcp 1/ccl2: monocyte chemoattractant protein-1; hgf: hepatocyte growth factor; g-csf: granulocyte-colony stimulating factor; gm-csf: granulocytemacrophage colony stimulating factor; am: amniotic membrane; cp: chorionic plate; uc: umbilical cord; lpc: liver progenitor cells; paf: platelet-activating factor; vla-4: very late antigen 4; bbb: blood brain barrier; hsc: hepatocyte stellate cells; jak: janus kinase. prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti-human monoclonal antibodies introduction to the revised american association for the study of liver diseases position paper on acute liver failure liver transplantation : official publication of the american association for the study of liver diseases and the 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visibility for your research: over ready to submit your research ? choose bmc and benefit from the concanavalin a model of acute hepatitis in mice tonsil-derived mesenchymal stem cells alleviate concanavalin a-induced acute liver injury adipose tissue derived stromal stem cell therapy in murine conaderived hepatitis is dependent on myeloid-lineage and cd4 + t-cell suppression met signalling: principles and functions in development, organ regeneration and cancer hgf/c-met signaling mediated mesenchymal stem cell-induced liver recovery in intestinal ischemia reperfusion model hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair cholesterol enhances the toxic effect of ethanol and acetaldehyde in primary mouse hepatocytes expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat immunomodulation of activated hepatic stellate cells by mesenchymal stem cells inhibition of hepatic 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increased binding, and target gene transcription during liver regeneration publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. key: cord-009987-biop7gyd authors: ali, muhammad; khan, tariq; fatima, kaneez; ali, qurat ul ain; ovais, muhammad; khalil, ali talha; ullah, ikram; raza, abida; shinwari, zabta khan; idrees, muhammad title: selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions date: 2017-10-19 journal: phytother res doi: 10.1002/ptr.5957 sha: doc_id: 9987 cord_uid: biop7gyd insight into the hepatoprotective effects of medicinally important plants is important, both for physicians and researchers. main reasons for the use of herbal medicine include their lesser cost compared with conventional drugs, lesser undesirable drug reactions and thus high safety, and reduced side effects. the present review focuses on the composition, pharmacology, and results of experimental trials of selected medicinal plants: silybum marianum (l.) gaertn., glycyrrhiza glabra, phyllanthus amarus schumach. & thonn., salvia miltiorrhiza bunge., astragalus membranaceus (fisch.) bunge, capparis spinosa (l.), cichorium intybus (l.), solanum nigrum (l.), sapindus mukorossi gaertn., ginkgo biloba (l.), woodfordia fruticosa (l.) kurz, vitex trifolia (l.), schisandra chinensis (turcz.) baill., cuscuta chinensis (lam.), lycium barbarum, angelica sinensis (oliv.) diels, and litsea coreana (h. lev.). the probable modes of action of these plants include immunomodulation, stimulation of hepatic dna synthesis, simulation of superoxide dismutase and glutathione reductase to inhibit oxidation in hepatocytes, reduction of intracellular reactive oxygen species by enhancing levels of antioxidants, suppression of ethanol‐induced lipid accumulation, inhibition of nucleic acid polymerases to downregulate viral mrna transcription and translation, free radical scavenging and reduction of hepatic fibrosis by decreasing the levels of transforming growth factor beta‐1, and collagen synthesis in hepatic cells. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. liver disorders have been classified in the high priority areas of health care. according to an estimate by the world health organization, approximately 500 million people of the world are suffering from a severe form of liver disorders, that is, chronic hepatitis (al-asmari et al., 2014) . medicine of herbal origin may serve as a feasible therapy for the prevailing liver problems because of their safety, easier availability, cost effectiveness, and environment friendliness (izzo, hoon-kim, radhakrishnan, & williamson, 2016) . medicinal plants have acquired importance in healthcare system throughout the world for their proven and effective therapeutic properties (helmstädter & staiger, 2014 ). an estimated 80% of the world's population is relying on medicines that contain compounds of herbal origin (ekor, 2013) . the international union for conservation of nature has suggested that approximately 50,000 to 80,000 flowering plants are used for medicinal purposes (chen, li, ren, & hu, 2016) . many factors regarding these medicines are important. herbal medicines are claimed to both treat and prevent diseases, which adds to a deep belief that these abbreviations: alt, alanine aminotransaminase; asp, angelica sinensis polysaccharides; ast, aspartate transaminase; egf, epidermal growth factor; hbv, hepatitis b virus; lbps, lycium barbarum polysaccharides; wf4, woodfordia fruticosa flower extract. treatments are safe because they are "natural and gentle" and therefore, a harmless alternative to the conventional medicine. moreover, the latter may sometimes cause disappointing results and undesirable side effects in patients (izzo et al., 2016) . in addition, the less expensive herbal products are often not subject to strict regulations and medication prescribed by a physician or other qualified practitioners (hunter & hegele, 2017) . although medicinal plants have been used globally, their wider usage is limited to a few countries like japan, india, china, pakistan, thailand, iran, and some african countries (bahmani et al., 2014; iwu, 2014; li, 2016; sivasankari, anandharaj, & gunasekaran, 2014) . other countries are also encouraging the use of plant-based medicinal products in their healthcare systems. for example, natural health product regulations of canada for the plant-based product in healthcare encourages usage of modern technology and evidencebased scientific support towards promoting medicinal plants and the associated products (tomlinson & akerele, 2015) . a major concern of scientists investigating herbal treatments is that the chemical composition of the plants contributing to their biological effects is mostly undetermined (ling et al., 2009 ). herbs and herbal medicines have been used for the treatment of liver diseases for a long time (dhiman & chawla, 2005) . there are many herbs having ingredients that are potential sources of medicine for the treatment of liver diseases having various modes of actions and bioactivities (babu, bhuvaneswar, sandeep, ramaiah, & rajendra, 2017; gnanadesigan, ravikumar, & anand, 2017; pereira, barros, & ferreira, 2016) . however, several of them are well-studied for their bioactive components and the mechanism of hepatoprotective activity. in the current review, we have selected some of these compounds for which elaborate detail about hepatoxicity is available in literature in the form of either in vivo studies, study into biochemical parameters and bioactive compounds. this article highlights the possible ways of inducing hepatoxicity in mice models and encompasses the mechanisms in which certain medicinally important plants perform their hepatoprotective activity. the article further aims to summarize studies conducted on the composition, pharmacology, and nature of the selected plants in the light of possible mechanism deduced from experimental trials. a thorough search was conducted on the electronic literature databases, google scholar, pubmed, scopus, and web of science. literature was retrieved using the key words and phrases "hepatitis c", "hepatoprotective activity", "mechanism of action", "medicinal plants", "herbal", and "treatment". about 100 relevant articles were extracted after a narrow search for a combination of the keywords and subsequent analysis per the inclusion criteria. there were two sets of criteria applied to articles for inclusion in this manuscript. according to the first, "general criteria", articles selected for this manuscript were those which (a) reported plants and their parts that were traditionally applied to hepatitis and liver disorders and any other type of hepatoprotective activity; (b) reported extract or pure compounds important for their hepatoprotective role; and (c) attempted to explain the mechanism of hepatoprotective action of these plants. the 2nd criteria were used for selecting those plants that are discussed in detail (shown in figure 3 ). for this purpose, seventeen plants were selected for which recent articles were available that (a) studied in vitro and in vivo hepatoprotective activities of herbal products, (b) reported active compounds from the plant, and (c) described the mechanism of action herbal hepatoprotective products. the plants described in detail were selected if literature available for them fulfilled at least two of the above 3-point criteria. each of the selected plants was discussed; mainly focusing its hepatoprotective activities, active compounds, and possible mechanism of action. in addition, featured hepatoprotective herbal combinations have been deliberated. toxicity and quality control issues associated with these herbs/herbal products have been debated. two of the authors independently reviewed all the full-text articles obtained during the electronic search. data from the eligible articles were extracted; all the disagreements were discussed and were referred to a third reviewer (one of the author) for a final decision. all the data were extracted in two tables (tables 1 and 2) , and the mechanisms of action were explained in respective subheadings and demonstrated through four different figures (figures 1-3, and 4 ). the figures were constructed in chembiodraw ultra (version 14.0) software package. furthermore, the plant database "plant list" was used for the taxonomic categorization of all the documented plant species (theplantlist, 2013) . the prerequisite to screen/study any medicinal compound for its hepatoprotective activity is to develop a model (animal model or cell culture model) in which hepatic injury is induced (salehi, karegar-borzi, karimi, & rahimi, 2016) . several studies have manipulated mice models to induce hepatotoxicity and then treat those induced liver diseases using herbs and herbal products (figure 1 ). this approach provides insight into how hepatitis and other liver diseases are caused. however, for many plants, their mechanism of action against hepatotoxic agents is not well-documented. the common prototype applied for hepatoprotective drug screening is the carbon tetrachloride (ccl 4 ) induced hepatic injury (rodrigues et al., 2016) . as ccl 4 have been reported for its damaging effects on the liver because on metabolism by p450, it produces free radicals (johnston & kroening, 1998) . these free radicals cause lipid peroxidation by binding to dna, proteins, or lipids (yasuda, izumi, shimada, kobayakawa, & nakanishi, 1980) . the degree of hepatic injury is evaluated by the higher level of biochemical parameters that is ascribed to the production of trichloromethyl free radicals which eventually causes lipids peroxidation present in cellular (dusheiko, 1996) calotropis procera (aiton) dryand. crude hydro-ethanol solution extract prevents of the depletion of gsh levels. c. procera contains flavonoids thus it also performs the antioxidant activity (mcomish et al., 1994) clerodendrum abilioi r. fern. ethanol extract decreased the serum enzyme alt, ast, alp, tgl, and total cholesterol and considerably increased the glutathione level (chamberlain, adams, saeed, simmonds, & elliott, 1997) ficus carica l. leaves crude petroleum ether extract reduction in the levels of alt and ast. the petroleum ether extract of ficus leaves repair the damaged liver cell (gond & khadabadi, 2008) glycyrrhiza uralensis -glycyrrhizin glycyrrhizin administered in plc/prf/5 cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes (sato et al., 1996) momordica dioica roxb. ex willd. alkaloids, phenolic compounds, glycosides, flavonoids oral administration of the extract significantly normalized and restored the elevated serum enzymatic levels of ast, alt, salp, and total bilirubin. its hepatoprotective activity is due to the antioxidant and free radical scavenging activity. (surai, 2015) nelumbo nucifera gaertn. leaves catechin glycoside, myricitrin-3-o-glucoside, hyperin, isoquercitrin, quercetin-3-o-rhamnoside, astragalin lotus leaf extract possess significant hepatoprotective and antioxidant activity in ccl 4 -induced toxicity rat model. free radicalscavenging and antioxidant activity due to the presence of some flavonoids and phenolic compounds results in the hepatoprotective activity. (theplantlist, 2013) paeonia lactiflora pall. and a. membranaceus (fisch.) bunge. -progression of ccl 4 -induced hepatic fibrosis was inhibited in rates by decreasing the level of tumor growth factor-β1 and inhibit collagen synthesis (sun et al., 2007) s. miltiorrhiza bunge. roots -s. miltiorrhiza could reverse the ccl 4 -induced fibrosis treatment by decreasing the levels of transforming growth factor-β1, procollagens i and iii, and metalloproteinase-1 and decreasing the levels of metalloproteinase-13 in liver of the affected rates (wasser et al., 1998) s. miltiorrhiza bunge. roots s. miltiorrhiza polysaccharides protects liver against immunological injury by adjusting the levels of alanine aminotransferase, aspartate aminotransferase, nitric oxide, tumor necrosis factor and interleukin-1 (zein et al., 1996) solanum nigrum l. total decoction crude aqueous extract inhibited thioacetamide-induced collagen (α1) and transforming growth factor-β1 mrna levels in the liver of mice with thioacetamide-induced liver fibrosis (hsieh et al., 2008) s. nigrum l. and cichorium intybus l. crude plant extract protect dna against oxidative damage in the reaction mixture containing calf thymus dna and free radical generating system (sultana et al., 1995) (continues) membrane (chen, yu et al., 2016) . figure 2 shows the different strategies applied for studying the in vivo effects of induced hepatotoxicity in mice models. the leaves are marked by distinct white "milky" veins that give the plant its common name (theplantlist, 2013) . historically, s. marianum was used medicinally to treat disorders of the gallbladder, spleen, and liver, but the most important medicinal application of s. marianum is its use as a hepatoprotective herbal treatment and as supportive treatment for chronic inflammatory liver disorders such as hepatitis, cirrhosis, fatty infiltration, and some other forms of liver damages due to toxic chemicals, poisonous mushrooms, and alcohol (freitag et al., 2015) . the most important component extracted from s. marianum is silymarin (lu, lu, chen, zhang, & wu, 2007; wu, wang, & que, 2006) , which is used to treat a variety of liver disorders, including chronic and acute viral or drug/toxin-induced hepatitis, alcoholic liver disease, and liver cirrhosis (lu et al., 2007) . silymarin is a combination of different ingredients with silibinin as the most active among them (surai, 2015) . silymarin has been approved for clinical studies in treating the hepatitis c virus infection (ferenci et al., 2008) . there are many studies on the mechanism of hepatoprotective effects of silymarin. recently, tunca et al. (2009) showed that silymarin has a protective action on pyridine-induced hepatic injury in syrian hamsters. the study concluded that it decreases the metabolic activation of pyridine (by decreasing the cytochromes p450 1a1 protein concentration) and control the elevation of inducible nitric oxide synthase expression. all these factors play a protective role in liver injury. in another study, farghali, kamenikova, hynie, and kmonickova (2000) concluded that in addition to inhibition of lipid peroxidation, the hepatoprotective activity against thioacetamide-induced hepatotoxicity (khatri, garg, & agrawal, 2009) tephrosia purpurea (l.) pers. decreased serum aspartate aminotransaminase (35% and 31%), alanine aminotransaminase (50% and 42%), gamma glutamyl transpeptidase (56% and 49%), alkaline phosphatase (46% and 37%), total bilirubin (61% and 48%), and liver mda levels (65% and 50%), and significant improvement in liver glutathione (73% and 68%) when compared with thioacetamide-damaged rats. (hosseinzadeh & nassiri-asl, 2015) vitex negundo l. administration of ethanol solution extract of vitex leaf caused a significant decrease in tb, ast, alt, and alp levels in rats. (abdulkarim et al., 1998) zanthoxylum armatum dc. bark berberine elevated serum enzymatic levels of serum transaminases, alkaline phosphatase. total bilirubin was considerably restored to a normal level. (cha et al., 1991) note. gpx = glutathione peroxidase; mda = malondialdehyde; ast = aspartate transaminase; alt = alanine aminotransaminase; ccl 4 = carbon tetrachloride; sod = superoxide dismutase; gsh = glutathione; tb = total bilirubin; alp = alkaline phosphatase hbsag = hepatitis b surface antigen; tgl = triglyceride lipase. inhibition of the increased intracellular ca 2 i plays a critical role in the hepatoprotective effect of silymarin. although, upadhyay, kumar, and singh (2007) showed that silymarin restores the changes in the expression and activity of cytochrome p450 (cyp) enzymes (cyp1a1, cyp1a2, and cyp2e1), glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. glycyrrhizin administered in plc/prf/5 cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes glycyrrhizin administered intraperitoneally inhibits the lipopolysaccharide-and d-galactosamine-induced liver injury by preventing inflammatory responses and il-18 production in mice glycyrrhizin inhibited anti-fas antibody-induced hepatitis in mice by acting upstream of cpp32-like protease administration of glycyrrhizin or glycyrrhetinic acid, significantly suppressed α2 (i) collagen gene promoter activation and progression of liver fibrosis induced by repeated ccl 4 injections in transgenic mice (liew, erali, page, hillyard, & wittwer, 2004; martell et al., 1992; ogata, alter, miller, & purcell, 1991; sato et al., 1996) phyllanthin phyllanthus amarus schum. et thonn. phyllanthin help in restoration of antioxidant potential of rat hepatocytes, level of gsh, and sod and gr activities reduced by ethanol (chirdchupunseree & pramyothin, 2010) p-methoxy benzoic acid capparis spinosa l. the compound alleviated the enzyme levels increased as result of administration of ccl 4 , and pcl (gadgoli & mishra, 1999) silymarin silybum marianum (l.) gaertn. silymarin attenuated the rifampicinand/or pyrogallol-induced hepatotoxicity by restoring the alterations in the expression and activity of cyp1a2 and cyp2e1, glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. silymarin suppresses n-nitrosodiethylamine induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals (farghali et al., 2000; upadhyay et al., 2007) note. hbsag = hepatitis b surface antigen; cpp32 = 32-kda putative cysteine protease; ccl 4 = carbon tetrachloride; gsh = glutathione, sod = superoxide dismutase; gr = glutathione reductase; cyp = cytochrome p450; pcl = paracetamol. g. glabra is a member of the glycyrrhiza genus (isbrucker & burdock, 2006) , an ancient genus that contains the most commonly used herbs in chinese traditional medicine (hosseinzadeh & nassiri-asl, 2015) . glycyrrhiza species are considered among the most important herbaceous plants for a diverse array of pharmacological activities (hosseinzadeh & nassiri-asl, 2015) . chemical structures of (1) cryptotanshinone, (2) phyllanthin, (3) quercetin, (4) glycyrrhizin, (5) silymarin, and (6) p-methoxybenzoic acid, also known as p-anisic acid. all the images were adopted from ncbi-pubchem with the compound ids; 160254, 358901, 5280343, 14982, 7073228, and 7478, respectively (pubchem, 2017) (mao et al., 2016) . one of the bioactive compounds from p. amarus is phyllanthin, which is a lignan compound and is traditionally applied in the treatment of many liver diseases (hanh, sinchaipanid, & mitrevej, 2014) . it was shown to have hepatoprotective effects on ethanol-induced oxidative damage in primary culture of rat hepatocytes through its antioxidant activity especially the activities of superoxide dismutase (sod) and glutathione reductase (chirdchupunseree & pramyothin, 2010) . previously, naaz, javed, and abdin (2007) 3.9 | c. intybus l. c. intybus l., commonly known as chicory, belongs to the lactuceae family and is typical mediterranean plant indigenous to western asia, europe, north america, and egypt, which varies in perianth color from white, red to blue (norbaek, nielsen, & kondo, 2002 3.10 | s. nigrum l. s. nigrum l., commonly known as "black nightshade", is a species in the family solanaceae ( ccl 4 -induced hepatic necrosis. this hepatoprotective effect might be due to its adjustment of antioxidant activity, detoxification enzymes, and its free radical scavenger effects. in another study, hsieh, fang, and lina (2008) induced liver fibrosis by administering thioacetamide in mice and treated them with distilled water and s. nigrum extract via oral administration for 12 weeks. this treatment alleviated the hepatic hydroxyproline and α-smooth muscle actin protein levels in mice and inhibited thioacetamideinduced collagen and transforming growth factor-β1 mrna levels in the liver. histological examination of liver also confirmed that this extract reduced the degree of fibrosis caused by thioacetamide treatment which is the probable reason for the reduction of hepatic fibrosis. 3.11 | s. mukorossi gaertn. s. mukorossi gaertn., commonly known as ritha or aritha, is abundantly found in india. its fruit is reported to have expectorant, purgative, antidotal, and abortifacient effects. additionally, it is used in epilepsy, extreme salivation, and chlorosis (suhagia, rathod, & sindhu, 2011) . the saponins extracted from this plant are spermicidal (in vitro) and due to this property, it has been used in contraceptive cream (rastogi & mb, 1999) . pharmacological studies of s. mukorossi have shown their potential effect as hepatoprotective agents (upadhyay & singh, 2012) . to assess the hepatoprotective activity of the s. mukorossi, wistar male rats were treated with ccl 4 . administration of ccl 4 to normal rats increased the serum levels of alt, ast, alp, and bilirubin. these enzymes eventually cause damage to the hepatic cells. the ccl 4 -treated liver cells cultured on petri plates were treated with the extracts of s. mukorossi and were reported to alleviate the levels of these enzymes. when histopathological studies of the ccl 4 -treated rats were performed, they showed that it also causes the demolition of architectural configuration of target cells. however, rats that were treated with s. mukorossi presented normal lobular structural design, which shows its reparative properties and thus its hepatoprotective effects. 3.12 | g. biloba l. g. biloba l. belongs to family ginkgoaceae (theplantlist, 2013) . it is one of the significant herbs of the chinese traditional medicine. g. biloba leaf extract has been reported to have therapeutic activities against age-related memory deficit problems, including alzheimer's and dementia; cardioprotective, antiasthmatic, antidiabetic, hepatoprotective, photoprotective effects, dna repair mechanism, antioxidant, and antiinflammatory activities (mohanta, tamboli, & zubaidha, 2014) . g. biloba has been associated with a strong hepatoprotective activity through numerous studies (parimoo et al., 2014) . g. biloba amplifies cellular antioxidant protection system consisting of glutathione peroxidase, glutathione s-transferase, glutathione reductase, nonprotein thiols, catalase, and antioxidant enzymes (sod). the binding of an individual part of herbal tracks to that of phosphatidylcholine produces phytosome having better efficacy compared with traditional herbal extracts (naik, pilgaonkar, & panda, 2006) . rifampicin is an antibiotic widely used in tuberculosis chemotherapy. it has been reported to cause hepatoxicity, the reason of which is unknown as it is always given in combined form with other antibiotics such as isoniazid and ethambutol. wistar albino rats were treated with rifampicin that caused hepatoxicity in them. their blood samples were taken, and assays of their blood samples were performed to know the levels of sgpt, sgot, and alp. the elevated levels of sgot, sgpt, and alp show liver damage as these enzymes escape from the liver into the blood in case of liver damage. with parallel treatment through ginkoselect phytosome® and the standard drug silymarin, the markers enzymes levels in serum were nearly at a normal level or marginally elevated. this suggests the hepatoprotective quality of g. biloba plant. it also elevates total protein levels and albumin, which shows its hepawith activities against chronic hepatic fibrosis (nitha, prabha, ansil, & latha, 2014) . it also shows antiinflammatory, antibiotic, antileprosy, and antihelminthic properties (arya et al., 2015; shoaib et al., 2016; syed & khan, 2016) . in an experiment designed to check the hepatoprotective effect of w. fruticosa flower extract (wf4), albino wistar rats were administered with ccl 4 which resulted in the increased level of alp, ast, alt, and lactate dehydrogenase. these enzymes leak from serum into the blood. thus, ccl 4 damage causes loss of enzymes which are responsible for drug metabolism (chandan et al., 2008) . these rats were administered with wf4. the extract reversed the elevated lipid peroxidation and regulated the liver glucose-6-phosphate and gsh levels. these results are in line with former information for other hepatoprotective agents . 3.14 | v. trifolia l. v. trifolia l., known generally as chaste tree, is a high-value medicinal plant that belongs to the family verbenaceae. its leaves are effective as plaster against pains, infections, and fever. its fruits are used in curing amenorrhea, and the flowers are effective against fever (chan, baba, chan, kainuma, & tangah, 2016) . the active constituents of this plant are essential oil (kvasnicka, biba, sevcik, voldrich, & kratka, 2003) , viterifolins, and diterpenes. it also possesses some important pharmacological qualities, that is, antipyretic (rani & sharma, 2013) , antibacterial (lawitz et al., 2014) , antiallergic, and antiasthmatic properties (lawitz & gane, 2013) . medical practitioners use this plant in the treatment of acute jaundice. however, literature study suggested that this plant is not well screened for its hepatoprotective activity. nonetheless, the tribal groups of western ghats use this plant leaf extracts in treating jaundice, and these results give some scientific evidence of hepatoprotective activity. 3.16 | s. chinensis (turcz.) baill s. chinensis (turcz.) baill is widely used in traditional and modern chinese medicine for the treatment of many disorders including insomnia, respiratory failure, and weakness. moreover, mental health improving ability along with fatigue reduction property is also validated for s. chinensis in russian medicine (szopa, ekiert, & ekiert, 2017) . in general, dibenzocyclooctadiene lignans found in s. chinensis are known to exhibit potent hepatoprotective activity (zheng et al., 2017) . in one of the study of individual lignin, gomisin a was found responsible for the acceleration of hepatocytes proliferation and increase hepatic flow (panossian & wikman, 2008) . furthermore, elevation of mitochondrial glutathione concentration was found to be linked with γ-schisandrin hepatoprotective mechanism. the increase in vitamin c concentration in the liver of test animals upon treatment with γ-schisandrin also validates its hepatoprotective ability. another individual lignin, schisandrin b was also found to counter oxidative harm to liver tissues (thandavarayan et al., 2015; xin et al., 2017) . in one scientific study, the hepatoprotective mechanism against acetaminophen-induced liver injury of six schisandra lignans (deoxyschisandrin, schisantherin a, schisandrin b, gomisin a, schisandrin c, and schisandrin) was elucidated. the hepatoprotective ability of these lignins was found to be associated with inhibition of cytochrome-mediated bioactivation (jiang et al., 2015) . furthermore, another mechanistic study investigated the hepatoprotective effect of schisandra polysaccharide in nonalcoholic fatty liver disease mice models. the results demonstrate potential down regulation of hepatic lipogenesis genes and lxrα/srebp-1c/ fas/acc and srebp-2/hmgcr signaling pathways in the liver (wang, song et al., 2016) . 3.17 | c. chinensis lam. c. chinensis lam. also known as chinese dodder is a parasitic plant having diverse traditional medicinal uses as a tonic, sex enhancer, and abortion preventer (zheng, dong, & she, 1998) . studies also have scientifically validated the hepatoprotective activity of c. chinensis (donnapee et al., 2014) . yen, wu, lin, and lin (2007) chinensis seeds ethanol solution extract was found to be more effective in rats with acetaminophen-induced hepatotoxicity (yen, wu, lin, cham, & lin, 2008) . the mechanism of hepatoprotective potential as demonstrated by ethanol solution extract of c. chinensis is proposed to be the elevated activities of antioxidant enzymes. 3.18 | l. barbarum l. l. barbarum l. berries are very famous in traditional chinese medicine for the treatment of inflammation, cancer, eye disorders, throat infection, and anemia. the use of these berries has been validated as food and also has gained great importance due to its significant antioxidant potential (cheng et al., 2015) . the major active components of l. barbarum berries are l. 3.19 | a. sinensis (oliv.) diels a. sinensis (oliv.) diels is reported in chinese herbal medicine for the treatment of cardiovascular disease, anemia, and hepatic disorders (bunel, antoine, nortier, duez, & stévigny, 2015) . the a. sinensis polysaccharides (asp) extracted from a. sinensis roots having the average molecular weight of 72,900 da is regarded as a potential active component of a. sinensis that exhibits a wide range of pharmacognostic properties (hsu, tsai, & tsai, 2014) . the hepatoprotective potential of asp in ccl 4 -induced liver injury and via using ischemia/reperfusion rat is widely established (zhang et al., 2010) . wang, wen, li, zhang, & yang (2016) the number of hepatoprotective products from plants is ever increasing. in addition to the hepatoprotective role of a general class of phenolics and flavonoids, many studies have defined specific compounds for their preferable role in hepatitis and other liver disorders (shehab et al., 2015) . some of the important plants and their products are highlighted in figure 3 . among the many different compounds, few are distinguished for their promising role in liver inflammation. we have, therefore, selected six important compounds for their hepatoprotective role (table 2) . quercetin, for instance, a major flavonol commonly found most of the plants, is a potent hepatoprotective agent. the first basis of quercetin-based potency has been attributed to the antioxidant activity of this compound. one of the specific mechanisms of quercetin supplementation was established through ethanol-induced cytotoxicity which affects the activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. for instance, quercetin supplementation restored the glutathione reductase activity which was affected by ethanol that in turn reduced the glutathione content of liver. quercetin supplementation has also been attributed to hepatoprotection against metals, pesticides, drugs, toxins, and viruses (miltonprabu et al., 2016) . rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) is another important hepatoprotective compound extensively found in medicinal herbs, such as rheum palmatum l., cassia tora l., polygonum multiflorum thunb., and aloe barbadensis miller. the mechanism of action through which rhein acts has been described as modulation of cyp enzymes in rat liver, attenuation of total cholesterol and triglyceride levels in serum, and amelioration of glucose and lipid metabolism. similarly, rhein downregulated the levels of serum alt, hyaluronic acid, procollagen type iii, and liver malondialdehyde and inhibited the expression of transforming growth factor beta 1 and alpha-smooth muscle actin in tetrachloride/ethanol-induced liver fibrosis rats . similarly, flavonoids, lignans, terpenoids, and steroids from vitex negundo l. have also been shown to demonstrate hepatoprotective activities (zheng et al., 2015) . extracts containing these compounds have been shown to improve biochemical and functional parameters and thus alleviate ccl 4 -induced damage in liver rats. negundoside, for instance, which is a glycoside, has been demonstrated to reduce calcium-mediated toxicity and ccl 4 -induced oxidative stress through regulation of calcium homeostasis and decreasing the production of ros and lipid peroxidation (zheng et al., 2015) . table 2 with their structures given in figure 4 . the present literature is not sufficient to assess the safety of most of the hepatoprotective and liver regenerative herbs and herbal products, as most of the studies focus on their antihepatotoxic effects only. however, some previous experiments on rats show that no adverse effects were observed by administering intraperitoneal injection of the a. membranaceus extracts at 0.5 g/kg for 30 days, whereas large doses (1 g/kg) of a. membranaceus root extracts resulted in mutagenicity in mice when injected directly into the stomach lining ). in another study, sato et al. (1996) observed no significant toxicity of various concentrations of glycyrrhizin on plc/prf/5 cells in vitro. gadgoli and mishra (1999) reported that p-methoxy benzoic acid extracted from c. spinosa was nontoxic at 1 mg/ml when applied to rat hepatocytes in vitro. this supports the claims made in the traditional system of medicine. similarly, silymarin is shown to have a lack of toxicity and side effects even at high doses (upadhyay et al., 2007) . isbrucker and burdock (2006) reported that no-observed-effect levels for purified glycyrrhizin are in the range of 15-229 mg/ kg/day and concluded that current levels of consumption of licorice extract products and glycyrrhizinate are safe. although, several herbals show potential activity for the treatment of acute and chronic liver diseases premarketing drug-testing, and pharmacovigilance is needed as with any other drug. so far, herbals to treat chronic liver diseases should not be recommended outside clinical trials as the evidence supporting its use is insufficient (stickel, patsenker, & schuppan, 2005) , and publications relevant to the cytotoxicity of medicinal plants should be encouraged (mukhtar et al., 2008) . moreover, there are issues like approval of the plant products/extracts as a drug from regulatory agencies such as the food and drug administration or any other equivalent agencies. extensive literature survey of hepatoprotective plants clearly indicates that herbal drugs have an enormous potential for the treatment of liver diseases. in this article, we reviewed the scientific merit of selected plants studied for their hepatoprotective mechanism of action. the major hepatoprotective mechanism identified by the majority of the studies is through combating the oxidative stress that damages the liver. we have summarized the effect of extracts and compounds from different herbs on liver injury considering changes in their biochemical parameters. we also presented the possible data available in the literature for different plants regarding their phytochemical constituents. we, therefore, conclude that herbs and herbal preparations are among the most important sources of hepatoprotective and liver regeneration medicines. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. moreover, a combination of the traditional herbal medicines with the modern and conventional medicine may be one of the best options for the treatment of liver disorders and other diseases and infections, soon. the importance of medicinal plants can be determined from world health organization's estimates, which states that up to 80% of the world's population fulfill their healthcare needs from medicinal plants (mukhtar et al., 2008) . there has been a significant rise in using overthe-counter medicinal plant products containing powerful medicinal drugs and are believed to have to produce progressive effects with reduced side effects. however, therapeutic failures or adverse effects have been observed in many cases as pharmacological mechanisms of the herbal mixtures/preparations are not well-studied. the most important concern involving the use of medicinal plants is to identify and standardize the exact method of preparation of an extract, identification of active ingredients and details of administration (yip & kwan, 2006) . in this relationship, the screening and characterization of other undiscovered herbal products in traditional medicine is needed. the integration of the therapeutic use of traditional chinese medicinal knowledge with the synthetic and traditional oriental medicinal knowledge is a key area of research (cho & leung, 2007) . however, medicinal plants cultivated in different geographical regions are believed to differ in therapeutic effects in different diseases and infections. for example, a. membranaceus used in chinese traditional medicine, from certain locality contains more favorable trace elements and fewer harmful trace elements than those from other localities. in this context, the use of new therapeutic strategies based on natural plants may be useful to provide minimal toxicity, higher effectiveness, and a wider therapeutic background for effective manipulation than existing pharmaceutical products (panico et al., 2005) . authors declare that they have no competing interests. animals not applicable. the current article is a review article and does not contain any studies with human participants performed by any of the authors. hepatitis c virus genotypes and hepatitis g virus in hemodialysis patients from syria: identification of two novel hepatitis c virus subtypes hepatoprotective activity of two plants belonging to the apiaceae and the euphorbiaceae family a review of hepatoprotective plants used in saudi traditional medicine. evidence-based complementary and alternative medicine: ecam a systematic 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states: epidemiology, pathogenicity, and response to interferon therapy. collaborative study group systematic review of the renal protective effect of astragalus membranaceus (root) on diabetic nephropathy in animal models extraction, chemical analysis of angelica sinensis polysaccharides and antioxidant activity of the polysaccharides in ischemia-reperfusion rats levistilide a inhibits angiogenesis in liver fibrosis via vascular endothelial growth factor signaling pathway phytochemical and pharmacological profile of vitex negundo modern study of traditional chinese medicine schisantherin a protects against liver ischemia-reperfusion injury via inhibition of mitogen-activated protein kinase pathway selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions key: cord-002272-c7f1l13s authors: sauter, kristin a.; waddell, lindsey a.; lisowski, zofia m.; young, rachel; lefevre, lucas; davis, gemma m.; clohisey, sara m.; mcculloch, mary; magowan, elizabeth; mabbott, neil a.; summers, kim m.; hume, david a. title: macrophage colony-stimulating factor (csf1) controls monocyte production and maturation and the steady-state size of the liver in pigs date: 2016-07-21 journal: am j physiol gastrointest liver physiol doi: 10.1152/ajpgi.00116.2016 sha: doc_id: 2272 cord_uid: c7f1l13s macrophage colony-stimulating factor (csf1) is an essential growth and differentiation factor for cells of the macrophage lineage. to explore the role of csf1 in steady-state control of monocyte production and differentiation and tissue repair, we previously developed a bioactive protein with a longer half-life in circulation by fusing pig csf1 with the fc region of pig igg1a. csf1-fc administration to pigs expanded progenitor pools in the marrow and selectively increased monocyte numbers and their expression of the maturation marker cd163. there was a rapid increase in the size of the liver, and extensive proliferation of hepatocytes associated with increased macrophage infiltration. despite the large influx of macrophages, there was no evidence of liver injury and no increase in circulating liver enzymes. microarray expression profiling of livers identified increased expression of macrophage markers, i.e., cytokines such as tnf, il1, and il6 known to influence hepatocyte proliferation, alongside cell cycle genes. the analysis also revealed selective enrichment of genes associated with portal, as opposed to centrilobular regions, as seen in hepatic regeneration. combined with earlier data from the mouse, this study supports the existence of a csf1-dependent feedback loop, linking macrophages of the liver with bone marrow and blood monocytes, to mediate homeostatic control of the size of the liver. the results also provide evidence of safety and efficacy for possible clinical applications of csf1-fc. lations in mice (24) , there were a number of clinical trials of applications in cancer and other indications (23) . the interest in csf1 as a therapeutic agent has been reinvigorated by evidence of the requirement for macrophages in tissue regeneration in multiple organs (7) and the finding that macrophages generated in response to csf1 have trophic roles (9, 43) . csf1 treatment has been shown to promote regeneration and repair in injury models in the kidney (1), brain (5, 37) , and bone (6) . the pleiotropic impacts of csf1 mutations in mice (12) suggest that repair in most tissues is reliant on macrophages that depend on this growth factor. applications of csf1 therapy were constrained by the very short half-life of the 150-amino acid active form of csf1. we developed a bioactive protein with a longer half-life in the circulation. we fused pig csf1, which is equally active in all mammalian species tested (20) , with the fc region of pig igg1a (csf1-fc) (21) . the expected increase in half-life was confirmed, and csf1-fc administration to mice produced substantial increases in circulating monocyte and tissue macrophage numbers, at much lower doses than the native protein. an unexpected effect of the treatment was a substantial increase in the size of the liver, associated with extensive hepatocyte proliferation (21) . this observation was consistent with previous data implicating csf1-dependent macrophages in hepatic regeneration. acute liver failure in human patients, for example due to paracetamol toxicity, is associated with the loss of clearance functions that protect the body against the contents of the portal blood. circulating csf1 levels in human patients upon admission to hospital with paracetamol poisoning were found to be predictive of subsequent prognosis (46) . administration of csf1-fc in mouse models produced both accelerated regeneration of the liver and, perhaps more importantly, very rapid restoration of clearance functions (21, 44, 46) . pigs have been used increasingly as models of human disease (17) . the gene expression profiles of stimulated mouse and human macrophages differ greatly, and those of pig macrophages are much more human-like (29) . aside from the possible applications as human disease models, pigs are a major livestock species. early weaning in pigs, when the mucosal barrier and innate immune systems are immature, is associated with susceptibility to a very wide range of mucosal and systemic bacterial and viral infections that produce significant losses (3) . the results obtained in mice cannot necessarily be extrapolated to large animals, including humans, in which the profiles of macrophage activation by both csf1 and microbial stimuli are very different (29) . our results confirm that, as in the mouse, csf1-fc can drive hepatocyte proliferation and modulate the size of the liver in a large animal model. the data support applications of csf1-fc in liver regeneration and provide further evidence for the role of csf1 in monocyte/ macrophage maturation. in combination with earlier data, they reinforce the conclusion that circulating csf1 is a central contributor to the homeostatic control of the size of the liver. animals. approval was obtained from protocols and ethics committees of roslin institute or agri-food and biosciences institute (afbi) for the trials. the experiments were carried out under the authority of a uk home office project license, under the regulations of animals (scientific procedures) act 1986. csf1-fc was made as previously described (21) and provided by zoetis. large white pigs ϳ8.5 wk of age from one litter were used. four days prior to the first injection each pig was weighed and had blood collected into an edta tube. pigs were injected subcutaneously once a day for a total of 3 days with the appropriate volume of csf1-fc (0.75 mg/kg; n ϭ 6) or pbs vehicle (n ϭ 5). pbs injection was used to control for the possible impact of stress and restraint associated with treatment. in experiments on weaners, large white ϫ landrace pigs ϳ4 wk of age from three litters were used. six days prior to the first injection each pig was weighed and an estimated weight was extrapolated for each for the first injection day. pigs were injected intramuscularly once a day for 2 days with the appropriate volume of csf1-fc (0.75 mg/kg; n ϭ 12) or pbs (n ϭ 12). on the second injection day the pigs were weaned. all pigs were sedated with ketamine and azaperone before being euthanized by captive bolt. neither subcutaneous nor intramuscular injection produced any side effects. isolation of pbmc and bmc. blood was collected into blood collection bags containing acid citrate dextrose (acd) (sarstedt) or into beakers containing acd (sigma). the buffy coat was layered onto lymphoprep (axis-shield) and centrifuged for 25 min at 1,200 g with no brake. peripheral blood mononuclear cells (pbmc) were retrieved and red cells were removed with cell lysis buffer (biolegend). pig bone marrow cells (bmc) were obtained by flushing the bone marrow from ribs with rpmi/5 mm edta followed by removal of red cells with cell lysis buffer. all isolated cells were suspended in pbs prior to counting and cryopreservation. flow cytometry analysis. cells were washed, pelleted, resuspended in blocking buffer (pbs/2% heat inactivated fcs), transferred to a 96-well plate (v-bottom), and incubated on ice for 15-20 min. the plate was centrifuged for 4 min at 400 g followed by removal of supernatant. cells were resuspended in 100 l of pbs containing the appropriate antibody or isotype control (table 1) . samples were incubated at 4°c in the dark for 30 min before being washed two times with 200 l pbs. cells were resuspended in 600 l pbs with 0.1% sytox blue (invitrogen) immediately prior to analysis using a bd fortessa lsr flow cytometer (becton dickinson). analysis was performed using flowjo software (flowjo). complete blood count analysis. an aliquot of blood from acd blood collection bags was analyzed for complete blood cell counts. total white blood cell (wbc) was measured on the siemens advia 2120 analyzer. wbc differential counts were performed by making a blood smear counterstained with giemsa stain prior to cells of each cell type being counted. the absolute value for each wbc type was determined by using the total wbc and % leukocytes. manual platelets counts were carried out using a hemocytometer slide (by the r(d)svs clinical pathology laboratory, university of edinburgh). plasma analysis. plasma was analyzed by r(d)svs clinical pathology laboratory for cortisol (performed on siemens immulite analyzer) as well as a large animal liver damage profile (performed on the il650 analyzer from instrumentation laboratories). tissue processing. tissues were dissected, weighed (liver, spleen, and kidney), and placed in 10% neutral buffered formalin or rnalater (ambion). for histology, tissues were processed overnight using an excelsior tissue processor (thermo fisher scientific). sections were embedded in paraffin wax prior to 4-m sections cut and mounted onto slides (superfrost plus, thermo fisher scientific). slides were dried overnight at 37°c before 60°c for 25 min. sections were stained with h&e or immunohistochemistry was performed by r(d)svs pathology department. immunohistochemistry for cd163. antigen retrieval was performed with proteinase k (dako s302030) for 10 min. nonspecific protein binding was blocked using 2.5% goat serum (vector laboratories) for 20 min. endogenous peroxidase activity was blocked using dako real peroxidase blocker (dako s202386) for 10 min. sections were incubated for 60 min using mouse anti-pig cd163 (serotec mca2311ga) diluted 1/30. visualization using secondary reagent dako envision mouse hrp (dako k4007) for 40 min followed by dab (newmarket scientific monosan dab substrate kit cat. no. mon-app177) for 10 min and dab enhancer for 3 min (newmarket scientific dab concentrate cat. no. co7-25) was performed by the r(d)svs pathology department. the staining was analyzed using image j (fiji). immunohistochemistry for ki67 and pcna. antigen retrieval was performed by boiling in 10 mm sodium citrate buffer. nonspecific protein binding was blocked using 2.5% goat serum (vector laboratories) for 20 min. endogenous peroxidase activity was blocked using dako real peroxidase blocker (dako s202386) for 10 min. sections were incubated for 60 min using rabbit anti-human ki67 (abcam ab15580) diluted 1/10,000. visualization was performed with secondary reagent immpress hrp anti-rabbit igg (peroxidase polymer; vectormp-7401) for 30 min followed by dab (newmarket scientific monosan dab substrate kit cat. no. mon-app177) for 10 min and dab enhancer for 3 min (newmarket scientific dab concentrate cat. no. co7-25). statistical analysis. data were analyzed by t-tests. results are presented as treatment group means ϯ se. all analyses were performed using graphpad prism 5.0 (graphpad software). a p value ͻ 0.05 was considered statistically significant. microarray. total rna was prepared from liver samples using trizol, prepared for hybridization using the ambion wt expression kit (life technologies), following the manufacturer's instructions, except for the input amount of rna (500 ng input instead of 100 ng) and hybridized in a random order to the affymetrix porcine gene 1.1 st array (performed by edinburgh genomics, university of edinburgh). statistical analysis of the array data utilized partek genomic suite (partek). for network analysis, the normalized array data were uploaded to the software biolayout express 3d (http://www.biolayout.org/) as described previously (18, 30) . the data from the microarray are available at gene expression omnibus ncbi (http://www.ncbi.nlm.nih.gov/geo/) accession code gse78837. we first examined 8-wk-old pigs of both sexes from the same litter, with control and treated groups weight matched in pairs. based on mouse data (21) we used a dose of 0.75 mg/kg for three daily treatments followed by cull 24 h after final injection. in previous studies, we have compared csf1-fc with the native, non-fc conjugate, form of csf1 as a control protein. native csf1 has a much shorter half-life and at the same dose had no effect on monocyte-macrophage numbers (21) . we have not compared csf1-fc directly with an irrelevant fusion protein, or with an isotype control for the fc component. however, an equivalent dose of igg1 (10 -20 mg depending on the size of the pig) would have no impact on the plasma igg concentration (15-30 mg/ml). csf1-fc treatment of macrophages in vitro did not induce proinflammatory cytokines (21) and, in keeping with the lack of intrinsic proinflammatory activity, there was no evidence of any reaction at the sites of injection in any treated animals. the most obvious effect of csf1-fc administration was hepatosplenomegaly. csf1-fc doubled the spleen/body weight ratio and increased the liver/body weight ratio by 40% after only 4 days (fig. 1a ). the total wbc count was significantly increased, mainly due to lymphocytosis in addition to the expected monocytosis (fig. 1b) . csf1-fc accelerates the maturation of macrophage populations in peripheral blood monocytes. csf1 has been implicated in the maturation of blood monocytes in both mice and humans, driving the formation of the nonclassical (cd14 low , cd16 high ) subset in humans (ly6c low in mice) (31, 33) . pig blood monocytes can also be separated into subsets based on expression of surface markers, although the distinctions are not as clear as in other species (16) . expression of various markers by peripheral blood monocytes was assessed by flow cytometry staining (fig. 2 ). in addition to the increase in total wbc seen in fig. 1 , the proportion of monocytes, detected by cd172a (sirpa) was increased around twofold (fig. 2b ). the proportion of cells expressing cd16 was also increased ( fig. 2a) . no increase was seen in the percentage of cd3 ϩ lymphocytes ( fig. 2c ) the best-characterized monocyte maturation marker in pigs is the haptoglobin receptor, cd163 (16) , which has also been implicated as a receptor for the major pig viral pathogen, porcine reproductive and respiratory syndrome (prrsv) (14) , and which varies inversely with cd14 expression. double staining with the two markers indicated that csf1-fc shifted the profiles of both markers, favoring expansion of the cd163 ϩ cells (fig. 2d ). the results are consistent with an impact of csf1-fc to promote both monocyte production/ release and maturation. impact of csf1-fc treatment on the bone marrow. we next investigated whether the ability of csf1-fc to promote monocytosis was associated with expansion of progenitor pools in the marrow (fig. 3) . figure 3 , a and b, demonstrates a substantial increase in large cd14 ϩ monocytes, and even greater increase in cd163 ϩ cells in the marrow of treated animals, consistent with the pattern seen in blood. aside from monocyte progenitors, a key population of macrophages in bone marrow forms the center of hemopoietic islands. in mice, these cells express sialoadhesin (cd169, which provides a receptor for immature erythrocytes) and are critical for suc-cessful engraftment in bone marrow transplantation (10) . as shown in fig. 3c , the csf1-fc treatment produced a substantial increase in the cd169 ϩ population in pigs. the csf1-fc treatment did not expand the small percentage of cells that express cd117 (kit), a marker of the stem cell population (fig. 3d) , suggesting that csf1-fc acts primarily to promote proliferation/expansion of committed progenitors. in bone marrow of pig, neither cd172a nor cd16 provides a useful marker of monocyte lineage cells, being detected on the large majority of the cells (fig. 4, a and b) and only marginally increased by csf1-fc. we also examined the expression of the csf1r (cd115) using either a recently described monoclonal antibody (39) or labeled csf1-fc. there was some evidence of expansion of the positive cell populations in each case, but the levels of labeling were very low (fig. 4, c and d) . we suggest that the receptor may be downregulated by csf1-fc. counts. pigs (8-wk-old males and females) were injected with pbs or 0.75 mg/kg csf1-fc for 3 days prior to euthanasia on day 4. blood was collected into edta tubes postmortem and complete blood count assessment was performed. graphs show means ϯ se. **p ͻ 0.01, ***p ͻ 0.001, ****p ͻ 0.0001 by t-test; n ϭ 5-6 pigs per treatment. a: liver weight/body weight ratio, spleen weight/body weight ratio, and kidney weight/body weight ratio. b: total wbc count, lymphocyte number, neutrophil number, monocyte number, and platelet number. origin of the increase in liver and spleen weight. csf1-fc treatment caused a substantial increase in macrophage numbers in both organs, detectable by immunolocalization of cd163. in immunostained sections of liver csf1-fc treatment increased cd163 ϩ area (quantified with imagej) from an average of less than 0.5% to an average of over 9% (fig. 5a) . in spleen csf1-fc treatment had an even greater effect, causing an increase of cd163 ϩ area from an average from ϳ1% to 16% (fig. 5b ). as in mice (21) , in the spleen the majority of the increase in size could be attributed to increased red pulp macrophages and also to expansion of the marginal zones. by contrast, the increase in the area apparently occupied by macrophages is not sufficient to explain the substantial increase in the size of the liver. sections of liver were stained for the figure 6 shows images of the liver from two control and two csf1-fc-treated pigs. the pigs are relatively young, and still growing, and accordingly there is significant ongoing proliferation evident from ki67 staining. the vast majority of ki67 ϩ nuclei in both control and csf-1-fc-treated pig livers were large and round, consistent with identity as hepatocytes. macrophage nuclei are more difficult to visualize in histological sections, because the cells and nuclei are much smaller and ramified in the sinusoids. very occasional smaller ki67 ϩ nuclei visible in the sinusoids suggested that some infiltrating monocyte-macrophages were also proliferative, as shown directly in the mouse system (46) . the images in fig. 6 also show an obvious increase in cellularity in response to csf1-fc. we counted the total nuclei and the proportion stained with anti-ki67 in representative large fields from each animal. as shown in fig. 6 , csf1-fc treatment almost doubled the total number of nuclei in each field and produced a threefold increase in the percentage of those nuclei stained with anti-ki67. essentially the same findings were made with staining for proliferating cell nuclear antigen (pcna) (not shown). the sections in fig. 6 show no evidence of pathology in the liver; notably, there are no pyknotic nuclei and granulocytes are absent. granulocyte infiltration is the hallmark of tissue injury, including injury to the liver (27) . changes in the liver might occur secondary to alterations in the gut. csf1 has been attributed indirect functions in control of proliferation and differentiation of gastrointestinal epithelium (26, 45) . treatment with csf1-fc in pigs produced a small but significant increase in the mean villus length of the mid jejunum but had no detectable effect in the ileum, cecal base, or ascending colon (fig. 7) . there was also no significant change in goblet cell number. effect of csf1-fc on liver function. a panel of biochemical tests to measure serum enzymes, bile acid, bilirubin, and protein concentrations was performed to assess hepatic function. the only change was a small increase in bile acids and bilirubin in serum from csf1-fc-treated pigs (fig. 8) , only marginally outside the normal range (50) . since standard enzymic indicators of liver injury (alkaline phosphatase, alanine aminotransferase, ␥-glutamyl transpeptidase) were unchanged, the increase in bile acids probably reflects the increased size of the liver. to examine the impact of csf1-fc on liver function in more detail, we profiled the transcriptome. the expression results were filtered to remove genes detected below an arbitrary relative intensity threshold and also genes that did not differ by more than 1.5-fold between the highest and lowest value in the nine samples. the second criterion removed around 30% of probes on the microarray, including many hepatocyte-specific gene products. figure 9c shows that the relative abundance of representative examples of these known hepatocyte gene products, albumin (alb), cd14, fetuin, and transferrin (tf), within the total liver rna pool was unchanged in response to csf1-fc. in other words, the infiltration of the liver by macrophages was insufficient to dilute the contribution of hepatocyte mrna to the total mrna pool. that finding is consistent with the histological observation above, that even in the csf1-fc-stimulated state the infiltrating macrophages appear to make up no more than 10% of the total area of the liver. hence, the 40% increase in total liver weight can be attributed primarily to an increase in using imagej software the total cd163 ϩ area was calculated from 2 representative images per pig per organ. graphs show means ϯ se. *p ͻ 0.05, **p ͻ 0.01 by t-test; n ϭ 4 -5 pigs per treatment. g539 csf1 controls the steady-state size of the liver hepatocytes, consistent with the extensive proliferation and increased cellularity shown in fig. 6 . we clustered the included probe sets based on expression pattern and displayed them using biolayout express 3d . the advantage of using the clustering approach is that genes that might appear regulated, but in only a subset of animals, appeared in separate smaller clusters. these may reflect the interanimal variation in macrophage-inducible gene expression that we documented previously in a study of pig breeds (30) . the gene lists of specific clusters are provided in supplemental table s1 . (supplemental material for this article is available online at the journal website.) functional annotations of the two large clusters were tested using david (supplemental table s2 ). cluster 1, the set of genes elevated in all csf1-fctreated pigs, was clearly enriched for genes involved in the cell cycle and innate immunity, whereas cluster 2, the set that was reduced in the csf1-fc-treated pigs, was enriched in genes involved in metabolism. importantly, there is no evidence among the induced genes of expression of apoptosis-associated genes, no induction of acute phase genes, and no appearance of classical granulocyte marker genes such as s100a8/s100a9 or mpo. figure 9 shows the expression profiles of a number of genes that highlight the biological processes involved. cluster 1 (fig. 9a ) includes many genes that were shown previously (18) to be restricted to macrophages, such as the transcription factor spi1; surface markers sirpa, emr1, and itgam; known csf1-inducible genes plau, mmp9, chi3l1, and c1q; endocytic receptors marco, msr1, and fcgr1a; and pattern recognition receptors tlr1, 2, 4, 6, 7, 8, and 9 . on average, the relative contribution of cluster 1, macrophagespecific genes, to the liver total rna increased by three-to fivefold in response to csf1-fc, again consistent with the fig. 5 . in mice, the recruited monocytes express the chemokine receptor ccr2 and apparently respond to ccl2 (49) . by contrast, ccr1 was enriched in the liver mrna of treated pigs, alongside three of its known ligands, ccl8, ccl14, and ccl3l1. as previously observed in mice (46) , monocytes recruited to the treated livers apparently responded to proinflammatory signals, since cluster 1 contained numerous known lps-inducible genes (29) such as inflammatory cytokines tnf, il1a, and il1b; interferon targets ido1; multiple type 1 interferon targets irf1, irf5, ifitm2, and ifit3; tgfb1; and costimulators of t cell activation cd40, cd80, and cd86. cluster 1 also contains numerous cell cycle-associated genes, including pcna; the key transcription factors foxm1, e2f4, e2f7, and e2f8; and several cyclin genes ccna2, ccna3, ccnb2, ccnb3, ccnd2, and ccnd3. the increased expression of enzymes of glycolysis hk1, hk2, hk3 pfkp, pgk1, pgd, pkm, gpi, gapdh, and ldha also reflects the requirement for aerobic glycolysis in proliferating cells (25) . cluster 2 (fig. 9b) , the set of genes reduced in the csf1-fc-treated pigs, most likely reflects the functional zonation of the liver between periportal and perivenous regions of liver lobules (8, 19, 48) and the selective proliferation of cells derived from portal progenitors that has been observed in regenerating liver (15, 34, 36) . it includes genes involved in xenobiotic metabolism and detoxification, notably p450 family (e.g., cyp1a1, cyp2e1), glutathione s-transferases (e.g., gstaa2) and aldo-ketoreductases (e.g., akr1c1), and the gluconeogenic enzyme pck2, that are known to be enriched in perivenous locations. the cluster contains the gene for the regulator of hepatocyte stem cells, sox9 (2), indicating that these cells are not expanded in the csf1-fc-treated livers. unexpected members of this cluster are genes for the growth hormone receptor (ghr) and the target, igf1, and both estrogen (esr1) and androgen (ar) receptors. also unexpected is the inclusion of the receptor for hepatocyte growth factor, met, which is implicated in regeneration (36), but this might reflect autoregulation in response to its ligand (52) . although models of acute liver failure in pigs have been described (32, 41) , and may be one path to clinical development of csf1-fc as a treatment, it is challenging to perform sufficient replicates to test a clinical intervention. we therefore considered an alternative in production pigs. commercial pigs are normally weaned at 4 wk, when the gut is immature. diarrhea and disseminated infections with organisms such as escherichia coli and streptococcus suis are relatively common (38) . in this respect, the pig has been studied as a model of early-life stress (42) . the biology of early weaning in pigs may also be relevant to intestinal failure-associated liver disease in neonates and children (40) . weaner pigs were treated with a higher dose of 1.0 mg/kg csf1-fc for two daily injections, immediately prior to weaning and on the day of weaning followed by euthanasia 24 h following the final injection. at this early time point, there was already a significant increase in the spleen/body weight ratio and a trend toward increased liver/body weight ratio (fig. 10a ). the number of cd163 ϩ cells was more than tripled in the bone marrow, from ϳ10% to over 30% (fig. 10b) , and increased numbers of cd163 ϩ macrophages were confirmed by immunostaining in liver and spleen (fig. 10c ). at this time point, there was not a significant monocytosis, indicating that both marrow expansion and tissue macrophage proliferation precede monocyte expansion and are likely direct effects of csf1-fc. we repeated the treatment in a larger cohort of weaned pigs. this study was conducted in a high health status research unit, which reflected commercial practice. we explicitly removed zinc from the feed, which is usually added to reduce weaningassociated infections. given the production of inflammatory cytokines and reduction in igf-1 in the liver of treated pigs, we measured weight gain daily in all animals. csf1-fc (0.75 mg/kg) was administered to pigs for two daily intramuscular injections on the day before and the day of weaning, and pigs were killed 5 days after the second injection. although some pigs showed evidence of mild postweaning diarrhea, all the animals in both groups continued to gain weight rapidly (fig. 11a) . the treated pigs, like the treated mice left for longer after the final injection, demonstrated hepatosplenomegaly (fig. 11b) , and the increased numbers of cd163 ϩ macrophages in the liver remained evident after 5 days (fig. 11c ). in this study we have extended previous studies in mice (21) to examine the impact of a sustained increase in csf1 activity on monocyte-macrophage homeostasis. all of the impacts we have observed are consistent with the known biological activity of csf1. in mice, the same impacts on monocyte-macrophage numbers and maturation can be generated by injection of very much higher doses of native csf1 (21) or injection of a much larger native form of human csf1 (24) . the doses of native csf1 required are prohibitive in a large animal. although we cannot entirely eliminate other functional contributions of the fc component, the increase in circulating half-life is the most obvious explanation for the increased efficacy compared with native csf1. the nature of the so-called hepatostat, which determines that the liver returns to a size that is strictly proportional to body size, has continued to be something of a mystery (34, 35) . although there are many candidates, including growth factors and inhibitors, extracellular matrix proteins and metabolites, and circulating hormones that can regulate hepatic regeneration, it is unclear how any of them functions as a sensor. in a previous study, we made the striking observation that csf1 treatment of mice (using an fc conjugate with an increased circulating half-life) was able to increase the size of the liver as well as the number of kupffer cells. this ability is quite unique. in the present study, we have extended the finding to the domestic pig, an animal that is considerably more humanlike in size and vascular biology. the data in fig. 6 show that a major impact of csf1-fc treatment in pigs is to increase the number of hepatocytes through extensive proliferation, so that the total cellularity of the liver is increased even more than the increase in total liver weight. hepatocyte proliferation, as opposed to hypertrophy, is also a feature of liver regeneration in response to partial hepatectomy (34 -36) . we have made the reciprocal observation in mice; namely, that prolonged depletion of kupffer cells with anti-csf1r treatment leads to a reduction in the size of the liver (45) . others have shown that liver regeneration is greatly impaired in csf1-deficient or anti-csf1r-treated mice (46) and in mice depleted of blood monocytes (15) and have promoted liver repair by infusing csf1-stimulated macrophages into the portal vein (47) . the impact of csf1-fc on hepatic growth in mice was dependent on monocyte recruitment, as evident from the impact of knockout of ccr2 (46) . the role of monocyte-macrophage products, including the inflammatory cytokines tnf, il1, and il6, in hepatocyte proliferation has been well recognized (22, 46) . csf1-fc action in mice was partly dependent on il6 (46) , which was also induced in all of the csf1-fc-treated pigs. the effect of csf1-fc treatment demonstrates that csf1-dependent monocyte recruitment is both necessary and sufficient to drive hepatic proliferation and can drive it beyond the homeostatic limits even in a large animal. the effect of csf1 treatment supports other evidence of the existence of a homeostatic feedback loop. macrophages, notably those of the liver (4) and blood monocytes (51) , together regulate the level of circulating csf1 via receptor-mediated endocytosis. this mechanism is evident from the massive increase in circulating csf1 seen in animals treated with anti-csf1r (33), and the importance of the liver is evident in patients following partial resection (46) . the csf1-fc treatment reveals that elevated csf1 can provoke expansion of the committed monocyte pool in the marrow (fig. 3) , maturation of the monocytes toward a resident phenotype (fig. 2) , and infiltration of the liver (fig. 5) . hence, the physiological hepatostat (34 -36) may actually be a "macrostat." of course, the increased macrophage numbers in the liver elicited by csf1 produce secondary impacts, not only removing potential toxins from the portal blood, but altering the balance of metabolism in the liver between portal and venous-associated functions. interestingly, resident kupffer cells are selectively located toward the portal vein in mouse liver lobules (13) , which might also serve to localize macrophage-derived hepatocyte proliferative signals. the expression profiling of the livers of the csf1-fc-treated pigs (fig. 9 ) closely parallels results obtained previously in the mouse system (21) . the newly recruited monocytes clearly respond to tlr-mediated and other signals to express the large majority of transcripts seen when csf1-stimulated bone marfig. 10 . effect of a short course of csf1-fc in weaning pigs. pigs (4-wk-old males and females) were injected with pbs or 1 mg/kg csf1-fc for 2 days prior to euthanasia on day 3. graphs show means ϯ se. *p ͻ 0.05, **p ͻ 0.01, ***p ͻ 0.001 by t-test; n ϭ 5 pigs per treatment. a: blood was collected into edta tubes postmortem and complete blood count assessment was performed. graphs show liver weight/body weight ratio, spleen weight/body weight ratio, and monocyte number. b: bm from ribs was collected as described in materials and methods. bm cells were analyzed via flow cytometry for expression of cd163 with exclusion of dead cells using sytox blue. representative flow cytometry plots are shown. c: formalin-fixed liver and spleen tissue was prepared and stained for cd163. representative images are shown. fig. 11 . long-lasting effect of csf1-fc in weaning pigs. pigs (4-wk-old males and females) were injected with pbs or 0.75 mg/kg csf1-fc for 2 days prior to euthanasia 5 days after the final injection; n ϭ 12 pigs per treatment. a: body weight was recorded at each of the time points shown and total body weight change over the duration of the experiment was graphed for pbs treated pigs (black) and csf1-fc-treated pigs (red). b: bar graphs show means ϯ se. ***p ͻ 0.001, ****p ͻ 0.0001 by t-test. graphs show liver weight/body weight ratio, spleen weight/body weight ratio, and kidney weight/body weight ratio. c: formalin-fixed liver tissue was prepared and stained for cd163. representative images are shown. scale bar ϭ 10 m. csf1 controls the steady-state size of the liver row-derived macrophages respond to lps (29) . however, classical neutrophil chemoattractants such as il8 were not detected; we found no evidence of neutrophil infiltration, no induction of classical acute phase response or apoptosis genes, and no evidence of damage to liver cells. furthermore, the pigs showed no adverse impacts, and in weaners the treatment did not impair their rapid growth (fig. 11) . this is consistent with earlier data, in which recombinant csf1 has previously been administered in phase 1 clinical trials by continuous infusion to humans and was well-tolerated (11, 28) , and indicates that the fc fusion protein does not produce any additional toxicity. the lack of severe consequences may be attributed in part to induction of the anti-inflammatory cytokines il10 and tgfb1 in the liver of the treated animals. whatever the mechanism, the outcome suggests that csf1-fc specifically promotes a proregenerative cellular response in liver, as it does in other organs (23) . in the final set of experiments (fig. 11) , we progressed toward the clinical application of csf1-fc in a pig model. the model may also reflect a practical application in pig production to bolster the innate immune system at weaning. we showed that only two doses, administered intramuscularly, were sufficient to produce a sustained increase in monocyte count, liver size, and liver macrophage numbers and produced no adverse local reaction. we propose that csf1-fc could provide protection against disseminated infections arising from the immature gut of early-weaned animals. similarly, clearance functions of the macrophages of the liver are crucial to prevent sepsis in acute liver failure in humans, and csf1-fc rapidly promoted clearance functions in mouse disease models (46) . 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under homeostasis regulation of the met oncogene: molecular mechanisms we thank the staff at the biological resource facility and dryden farm (at the roslin institute) and staff at the afbi-ni for their assistance in planning and executing the animal studies. we thank colleagues at zoetis for providing the csf1-fc, specifically the authors listed in our previous publication (21) . we also show our gratitude to iveta gazova, anna raper, and jun hu for helping to process samples and deliver them to their appropriate locations.present address for g. m. davis: the university of manchester, oxford road, manchester m13 9pl, united kingdom. key: cord-018318-vzzrsqsn authors: naidu, c. sudeep; sarin, arti title: postoperative liver failure date: 2017-02-08 journal: gi surgery annual doi: 10.1007/978-981-10-2678-2_3 sha: doc_id: 18318 cord_uid: vzzrsqsn technical innovations in surgical techniques, anaesthesia, critical care and a spatial understanding of the intra-hepatic anatomy of the liver, have led to an increasing number of liver resections being performed all over the world. however, the number of complications directly attributed to the procedure and leading to inadequate or poor hepatic functional status in the postoperative period remains a matter of concern. there has always been a problem of arriving at a consensus in the definition of the term: postoperative liver failure (plf). the burgeoning rate of living donor liver transplants, with lives of perfectly healthy donors involved, has mandated a consensual definition, uniform diagnosis and protocol for management of plf. the absence of a uniform definition has led to poor comparison among various trials. plf remains a dreaded complication in resection of the liver, with a reported incidence of up to 8 % [1], and mortality rates of up to 30–70 % have been quoted [2]. several studies have quoted a lower incidence of plf in eastern countries, but when it occurs the mortality is as high as in the west [3]. in 2005, balzan et al. [ 5 ] published their results of 775 elective liver resections over a span of 5 years. they focussed on serum bilirubin (sb) and prothrombin time (pt) as important prognostic markers of postoperative liver functional status and proposed the '50-50' criteria for the defi nition of plf, i.e. the combination of pt >50 % of baseline normal and sb >50 μmol/l on postoperative day (pod) 5 (the '50-50' criteria) was found to be strongly predictive of mortality. the fact that this 'criteria' was a precursor of clinical complications 3-8 days before they appeared lent it a strong base for life-saving interventions. the criticism of this simple calculation has been that it relies only on two laboratory tests and does not factor in the existing clinical status of the patient. the '50-50' rule is limited by the fact that it cannot be applied before pod 5, does not stratify patients and though it predicts death in up to 70 % of patients, it is not based on the clinical severity of the patient. in 2010, rahbari et al. [ 2 ] , of the international study group of liver surgery (isgls) defi ned plf as 'a postoperative acquired deterioration' in the functions of the liver: synthetic, excretory and detoxifying. this deterioration is evident by an increase in international normalized ratio (inr) (there may be a need of clotting factors for abnormal inr) and sb, on or after pod 5 (compared with the values of the preceding day). biliary obstruction needs to be ruled out as a cause for the deranged sb. they also stratifi ed plf into three grades: a, b and c, depending on up-scaling of the required level of care. traditionally, surgeons have resorted to keeping an eye on deteriorating liver function by charting rising inr (coagulopathy), rising sb (hyperbilirubinaemia) and the advent of hepatic encephalopathy (failure of detoxifi cation) as surrogate markers for the functions of the liver. other scores such as the child-pugh score (ctp) and the model for end stage liver disease (meld) have also been used by various authors for defi ning plf, but a uniform consensus has evaded clinicians due to the multifactorial and diverse aetiology and pathogenesis of plf. following liver resection the patient has multiple pathophysiological mechanisms at work. there is the trauma of surgery, the anaesthetic and haemodynamic changes, the metabolic demands of wound healing and especially in case of the liver: the pathophysiology of ischaemia-reperfusion injury (iri), liver regeneration and the small for size syndrome (sfss). not only do the number of hepatocytes have to be adequate for body homeostasis, they should be functioning optimally and retain their capacity for regeneration. the liver cells suffer mainly from a combination of iri, hepatic venous congestion and sepsis. the 'hyperfusion theory' is widely accepted and postulates that the relative spike in sinusoidal perfusion of the decreased cell mass precipitates a vicious cycle. a cascade mechanism of which is a combination of congestion, infl ammation, cholestasis and cell death taking place, preventing the normal function of a hepatocyte: uptake, secretion and excretion [ 6 ] . in addition cell proliferation and regeneration are impeded. a standard liver resection for a liver tumour has to deal with iri, congestion, portal hypertension and sepsis, while, in transplantation denervation and immunosuppression are added as precipitating factors. iri persists even after parenchymal damage during liver resection. after a period of ischaemia the infl ammatory response in the form of the complement cascade is activated. activated kupffer cells generate reactive oxygen species (ros) which cause endothelial cell damage [ 7 ] . later in the reperfusion phase, these metabolites are swept around leading to a cycle of microvascular injury and microcirculatory changes resulting in apoptosis and cell necrosis with resultant hepatocyte death. sepsis may intervene in as high as 50 % of patients after liver resections and may be related to loss of kupffer cell volume with impaired immune function. the relationship between infection and plf has not been fully explained [ 8 ] . patients with alf are particularly prone to developing sepsis. it has been shown that 73 % of patients with plf develop postoperative sepsis compared with 18 % of patients without [ 9 ] . sepsis has a triad of detrimental effects on liver synthetic function, hepatocyte regeneration and apoptosis by inducing a relative hepatic ischaemia due to systemic hypotension. it induces kupffer cell dysfunction, releases proinfl ammatory cytokines, and diminishes detoxifi cation of liver endotoxins thereby leading to diminished hepatocyte proliferation and regeneration [ 10 ] . liver surgery by itself may increase the incidence of infection [ 8 ] as major resections are associated with enteric bacterial translocation, which is enhanced by the prolonged infl ow clamping and duration of surgery. a major liver resection involving multiple segments signifi cantly impedes the function of the reticuloendothelial system, which is crucial in immune defence against sepsis. the precipitating factors for development of plf can be broadly divided into patient factors, surgery-related factors and postoperative complications and their management (table 3.1 ) . patients with diabetes have a signifi cantly poorer outcome after elective liver resections compared to those who do not have diabetes [ 11 ] . apart from being at higher risk for infections due to decreased immune tolerance, patients with diabetes may also have a higher incidence of fatty liver with insulin resistance and concomitant poorer functional reserve. preoperative diabetes mellitus is also an independent predictor of 90-day mortality [ 12 ] . patients with cholangitis and active viral hepatitis also do poorly after surgery. in a study, mortality was signifi cantly higher in patients who had resection of hepatocellular carcinoma (hcc) in cirrhosis associated with active hepatitis (8.7 versus 1.5 %; p < 0.05) [ 13 ] . however, increased risk of plf with raised sb has been controversial [ 14 ] . cherqui et al. showed that patients with a raised sb level had a morbidity rate of 50 % compared to 15 % in patients with normal sb (p < 0.01). however, the incidence of plf or mortality did not rise when compared with matched controls [ 14 ] . most patients being planned for liver resections have colorectal liver metastasis and receive chemotherapy with 5-fl uorouracil, oxaliplatin and irinotecan, and newer monoclonal antibodies cituximab and bevacizumab [ 7 ] . chemotherapy induces marked histopathological changes in the liver parenchyma including fatty liver, chemotherapy-associated steatohepatitis (cash), or sinusoidal injury (sinusoidal obstruction syndrome, sos). cash is pathognomonic of patients treated with irinotecan and is characterized by 'steatosis, lobular infl ammation and ballooning of hepatocytes': also called the 'grey liver syndrome'. sos is caused by the use of oxaliplatin and the syndrome is called 'blue liver syndrome' because of the characteristic bluish-red hue of the fi rm liver [ 15 , 16 ] . irinotecan-induced cash has been shown to be an independent risk factor for postoperative mortality and plf. oxaliplatin induced sos develops after more than 6 cycles and is a risk factor for increased hospital stay and postoperative complications [ 17 , 18 ] . hepatic steatosis is a major determinant of postoperative outcomes. over 20 % of patients undergoing a major liver resection have some degree of steatosis, significant enough to alter postoperative recovery [ 19 ] . patients with biopsy-proven hepatic steatosis have a higher incidence of plf (14 %) than patients with healthy livers (4 %) [ 20 ] even though the steatosis was of moderate grade. belghiti et al. [ 21 ] , reported a series of 478 patients who underwent liver resections, of which 37 patients had steatosis. steatosis was an independent risk factor for postoperative complications (8 % of patients with steatosis), while only 2 % of patients with a normal histology developed complications. the increasing incidence of cash, nash and sos has sparked interest in the use of preoperative liver biopsy as an assessment of the liver function and structure. screening of high-risk patients (obese, high body mass index or those on chemotherapy) has been proposed. if a moderate degree of steatosis is established, these patients could benefi t from manipulation of the volume, or surgery could be deferred till the acute changes subside either by treating the underlying aetiology or withdrawing the inciting agent. hepatitis b and hepatitis c virus infection-associated hcc develops along with the presence of fi brosis in the liver parenchyma. there is no correlation with fi brosis and liver resection, but when cirrhosis is established by a biopsy, it remains an independent predictor of poor outcomes in terms of overall and tumour-free recurrence. shen et al. used markers of liver fi brosis preoperatively to predict plf in patients with hcc undergoing liver resection [ 22 ] . they evaluated preoperative hepatitis b virus (hbv) dna levels, serum prealbumin (pa), hyaluronic acid (ha) and laminin (ln) levels and correlated these with plf. a prospective model was used with these four laboratory results and validated in 89 hcc patients with a sensitivity and specifi city of 62 % and 91 %, respectively. malnutrition is commonly prevalent among cirrhotics and leads to increased morbidity and mortality [ 23 ] . malnutrition causes 'disordered mitochondrial function' which alters the immune response and thus reduces the hepatocyte regenerative capacity when exposed to ischaemia [ 24 ] . it is thus essential to objectively evaluate the nutritional status of all patients with liver disease, and to intervene with supplements as indicated. plf and postoperative renal dysfunction are independent predictors of 90-day mortality following liver resection but the predictive value for mortality is significantly higher when both systems fail simultaneously. renal dysfunction following liver surgery may occur because of liver failure and hepatorenal syndrome but also due to hypovolaemia and release of free radicals and pro-infl ammatory mediators during surgery [ 25 ] . advanced age is no longer a deterrent to hepatic resections, which is now increasingly performed in elderly people with an acceptable morbidity and mortality [ 26 ] . advancing age reduces the capacity of the liver to regenerate. the american society of anaesthesiology (asa) and acute physiology and chronic health evaluation (apache) scores have proved useful in anticipating complications following major liver surgery [ 27 ] . assessment of the true functional status of the liver is fraught with complexities and routine blood tests have proved unreliable predictors of plf. notwithstanding this, all patients planned for major liver resections should have complete liver biochemistry, a complete blood count and a prothrombin time as baseline investigations [ 10 ] . experience in liver surgery/high volume centres show an inverse relationship with outcomes. it has been suggested that patients needing liver resections be referred to centres that perform 10-17 liver resections per year [ 28 ] . massive estimated blood loss (ebl) remains a key prognostic factor for a safe resection. ebl correlates with the extent of resection and the number of segments resected [ 29 ] and this correlates with the incidence of plf. massive ebl during a major liver resection should be anticipated in tumours abutting the inferior vena cava or major hepatic veins, or if there is injury to the middle hepatic vein during resection, and not by patient age, tumour size alone, or type of hepatectomy. cirrhosis creates a hyperdynamic milieu with increased cardiac output and decreased systemic vascular resistance. the hepatic buffer response of the cirrhotic liver is altered: portal blood fl ow is reduced/shunted as a result of collaterals in portal hypertension, and arterial blood fl ow is impeded because of hepatic fi brosis and sinusoidal resistance. this altered fl ow renders the cirrhotic liver relatively anoxic and less tolerant to hypotension and hypoxia. an intraoperative blood loss greater than 1000 ml increases the risk of plf [ 30 ] . this effect may be caused by a massive fl uid shift secondary to ebl causing global infl ammation because of bacterial endotoxins, peripheral vasodilation and pooling in third spaces. coagulopathy following blood loss with the inability of the liver to catch up, seems to increase the potential for intra-abdominal collections and bacterial infections. avoiding prolonged hypotension and hypothermia by using judicious and timely transfusion, rapid infusion devices and safe surgical techniques is the key to salvage these patients. in fact, even major blood loss may be tolerated, if adequate efforts have been made to maintain euthermia, perfusion, avoid metabolic acidosis and provide an adequate and timely buffer against the dangerous triad of acidosis, hypothermia and coagulopathy resulting from ebl [ 30 ] . prolonged operating time also leads to a poorer outcome and is extended in patients with ebl, vascular reconstructions and diffi cult surgery due to adhesions and tumour extensions into unsafe areas. the functioning liver remnant (flr) in patients has become a topic of much debate especially with the popularity of living donor liver transplantation. in a liver with normal parenchyma <25 % flr is associated with a poor outcome, compared to that of patients with ≥25 % flr [ 31 ] . the risk of plf is 3 times greater. the flr and the method of calculating it are even more important in livers with steatosis and fi brosis as in these livers functional reserve is markedly reduced. patients with histologically proven parenchymal changes of steatosis, fi brosis or cirrhosis, mandate a flr of up to 40 % [ 32 ] . postoperative management has an important bearing on outcome. the fi rst 48 h after a major hepatic surgery are crucial for a successful outcome. metabolic, functional and haemodynamic alterations after hepatic resection are unique to each patient and demand specifi c management protocols. a multidisciplinary team approach is required with goal-directed therapeutic options. it is mandatory to have invasive haemodynamic monitoring, mechanical ventilation, critical parameter monitoring, strict antisepsis measures, metabolic control and optimal nutritional support. some degree of coagulopathy is the expected norm after a major hepatic resection and can be assessed by markers such as pt/inr, platelet counts and partial thromboplastin time (ptt). postoperative coagulopathy peaks 48-96 h postoperatively and can be best monitored by a thromboelastograph (teg) [ 33 ] . the underlying coagulopathy leads to postoperative haemorrhage and blood collecting in the abdomen, leading to postoperative infection. postoperative antibiotic prophylaxis does not control infections [ 34 ] and no effort should be spared in obtaining meticulous haemostasis and following strict infection control protocols. preoperative risk assessment involves a thorough evaluation of all the factors mentioned above. a physical examination followed by appropriate clinical tests will identify patients at risk of developing plf. the patient's liver status, hepatic reserve potential and functional aspect need to be investigated along with the metabolic and haematological derangements, which may lead to plf. pre-existing liver disease can be determined by a thorough clinical history, recording previous blood transfusions, hidden illicit drug use, excessive ethanol use, history of jaundice, family history of familial cholestasis as well as history of adverse drug reactions. in india, it is important to take a history of use of complementary and alternative medicines (cam) as many of them maybe hepatotoxic [ 35 ] . physical examination should be able to pick up subtle signs of incipient liver failure and decompensation such as proximal muscle wasting, spider naevi, ascites and other signs. though routine liver tests have a low yield, they are of value in liver resections as the major indications for hepatectomy in eastern countries are hcc and in the west metastatic disease arising in either normal or cirrhotic livers. hccs are usually sequelae of hbv/hcv disease and it is prudent to evaluate for active disease as well as cirrhosis. though colorectal metastasis can occur in a liver with normal parenchyma, the widespread use of preoperative chemotherapy, as mentioned earlier, mandates a more thorough evaluation of steatohepatitis and fi brosis. in patients with hcc, cirrhosis is present in 64-74 %, but conversely in patients with cirrhosis there is only an 11 % incidence of hcc [ 36 ] . the rest would be divided equally between alcohol-related disease and hbv/hcv, with about 5 % due to metabolic factors. in a cirrhotic patient with hcc, it is the underlying liver parenchymal disease which precludes a safe liver resection and this needs to be addressed. a cirrhotic liver is much less rarely involved with metastasis in comparison to normal livers. liver tests may be labelled as 1. screening tests to indicate the presence of liver disease 2. diagnostic tests to discern aetiology 3. quantitative tests to measure functional reserve. 1. serum bilirubin evaluates conjugation and excretion functions. total bilirubin is a poorly sensitive and specifi c test for liver disease. direct bilirubin does not differentiate between extra-and intrahepatic cholestasis. however, it is an important factor of scores such as ctp and meld for prognostication. miyagawa et al. [ 37 ] found no signifi cant differences in morbidity and mortality after major hepatectomies in spite of a raised sb. postoperatively, sb is often increased; however this does not always indicate impending plf. 2. serum bile acids evaluate excretion and shunting. elevated bile acids are a good marker for portosystemic shunting with a sensitivity of over 90 % in detecting cirrhosis in patients with normal transaminases [ 38 ] . 3. alkaline phosphatise (alp) evaluates cholestasis. it is also synthesized in the bone and placenta and is elevated in metastasis and thus is not a specifi c hepatic marker. alp also has a lag curve in acute obstruction. high preoperative alkaline phosphatase level is indicative of metastatic disease and may be associated with increased mortality after major resections [ 39 ] . concomitant with a raised carcinoembryonic antigen (cea) level, it should raise a strong suspicion of liver metastasis. 4. gamma glutamyl transpeptidase (ggt) evaluates cholestasis, enzyme induction, alcohol abuse, renal failure, myocardial infarction, diabetes and pancreatic diseases, and intake of enzyme inducing agents. elevation of both ggt and alp indicates a hepatic origin for alp elevation. 5. transaminases evaluate necrosis and parenchymal damage. raised serum levels do not correlate with the extent of parenchymal necrosis and have no prognostic value. both enzymes are evaluated together as alt is more liver specifi c and ast more sensitive to changes. after full course chemotherapy, transaminases can be elevated up to 2.5 times of normal. partial hepatectomy induces only a mild-to-moderate increase in serum enzymes [ 40 ] . 6. coagulation factors and pt evaluate synthetic functions. pt is a routine investigation and is used in prognostic scores. as its half-life is shorter, it is a better index of the synthetic function than serum albumin, which has a half-life of 20 days. fibrinogen levels in mild liver disease are normal/slightly elevated but markedly decreased in massive hepatocellular damage. if the prothrombin time is prolonged, a detailed evaluation of the coagulation system is warranted. massive hepatectomy invariable leads to a fall in platelet counts and a depression of coagulation factors such as factors i, ii, v, vii, x and plasminogen [ 40 ] with resultant disseminated intravascular coagulation (dic). 7. albumin evaluates synthesis. it is not only a part of the ctp score but is associated with many non-hepatic diseases such as renal disease, and nutritional entities such as protein malnutrition, protein-losing enteropathy and burns. patients with preoperative albumin <3.0 g/dl are at risk of increased operative morbidity [ 41 ] . the serum concentrations of the above tests may not be truly refl ective of liver function alone as many extrahepatic causes may also alter the results, e.g. transfusion associated haemolysis, resorption of haematomas, extrahepatic loss of albumin in bowel-related diseases, or altered pt due to a lack of absorption of vitamin k in either resected small bowel or due to absence of bile in the gut, i.e. obstructive jaundice. moreover the production, excretion and absorption of these factors are varied and laboratory techniques also result in variances. p450-mediated n-demethylation of a 14 c-or 13 c-labelled methyl group of aminopyrine is measured. the formed 14 co 2 or 13 co 2 are trapped and measured. merkel et al. [ 42 ] studied abt and ctp scores and reported that both reliably predict death from liver failure in patients with cirrhosis. abt had 94 % sensitivity and 88 % specifi city and this was independent of the child's classifi cation. though the test is easy to do, it has not become popular due to the need for expensive equipment. 2. organic anionic dyes assess hepatic perfusion and excretory function. normal liver cells take up sulphobromophthalein (bsp), conjugate it with glutathione and excrete into the bile. bsp clearance differentiates cirrhotic from noncirrhotic livers and provides the status of hepatic uptake and biliary excretion. however, bsp is metabolized in the liver and has been reported to cause anaphylaxis, which has restricted its use. in contrast icg is not metabolized in the liver. the 15-min retention rate for indocyanine green (icg15) is the most common preoperative test for evaluating hepatic reserve [ 43 ] . when a hepatectomy is done in a patient with a high icg15 retention, the volume of non-tumorous liver resected must be minimized. hepatic function is estimated by icg15 or of its maximal removal rate (icg-rmax). the icg15 should be approximately 10 % in normal persons. the threshold value for a safe major hepatectomy is set at 14 %, although the cut-off of icg clearance has shown signifi cant reduction in cirrhotic patients who underwent resection and died subsequently. this was most accurate on day 3 following surgery. when icgr15 exceeds 20 %, a major hepatectomy should be deferred [ 43 ] . patients with icgr15 between 14 and 20 % benefi t from volume manipulation to achieve a viable flr. preoperative icg clearance may predict 30-day hospital mortality in patients with cirrhosis [ 44 ] . with an accuracy of 100 % for prediction of long-term prognosis in both retrospectively and prospectively evaluated cases, noguchi et al. [ 45 ] reported a ratio of icg-rmax relative to the flr after hepatectomy, which could reliably predict outcome. mainly researched by japanese surgeons, icg clearance has not been popular with other centres. an icgr15 value of 14 % has been proposed as a cut-off for identifying patients who will have high postoperative morbidity following a major hepatic resection [ 45 ] . recently, icg has been investigated again. fung et al. [ 46 ] studied liver stiffness (fi brosis) using a fi brosis measuring impedance elastograph. although icg extraction is unique to the liver with minimal extrahepatic elimination, the clearance rate is dependent on local and systemic haemodynamics. any change in hepatic fl ow or systemic perfusion causes variances in icg rates. therefore, they correlated liver stiffness with icgr15 and liver biochemistry, to determine its reliability in predicting postoperative outcomes. liver stiffness correlated well with icgr15 in liver resection patients, and predicted early postoperative complications and was recommended, to provide 'better prognostic information for patients undergoing resection.' 3. megx test: evaluates microsomal function and is a measure of the formation of the metabolite monoethylglycinexylidide (megx) after injection of a bolus of lidocaine and is evidence of the conversion rate of lidocaine by hepatic cytochrome p450. a value ≤25 μg/l is related to plf in patients with cirrhosis [ 47 ] . 4. technetium-99m galactosyl human serum albumin (99m tc-gsa): gsa scintigraphy studies [ 48 ] have been reported to be useful for predicting the functional reserve of the liver and superior to icg. 99m tc-gsa is a scintigraphy agent that binds specifi c hepatic receptors, and can be used to assess the functional hepatocyte mass and thus the hepatic functional reserve in various physiological and pathological states. unlike icg it is not affected by the haemodynamic status. various scoring systems are in vogue to assess the suitability and risk stratifi cation of hepatic resections in patients with cirrhosis. the ctp and meld score were initially designed for other prognostications, and their validity in predicting plf has been the objective of many trials. the results are inconsistent [ 1 , 2 , 5 , 49 ] . in general, it is well accepted that a ctp class c patient is not suitable for any liver resection and those in class b are suitable for only minor liver resections [ 49 ] . schroeder et al. [ 50 ] reported the superiority of ctp over the meld score in predicting 30-day morbidity and mortality after hepatic resections. however, other studies validate the meld score as a reliable predictor. a meld score above 11 in patients with cirrhosis could predict plf accurately [ 51 ] . 3d ct reconstructions or magnetic resonance imaging (mri) reconstructions are now used exclusively for volumetric analysis and predicting flr [ 52 ] . calculation of flr however remains a cause of disagreement and various techniques and calculations are in vogue. 3d reconstructions allow delineation of the hepatic veins, congestion volumes, exact tumour localization and facilitates virtual resection planning. however, imaging at present over estimates the flr, and different formulae are in use, in an attempt to account for this error. the crux of any imaging or formula used is to ensure that the flr is compatible with a smooth recovery and it is vital to assess the functional status of the flr. addition of preoperative hepatobiliary scintigraphy and ct volumetric measurement were performed by dinant et al. in preoperative patients [ 48 ] to assess the accuracy of risk assessment for postoperative morbidity, liver failure and mortality. they concluded that using hepatobiliary scintigraphy with preoperative measurement of 99mtc-mebrofenin uptake in the flr, proved more valuable than measurement of the flr on ct alone in assessing the risk of plf. keeping the pathophysiology in mind, plf can be prevented by a two-pronged strategy: protect the parenchyma against damage and increase the parenchymal volume. 1. ischaemic preconditioning: after a brief period of infl ow clamping, reperfusion is allowed, prior to the prolonged infl ow clamping ischaemic insult (10 min of ischaemia and 10 min of reperfusion). it increases tolerance to the subsequent prolonged ischaemia and adenosine 5-triphosphate (atp) depletion by exposing the parenchyma to brief intervals of anoxia and reperfusion before the fi nal resection. it acts by presumably controlling iri and retards the complement cascade. this reduces reperfusion injury particularly in steatotic patients. clavien et al. [ 53 , 54 ] did the initial trials and demonstrated a two-fold reduction in the postoperative serum transaminase levels. a reduced mass of apoptotic cells was noted on histopathology. a randomized trial by chouker et al. [ 55 ] comparing ischaemic preconditioning and continuous clamping, showed stable cardiovascular haemodynamics, lowering the need for adrenaline/noradrenaline after liver reperfusion. additionally, a recent cochrane analysis observed no statistically signifi cant difference in the mortality, liver failure, blood loss or haemodynamic changes [ 56 ] . however, length of hospital stay was signifi cantly lower in the ischaemic preconditioning group. 2. intermittent vascular clamping: consists of repeated periods of 15 min of infl ow clamping followed by 5-min reperfusion phases. belghiti et al. [ 57 ] reported that in contrast to the presumption, blood loss was signifi cantly more in the intermittent clamping group. acute phase liver enzymes and transaminase levels were signifi cantly higher in the continuous portal triad clamping group than in the intermittent portal infl ow clamping group when livers with chronic liver disease were included. postoperative sb levels in cirrhotics were also signifi cantly higher in the continuous infl ow clamping group compared to the intermittent portal infl ow cohort. they concluded that livers with chronic disease do not tolerate continuous vascular clamping as well as normal livers. 3. avoiding infl ow clamping: the cochrane meta-analysis published in 2009 [ 56 ] , based on three randomized trials, revealed statistically insignifi cant decreased blood loss with vascular clamping, when compared with no clamping. total vascular occlusion is to be avoided unless resection is required at the cavohepatic junction when it cannot be avoided. 4. hypothermic liver preservation: interest in decreasing warm ischaemia of transported livers has spawned experiments into isolating the infl ow and perfusing cold preservative into the future liver remnant, which is immersed in crushed ice to maintain the core temperature of the liver at 4 °c. hypothermic liver preservation when combined with total vascular exclusion attenuates iri. in situ cold isolation techniques are still in their infancy and remain isolated case reports used in special situations with total vascular exclusion/cardiopulmonary bypass [ 58 ] . 5. pharmacological preconditioning: it has been reported in a clinical study that the use of isofl urane before clamping the infl ow protected the liver from iri [ 59 ] . preconditioning with sevofl urane also signifi cantly reduced postoperative transaminase levels and the overall incidence of postoperative complications was reduced especially in patients with fatty livers. inhaled nitric oxide has also been cited to 'signifi cantly decreasing the length of hospital stay, improving serum transaminase levels and coagulation times, and reducing the number of apoptotic hepatocytes.' a similar effect has been demonstrated with preoperative administration of 500 mg of methylprednisolone [ 59 ] . during major resections, intraoperative preconditioning with 600 mg of alpha-lipoic acid also reduced markers of hepatic damage by infl ow occlusion. lesurtel et al. [ 60 ] have made the following recommendations: with better understanding of intrahepatic anatomy, newer energy devices and maintenance of a low central venous pressure during parenchymal transection, vascular clamping cannot be systematically recommended (level a). portal infl ow clamping reduces blood loss and use of blood products but does not infl uence morbidity (level a). among various methods of infl ow exclusion, they support intermittent clamping as better tolerated especially in patients with chronic liver disease (level a). ischaemic preconditioning has been recommended for steatotic patients (level a). intermittent clamping is preferred over ischaemic preconditioning in major liver resections and prolonged surgery (level a). currently no evidence supports or refutes the use of ischaemic preconditioning in donor liver retrievals during living donor transplants. portal vein ligation (pvl/pve) by ligation/embolization : pvl is usually performed by surgical ligation or percutaneously by transhepatic portal vein embolization (pve). pve induces apoptosis in the ipsilateral lobe, and hypertrophy and hyperplasia of the contralateral lobe. this increases the functional volume of the flr, thus obviating hyperperfusion in a sfss scenario. it is also a precursor phenomenon and predicts the regenerative response in the future remnant. pve can increase contralateral lobe mass by up to 20 %, with the peak in growth occurring within 2-4 weeks of the procedure [ 61 , 62 ] . the failure of the liver to enlarge after pve is indicative of patients with impaired regenerative capacity in whom major resection should be avoided [ 62 ] . to prevent a surge in tumour growth due to enhanced differential hepatic artery fl ow to the tumour, local control by ablation/chemotherapy are also added as well as biliary drainage. hyperperfusion of a small for size graft is often modulated by splenic infl ow control by ligation/embolization or shunting. plf is determined by haemodynamic parameters of the hepatic circulation and, specifi cally, by a portal blood fl ow that, when excessive for the volume of the liver parenchyma leads to an infl ow/outfl ow mismatch causing pressure build up in sinusoids with a leaking capillary bed in the liver. perisinusoidal and periportal haemorrhage occurs within a few minutes in a major hepatic resection as well as after the reperfusion of a sfss graft. late effects occur due to hepatic arterial and biliary epithelial hypoxia [ 6 ] . alpps--the 'associating liver partition and portal vein ligation in staged hepatectomy' (alpps) strategy is one of the surgical innovations used to manage flr volumes [ 63 ] . the alpps approach is proposed to induce rapid hypertrophy of the flr in patients with hcc and whose preoperative volume does not allow a safe resection. the procedure entails the combination of in situ splitting of the liver along the cantlie's line and ligating the portal vein on the side of the tumour. subsequently the second stage hepatectomy is done. the median flr volume increase was 18.7 % within 1 week after the fi rst phase and 38.6 % after the second [ 64 , 65 ] . recently, a number of trials comparing allps and post-pve liver resections have been published [ 66 -68 ] . alpps has shown higher hypertrophy rates compared to pve/pvl (40-80 % within a week compared to 8-27 %). however, alpps has higher sepsis rates (16-64 % of patients) and mortality rates (12-23 %). alpps facilitates an early removal of tumours whilst waiting for an adequate flr. due to the high morbidity rates there has to be a strict criteria for selecting a patient for alpps as pve has shown comparable flr hypertrophy rates. downsizing tumours with chemotherapy, local ablative techniques and embolization is yet another strategy to gain functional reserve volume when planning resections in livers, which are likely to have a low flr. the typical clinical features of plf parallel the clinical picture of alf: coagulopathy, raised sb and encephalopathy. in addition renal failure, respiratory compromise, hypotension and features of sepsis may be present. this clinical presentation parallels the presentation of alf, but is closer to that of subacute liver failure than to that of hyperacute liver failure [ 69 ] . with deteriorating liver function the patient will develop hyperbilirubinaemia and coagulopathy which in particular is a poor prognostic marker [ 70 ] . if plf is detected in a patient, it should be scored by the isgls system [ 2 ] . plf grade a: should be monitored well, but may not require specifi c treatment. plf grade b: it has to be evaluated if the patient should be placed in an intensive care unit (icu) plf grade c: need icu care rahman et al. [ 71 ] have cited a daily measurement of serum c-reactive protein (crp) as an early warning indicator of patients likely to develop plf. patients prone to developing plf had a lower crp level on pod 1 than patients who did not develop plf. a prognostic utility of postoperative crp was a serum crp <32 g/dl, which was an independent predictor of plf. initial treatment of plf is supportive: ventilatory support, vasopressors, renal replacement therapy and anti-sepsis protocols. controlling coagulopathy and supporting nutrition are the other mainstays. patients of liver resections are normally monitored closely in the intensive care or high dependency units. it is normal for sb levels and the inr to rise in the fi rst 2-3 days postoperatively. sb concentration above 50 μmol/l (3 mg/dl) or inr greater than 1.7 on or beyond 5 days suggests liver dysfunction. sepsis is indicated by raised serum lactate. the use of antibiotics in patients suffering from alf is associated with a signifi cant decrease in sepsis and this may also be of benefi t in patients suffering from plf [ 72 ] . overall the management of plf is along the lines for alf. identifying and controlling sepsis is the key to managing plf [ 73 ] . trials have shown that prophylactic antibiotics after liver resection do not lead to a reduction in plf or sepsis [ 74 ] . alf management guidelines propose that broadspectrum antibiotics should be administered empirically to patients who progress to grade 3 or 4 hepatic encephalopathy, renal failure and/or worsening systemic infl ammatory response syndrome (sirs) [ 73 , 75 ] . many clinicians strive to provide heptoprotection with n-acetylcysteine (nac) [ 76 ] . however, no evidence exists that it has any benefi t in alf. nac is advocated in the management of paracetamol-induced alf and its use in non-paracetamol hepatic failure remains controversial. sporadic papers do mention a benefi t for nac and it is used empirically for its anti-oxidant role. early stage non-acetaminophen patients with alf benefi t from intravenous nac. patients with encephalopathy grades 3 or 4 do not benefi t from nac and will require emergency liver transplantation. nac is commenced in a loading dose of 150 mg per kg per hour for 1 h followed by 12.5 mg per kg per hour for 4 h and 6.25 mg per kg per hour for the remaining 67 h. the rest of the management is along supportive care protocols as shown in table 3 .2 . hepatocyte transplantation has been used in trials as an effort to rejuvenate existing liver function. intrahepatic hepatocyte infusion [ 77 ] has been used successfully to treat patients with certain metabolic disorders of the liver. results in liver failure (alf and plf) have been poor due to insuffi cient delivery of viable and sustainable functional hepatocytes. though, liver support systems have been available for some years now, their high operational cost and sepsis rates have not improved. these include: 1. molecular absorbent recirculating system (mars) 2. modifi ed fractionated plasma separation and adsorption (prometheus) 3. bioartifi cial liver (bal) and extracorporeal liver assist device (elad). extracorporeal systems are predominantly sustained on albumin dialysis, and bioartifi cial devices are bioreactors with permeable membranes containing hepatocytes, either synthetic or natural. very few trials exist in the setting of plf, with the exception of one case series which showed no signifi cant benefi t [ 78 , 79 ] . they are not currently recommended in the medical management of alf. because their actual place in the global fi eld of acute or acute on chronic liver failure remains to be determined, their role in plf is undefi ned. however, because outcomes in plf are morbid, it is worthwhile to continue to investigate the benefi cial roles of these devices [ 80 -82 ] . the use of a rescue hepatectomy (removal of necrotic portions or segments) in patients suffering from plf may be of value when faced with a very sick patient. it is based on the concept that the 'necrotic liver' is the source of unknown humoral substances that contribute to sirs [ 83 ] . the effi cacy of orthotopic liver transplantation (olt) for plf has recently been reported [ 84 ] . in this paper, otsuka et al. did a retrospective review of 435 patients who had a liver resection between 1990 and 2004. nine of them (2 %) developed plf of which seven were offered olt at a mean of 25 days post resection. indications for resection included malignancies and benign disease. patients developing plf had signifi cantly altered biochemical and coagulation parameters manifesting on pod 2 and had the classical triad of coagulopathy, hyperbilirubinaemia and encephalopathy. there was no mortality following olt, though one patient required a retransplant. the mean survival with and without olt was 42.2 and 1.4 months, respectively (p = 0.03). they concluded that all patients ( n = 4) who suffered from plf but were not considered suitable for liver transplantation, died, while all those undergoing olt survived ( n = 7). olt allows salvage of an otherwise fatal condition. however, no defi nitive criteria are available for emergency liver transplantation for plf. olt as a rescue for plf has been gaining in popularity, governed only by the availability of organs or suitable donors in an emergency. the principles of transplantation should however be adhered to: in most instances the indications for olt after plf should be limited to patients who fulfi ll the primary indication for hcc-pre-resection tumour burden within the milan criteria [ 85 ] . patients with metastatic disease, beyond milan criteria, advanced medical and anaesthetic comorbid conditions, and poor functional status, should not be candidates for olt. olt is the only radical surgical remedy that improves survival in patients with end-stage liver disease. however, patients suffering from plf are rarely eligible for liver transplantation because of tumour characteristics or comorbid conditions. the incidence of plf after a major hepatic resection averages 8 %. an abnormal flr is the main cause for the pathogenesis of plf. other reasons, which worsen plf, are hepatic parenchymal congestion, iri and postoperative sepsis. these can exist singly or in combination. risk factors for the development of plf are small flr, blood loss, malnutrition, diabetes mellitus and active liver disease. a comprehensive preoperative assessment includes evaluation of liver volume, anatomy and function. a physical examination followed by appropriate clinical tests will identify patients at risk of developing plf. the patient's liver status, hepatic reserve potential and functional aspect need to be investigated along with the metabolic and haematological derangements, which may lead to postoperative liver failure. corrective measures should be applied when-ever possible, as curative treatment options are limited. the risk of plf is high when flr is below 25-30 % in livers without cirrhosis and below 40 % in livers with preexisting liver disease. pve and/or two-stage hepatectomy are options when surgery cannot be deferred. additional liver damage may be prevented by intermittent clamping techniques, though used with caution in steatotic livers. management principles are on the lines of management of alf with support of liver, cardiorespiratory and renal support function. control of sepsis is an important aspect. emergency liver transplantation has shown promise as a remedy for plf. postoperative liver failure after hepatectomy is a potentially life-threatening complication. fortunately, even though the rates of liver resection are increasing, the mortality from the procedure is decreasing. the operative mortality at specialized centres varies from 0 to 6 % [ 86 ] . however, the morbidity continues to be high. the defi nition of post-hepatectomy liver failure has not been standardized. three defi nitions are currently being followed. two of these (the 50-50 criteria and the international study group of liver surgery [isgls] criteria) have been mentioned by the authors [ 2 , 5 ] . the latter not only diagnoses the condition, it stratifi es the severity into 3 categories. grade a does not need any change in management strategy, grade b may be managed without any invasive intervention while grade c needs alteration in management including invasive intervention. the isgls criteria have been validated by one study [ 87 ] . the study detected post-hepatectomy liver failure using this criteria in 11 % of patients-8 % had grade a, 72 % grade b and 20 % grade c. the mortality in these patients was 0 % in grade a, 12 % in grade b and 54 % in grade c. thus, the grading has been shown to correlate with mortality. however, in 2 separate reports, 41 and 67 % of patients fulfi lling the isgls criteria recovered completely highlighting the importance of adequate management rather than just using the defi nition [ 4 , 5 ] . the third defi nition described by jarnagin et al. is simple. they defi ne post-hepatectomy liver failure as high bilirubin in the absence of biliary obstruction or leak occurring with ascites and coagulopathy with or without encephalopathy [ 88 ] . the authors have discussed the possible pathophysiological factors in detail. to this i may add the possibility of hepatic venous outfl ow tract obstruction as described by lhuaire et al. [ 89 ] the authors documented outfl ow obstruction with contrast enhanced ct, doppler ultrasound, cavography and assessment of pressure gradient between the hepatic vein and inferior vena cava. they managed the patient successfully with placement of a metallic stent in the left hepatic vein and documented reduction in the hepatic veininferior vena cava pressure gradient. the patient improved thereafter and was discharged. hepatic venous outfl ow obstruction following hepatectomy has been reported in experimental studies in rats [ 90 ] . various risk factors have been indentifi ed and have been discussed elaborately by the authors. this should allow surgeons to select proper patients for extensive resection to avoid post-hepatectomy liver failure. the main issue is the adequacy of the residual liver volume and presence of cirrhosis. while 30 % of residual volume is adequate for extended resections in a normal liver, it is grossly inadequate for a cirrhotic patient in whom at least 40 % of liver volume is required. this brings us to the question of increasing future liver remnant by various strategies. both portal vein embolization (pve) and liver partition with portal vein ligation are effective. however, the simplicity of pve makes it the most used approach. it has the advantage of the tumour biology being assessed during the waiting period of 4-6 weeks. if the tumour progresses especially with chemotherapy the patient should not undergo liver resection. on the question of properly selecting patients for major resection in cirrhosis the prevailing guidelines should be followed. these are child's a status, platelet count above 100,000/cm, absence of clinically signifi cant portal hypertension, a future liver remnant of 40 %, and the 15 min indocyanine green (icg) clearance of no more than 15 %. a number of tests are available to assess various functional aspects of the liver but they are cumbersome, not available at most centres and more importantly are not accurate with the exception of 15 min icg clearance. even this is not done routinely in the west. patients with features of postoperative liver failure by either the '50-50' criteria or the isgls criteria have circulatory changes as seen in septic shock such as vasodilatation, increased vascular permeability resulting in accumulation of fl uid in the third space, tachycardia and increased cardiac output leading eventually to hypotension. coagulapathy too is seen commonly. altered kidney function usually occurs due to hepatorenal syndrome or acute tubular necrosis because of sustained hypotension causing compromised renal perfusion. with worsening renal function, fl uid accumulates in the periphery or pulmonary bed. this often necessitates renal replacement therapy. with improvement in renal function the liver function also improves. hepatic encephalopathy is seen more commonly in patients with renal failure because serum ammonia cannot be cleared by either the kidney or the liver. the presence of sepsis is another problem. hypotension resulting from sepsis is detrimental to liver regeneration essentially due to ischaemia. endotoxins produced in sepsis interfere with kupffer cell activation and its function. one should not forget that following hepatic resection there is a depletion of kupffer cells in the liver. thus following hepatectomy there is a higher incidence of sepsis, and sepsis interferes with hepatic regeneration [ 91 ] . therefore, every attempt must be made to avoid sepsis. execution of the surgical procedure with utmost care avoiding excess blood loss, tissue necrosis, haematoma formation, prolonged ischaemia, etc. should minimize infection. management of post-hepatectomy liver failure has been duly addressed. i will emphasize on the fl uid and nutrition therapy. following hepatectomy the urine output may be low but it is expected. it is not necessary to give bolus fl uid therapy in these patients unless they are grossly oliguric. cirrhotic patients should probably be given parenteral nutrition in place of dextrose solutions because parenteral preparations have branch chain amino acids, dextrose, medium chain triglycerides, phosphates and vitamins. usually up to 2 l of such fl uid is necessary. if volume defi cit exists, it should be managed with additional fl uids. parenteral nutrition in post-hepatectomy liver failure helps liver regeneration and decreases septic complications. the addition of phosphate is extremely important because it is necessary for both energy production and liver regeneration. moreover the phosphate level goes down considerably after major liver resection [ 92 ] . since the available phosphate solutions contain potassium, these may need to be stopped if the serum potassium level is high. the phosphate will then need to be replaced orally and the oral forms have sodium. hence, the serum sodium would need to be monitored. posthepatectomy liver failure posthepatectomy liver failure: a defi nition and grading by the 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dialysis: comparison of albumin dialysis and fractionated plasma separation liver derived pro-infl ammatory cytokines may be important in producing intracranial hypertension in acute liver failure postresection hepatic failure: successful treatment with liver transplantation management of hepatocellular carcinoma defi ning postheptectomy liver insuffi ciency: where do we stand? post operative course and clinical signifi cance of biochemical blood tests following hepatic resection improvement in perioperative outcome after hepatic resection: analysis of 1,803 consecutive cases over the past decade post-hepatectomy liver failure: should we consider venous outfl ow? impact of hepatic vein deprivation on liver regeneration after major hepatectomy loss of nf-kb activation in kupffer cell-depleted mice impairs liver regeneration after partial hepatectomy the clinical implications of hypophosphataemia following hepatic resection or cryosurgery key: cord-271635-tydlyc1q authors: abdel-hamid, nabil m.; abass, shimaa a.; mohamed, ahmed a.; muneam hamid, daniah title: herbal management of hepatocellular carcinoma through cutting the pathways of the common risk factors date: 2018-11-30 journal: biomedicine & pharmacotherapy doi: 10.1016/j.biopha.2018.08.104 sha: doc_id: 271635 cord_uid: tydlyc1q abstract hepatocellular carcinoma (hcc) is considered the most frequent tumor that associated with high mortality rate. several risk factors contribute to the pathogenesis of hcc, such as chronic persistent infection with hepatitis c virus or hepatitis b virus, chronic untreated inflammation of liver with different etiology, oxidative stress and fatty liver disease. several treatment protocols are used in the treatment of hcc but they also associated with diverse side effects. many natural products are helpful in the co-treatment and prevention of hcc. several mechanisms are involved in the action of these herbal products and their bioactive compounds in the prevention and co-treatment of hcc. they can inhibit the liver cancer development and progression in several ways as protecting against liver carcinogens, enhancing effects of chemotherapeutic drugs, inhibiting tumor cell growth and metastasis, and suppression of oxidative stress and chronic inflammation. in this review, we will discuss the utility of diverse natural products in the prevention and co-treatment of hcc, through its capturing of the common risk factors known to lead to hcc and shed the light on their possible mechanisms of action. our theory assumes that shutting down the risk factor to cancer development pathways is a critical strategy in cancer prevention and management. we recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid hcc progression and decrease its global incidence. the mortality rate due to hepatocellular carcinoma (hcc) increased rapidly during the past decade. unluckily, the clinically satisfactory and successful treatment for hcc patient is still absent [1] . several risk factors are involved in hepatocarcinogenesis like non-alcoholic fatty liver disease (nafld), hepatitis b virus (hbv) and hepatitis c virus (hcv) infection, alcoholism, obesity, aflatoxin b1, iron accumulation and diabetes [2] . there are several protocols used in the treatment of hcc, including, surgical resection, ablation, chemotherapy and embolization. the use of these methods is limited due to their side effects and the development of resistance to the available chemotherapy and their complexities. due to the limited treatment options to hcc, other than surgery and the poor prognosis of the disease, there is a critical need for additional therapies to enhance the survival or the quality of life. complementary and alternative medicine (cam) is considered as one way that may improve the anticancer drug efficacy and reduce their toxic effects [3] . the use of herbal medicines can be traced back to more than 4000 years ago in ancient china and egypt. over recent decades, an increasing number of herbal products, including medicinal herbs and phytochemicals, have been used for treating chronic liver diseases worldwide due to cost effectiveness, higher safety margins, long-lasting curative effects and few adverse effects. according to the previous studies, medicinal herbs and phytochemicals could protect the liver by several mechanisms such as eliminating the virus, blocking fibrogenesis, inhibiting oxidative injury and suppressing tumorigenesis [4] . in this review, we discuss several factors that lead to hcc development, focusing on the role of different herbal medicines that used in the treatment of hcc by alleviating these risk factors. is similar to the fatty liver damage that caused by alcoholism, but, it happens in non-alcohol abuse people. nafld is characterized by the accumulation of triglycerides within hepatocytes, which is usually associated with metabolic syndrome and obesity [5] . the prevalence of nafld was established by the histological features found in ∼70% of obese individuals suffering from steatosis, while steatosis was present in ∼ 35% of lean individuals. nafld was also present in about18.5% of obese individuals and in about 2.7% of leans [6] . fatty liver is also found in about 13-22% of lean non-alcoholic individuals through several studies based on ultrasound imaging [7, 8] . nafld is present in 20-35% of adult individuals and 5-17% of children in the western world [9] . nafld is considered as one of the important reasons leading to chronic liver diseases in hong kong (∼27.3%) [10] and china (∼15%) [11] . this is because of the high-fat content in the modern diet and individual's lifestyle. nafld is considered as one of the most important reasons that cause chronic liver disease in developing and developed countries. nafld increases the risk of hepatocarcinogenesis similar to other pot cirrhotic liver diseases. hcc is now the end stage as a leading cause of obesity-related cancer deaths in middle-aged men in the usa [12] . an increasing number of case reports showed that hcc arises in non-cirrhotic individuals suffering from nafld [13] . other hcc risk factors may be synergistically involved in hcc development besides nafld, such as alcoholic liver injury and chronic hepatitis c. several mechanisms are involved in nafld-related hcc development ( fig. 1 ) [14] [15] [16] . obesity participates in increasing the risk of cancer development through a low-grade, chronic inflammatory impact [17, 18] . the expansion of adipose tissue stimulates the generation of pro-inflammatory cytokines such as tumor necrosis factor (tnf) and interleukin-6 (il-6) [19] . tnf and il-6 derived from adipose tissue play an important role in hcc development. this role has been supported in an experimental model, assuming that obesity enhances the growth of diethylnitrosamine-induced malignant liver tumor in mice [20] . the prevalence of hcc due to nafld is increasing around the world [21] , where, 4-22% of hcc patients in western countries are attributable to nafld [22] . in asia, viral hepatitis remains endemic, so that, 1-2% of hcc patients are attributed to nafld [23, 24] . wolfberry, a famous traditional chinese supplement or drug, is the fruit part of lycium barbarum plant, family solanaceae. it has valuable benfits in both eyes and liver [26] . the most important part of wolfberry is the lycium polysaccharide portion(lpp), which has a numerous biological actions, like immunoregulation, antioxidant effect, neuroprotection, control of glucose metabolism and anti-tumor activities. the lymphocyte number, interleukin-2 and immunoglobulin g level, were found to be increased upon the intake of polysaccharide juice in human beings in one of the clinical trials. it was reported also that it increases the levels of serum antioxidants and decreases the lipid peroxide level [27, 28] . in the liver, lpp was found to inhibit hepatocyte proliferation and induce apoptosis of hepatoma cells, which indicate its possible application as anti-tumor [29, 30] . another study demonstrated that lpp causes a restoration of the activities of antioxidant enzymes and reduction of oxidative stress products caused by high-fat diet induced liver injury [31] . the co-treatment of lpp with ethanol administration markedly enhanced the liver injury in an alcohol-induced liver injury rat model by decreasing the oxidative stress and the accumulation of lipid in the liver [32] . in acute liver injury, lpp was found to keep the normal hepatic histology, decrease the hepatic inflammation/chemoattraction, stimulate the partial regeneration of the liver through the nuclear factor kappa b (nf-κb)-dependent pathway, and reduce the oxidative stress when used prior ccl 4 intoxication in mice [33] . lpp is helpful in nafld due to its useful properties in decreasing the inflammation and the oxidative stress. the co-treatment with lpp, orally, in nafld in rats, showed a significant improvement in the hepatic histology, reduction in the fibrosis, oxidative stress, inflammation, accumulation of fats and apoptosis, through modulating the transcriptional factors nf-κb and activator protein-1 (ap-1). furthermore, the uptake of lpp for long-term did not have any unwanted side effects on the liver of healthy rats. so lpp can be useful in nafld treatment with minimal side effects [25] . green tea (camellia sinensis) leaves, has been reported to be used in the prevention of liver diseases. the origin of green tea is china, which then distributed to asian countries, including korea, vietnam and japan. in the last years, green tea also spread to western countries that traditionally consume black tea. the beneficial or therapeutic properties of green tea extracts have been reported by several studies. the major polyphenol of green tea, epigallocatechin-3-gallate (egcg), was used in ccl 4 -treated mice and showed a significant therapeutic potential in hepatic damage, inflammation and oxidative stress induced by ccl 4 in a dose-dependent manner at both biochemical and histological levels [34] . it was also reported that co-treatment of the whole green tea extract with alcohol administration showed an effective reduction of the hepatic oxidative stress and reduced form of nicotinamide adenine dinucleotide phosphate (nadph) oxidase systems in experimental alcohol-induced liver injury [35] . green tea extract also showed an enhancement of the nash features, such as oxidative stress, inflammation and lipid accumulation in obese mice [36] . a recent study also demonstrated that green tea extract showed significant improvement in inflammatory, chemical, metabolic, and radiological parameters of nafld patients who were dyslipidemic and non-diabetic [37] . it also showed an improvement in the liver enzymes of nafld patients in another study [38] . numerous in vitro studies demonstrated the chemoprevention and anti-tumor properties of green tea in hcc [39] . the growth and cell proliferation of hcc-derived cells are inhibited by egcg by induction of apoptosis [40] . the human hcc cell line, hepg2, growth was also inhibited by egcg, through suppressing the phosphorylation of insulinlike growth factor-1 receptor (igf-1r) and reducing the activation of its signaling molecules like extracellular signal-regulated kinase (erk), stat-3, akt and gsk-3β [41] . drinking of egcg caused a significant inhibition of liver cell adenoma development in contrast to the egcguntreated control group. this is related to decreased phosphorylation of erk, akt and igf-1r proteins, activation of the hepatic amp-activated kinase protein and the treatment of liver steatosis. the administration of egcg in drinking water also caused a significant reduction of serum levels of insulin, igf-1, igf-2, tumor necrosis factor (tnf)-α, and free fatty acid. it also caused a decrease in the hepatic expression of tnf-α, interleukins(il-1β,il-6), and il-18 mrnas [42] . the development of glutathione s-transferase placental form (gst-p + ) foci was also inhibited after the administration of egcg in drinking water through the reduction of hepatic fibrosis, triglyceride content, inflammation, oxidative stress and inhibition of excessive hepatocyte proliferation [43] . resveratrol (rsv; 3, 5 4′-trihydroxystilbene), a phytoalexin that extracted from red grapes, is considered as one of the most accepted and recognized herbal derivatives worldwide as it has a powerful antioxidant and anti-inflammatory properties [44] . recently, rsv has been found to be helpful in the treatment of nafld. when rsv is given after nafld induction by using high fat-diet, it causes a reduction of lipogenic genes as srebp-1c and fas [45] . the treatment with rsv also reduced the inflammation and oxidative stress in rats [46] . rsv causes a dysregulation in the metabolism of lipids in nafld through sirtuin 1 (sirt1) pathway [47] and the up-regulation of hepatic low-density lipoprotein receptor [48] . rsv increases apoptosis in hcc cells, which is associated with the reduction of hexokinase 2 expression. additionally, rsv improved the inhibition of cell growth induced by sorafenib in aerobic glycolytic hcc cells. the inhibition of hexokinase 2 by rsv can be considered to be a new trend to treat hcc and prevent its progression [49] . rsv was also found to decrease the expression of myosin light chain kinase (mlck), which inhibited liver tumorigenesis and promoted cell apoptosis in hcc rats induced by dena. the expression of mlck was found to be higher in hcc rats than normal rats, which is responsible for the proliferation and anti-apoptotic properties [50] . the latin word "inflammation" means set a light or ignite, which describes exactly its effect on cancer. inflammation increases the resistance to chemotherapy and promotes oncogenes and genes that convert healthy cells to tumors. it also stimulates cancer cell spreading and improves the cancer cells' ability for angiogenesis. because cancer is defined as an inflammation, the anti-inflammatory drugs can be useful in treating cancers as the relation between cancer development and inflammation has been appreciated [51, 52] . the hepatic damage may be due to either chronic or acute inflammation. in response to inflammation, several kinds of hepatic cells are activated, such as hepatic stellate cells (hscs), liver sinusoidal endothelial cells (lsecs), kupffer cells (kcs) and dendritic cells (dcs) to produce several types of cytokines, immune mediators and chemokines. one of the important pro-inflammatory cytokines is interleukin-6 (il-6) that can inhibit apoptosis and tissue inflammation [53] . the inflammatory response mediates a defense mechanism against the microbial infection and stimulates tissue regeneration and repair. there is a relation between the inflammation and cancer development as chronic inflammation stimulates the development of dysplasia. about 15% of cancer occurrence is associated with microbial infection. in immunocompetent patients, chronic inflammation, like hepatitis b and c viral infection or human papilloma virus may result in the development of hepatocellular and cervical carcinoma, respectively [54] . cancer also may result from an opportunistic infection like kaposi's sarcoma, which results from human herpes virus (hhv)-8 infection. inappropriate immune responses to microbes may also lead to cancer development as gastric cancer, which may result from chronic inflammation due to helicobacter pylori colonization. the long-standing inflammatory bowel disease may lead to colon cancer. long-term exposure to asbestos, silica and cigarette smoke, may lead to chronic irritation and subsequent inflammation that result in cancer development [51, 52] . the promotion of tumor cells requires both the survival of the initiated cells and their expansion. numerous inflammatory mediators like chemokines, eicanosoids, and cytokines have the ability to stimulate the proliferation of both untransformed and tumor cell [51] . inflammation plays an important role in tumor growth through angiogenesis mediation. it also plays a critical role in other aspects of tumor progression like metastasis and tissue invasion. matrix metalloproteinases and their inhibitors are important for remodeling of extracellular matrix and angiogenesis, which enhances vascular invasion of migrating cells [55] .the mechanisms contributed to the role of inflammation in hepatocarcinogenesis was showed in fig. 2 [56] . xanthorrhizol (xnt), a sesquiterpenoid complex, obtained from curcuma xanthorrhizza rhizome, family zingiberaceae. the anti-inflammatory effect of xnt was reported for the first time in lipopolysaccharide-mediated mouse leukemic monocyte macrophage cell raw 264.7. it caused a significant reduction in the activities of inducible nitric oxide synthase (inos) and reduced cyclooxygenase-2 (cox-2), through the inhibition of nitric oxide (no) and prostaglandin e2 (pge 2) production [57] . xnt was also found to inhibit pro-inflammatory cytokine interleukin-6 (il-6), tnf-α, inos and cox-2 in activated microglial cells [58] . the anti-inflammatory property of xnt was also postulated in another study as it blocks the inflammatory and neurogenic pain response in pain test that induced by formalin in rats [59] . xnt has also anticancer activities, which may be due to its antiinflammatory effect by inhibiting the activity of nf-kb, cox-2 and inos release [60] . it also inhibited the tumor development and formation in different in vivo studies. it decreased the expression of cox-2, ornithine decarboxylase and suppressing nf-kb signaling activity. an in vivo study also demonstrated that xanthorrhizol has anti-metastatic activity through inhibiting matrix metallopeptidase 9 and cox-2 in a mice lung metastasis model [61] . it has a potent anti-proliferative effect on hepg2 cells through apoptosis induction via bcl-2 family members [62] . berberine is a small alkaloid molecule isolated from coptidis rhizome. it possesses anti-inflammatory and anticancer activities [63] . it down-regulates numerous hepatic pro-inflammatory genes such as il-6, serum amyloid a3 (saa3), nf-kb and tnf-α. these genes play a vital role in steatohepatitis development [64] . it was reported that berberine has an anti-inflammatory activity for hepatic cells in different animal models. it has also been showed to decrease tnf-α and cox-2 expression in cyclophosphamide-induced hepatotoxicity in a rat model [65] . it also has the ability to inhibit tnf-α and il-6 production in hepg2 cells. the anti-inflammatory effect of berberine may be due to the inhibition of erk1/2 activation [66] . its anti-inflammatory effect was postulated through the inhibition of lps-induced inflammatory response in macrophages [64] . berberine has anti-cancer activity on the human hcc cell lines through the induction of apoptosis and inhibition of tumor cell proliferation [67] . berberine induces both cell death and apoptosis in hepg2 cells. this is related to the down-regulation of cd147, which is highly expressed in hcc cells [68] . it was shown to selectively inhibit the human hepatocellular cancer cell growth through the induction of ampk-mediated caspase-dependent mitochondrial pathway cell apoptosis in addition to suppressing p53 [69] . the expression of p53 was found to be up-regulated by berberine through suppression of mdm2, the inner p53 inhibitor, at the post-transcriptional level [70] . the combination of berberine and vincristine showed a helpful effect against hepatoma cell lines through the potentiation of the pro-apoptotic effect of the single drug [71] . alpinia officinarum, known as lesser galangal, belongs to family zingiberaceae. it has a variety of pharmacological actions as antioxidant, anti-inflammatory, antimicrobial, antiemetic and cytotoxic properties [72] . different mechanisms are involved in the anti-inflammatory effect of alpinia officinarum, including the regulation of nf-κb, mapks pathway and the inhibition of prostaglandin e2 synthase and cox-2, the important enzymes involved in the inflammation. this effect is usually due to its content of diarylheptanoids and flavonoids [73] . two compounds of alpinia officinarum rhizome extract, galangin and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone, exhibited antioxidant and anti-inflammatory activities due to their phenolic content. these compounds showed a high affinity toward cox-2 active site through a molecular docking study [74] . our recent study also demonstrated that five compounds isolated from the alpinia officinarum rhizome extract showed a powerful anti-inflammatory effect on hepg2 cells stimulated by lipopolysaccharide. these compounds are galangin, isorhamnetin, two diarylheptanoids and kaempferide. these compounds down-regulated the gene expression of il-6, il-1β, pro-inflammatory cytokines and tnf-α in a dose-dependent manner. this indicates that these isolated compounds may be a promising treatment for other inflammatory diseases [75] . alpinia officinarum rhizome extract and its components showed anticancer activities against numerous cancer cell lines, such as breast, neuroblastoma, lung, and liver [76] [77] [78] . our previous study suggested that alpinia officinarum rhizome extract can be used as a promising natural chemopreventive agent against hcc in rats. it is also a helpful chemosensitizing agent when used in combination with cisplatin in the treatment of hcc. additionally, alpinia officinarum rhizome extract improved hepatic functions and decreased alpha-fetoprotein concentration in experimental hcc model. it also protected the hepatic tissue of the treated rats [79] . oxidative stress (os) occurs when the body is exposed to either a harmful exogenous or endogenous per-oxidative stimuli. one of these stimuli is free radicals, including reactive nitrogen species (rns) and reactive oxygen species (ros). free radicals are produced during numerous oxidation-reduction (redox) reactions that happened in the cells. os is usually associated with the development of various diseases as cardiovascular and nervous system diseases, diabetes and cancer through the induction of oxidative damage of dna and abnormal expression of proteins [80] . os may be a risk factor for hcc as it elevates hepatocyte dna oxidative damage [81] . chronic viral infections may increase the production of ros and rns by causing inflammation and necrosis of hepatocytes that accompany immune cell infiltration [82] . the main cause of dna damage is mutation, caused by increasing ros and the failure in dna repair resulting in an increase in the mutation of cancer-related genes, meaning that chronic inflammation is considered the main risk factor for hcc [83, 84] . increasing os and the degree of dna damage has elevated the incidence of hcc related to viral infection [85] . the elevated level of transforming growth factor beta (tgfβ) and also tnf-α in patients with chronic hepatitis c is mainly related to oxidative stress. tgf-β is used as an indicator of how tissues are damaged and the extent to which they are injured [86] . ros is elevated due to increased oxidative stress, which stops the electron transport chain in damaged mitochondria. the excessive release of tnf-α not only causes severe damage to the mitochondrial respiratory chain but also causes damage to cytochrome oxidase. the high level of ros increases lipid peroxidation and inhibits the respiratory electron transport chain [87] . ros not only alter the mitochondrial metabolic activity but also affects the apoptotic pathways, changes the membrane permeability and causes damage to mitochondrial dna [88] . telomere shortening is accelerated by os that leads to an increase in the cytoplasmic migration of reverse transcriptase telomerase subunits [89] . damaging of the cells, mainly by os, causes the release of a unique type of dna that is called free circulating dna (fcdna), produced as oxides released by dna from dead cells. it can be used as an indication for a wide group of diseases like cancer. fc dna also can be present by low value in normal cells [90] . during the exposure to os, different types of genes and immune system mediator expression is altered. among these mediators, is micro rna (mirna), which is a small non-coding rna molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in rna silencing and post-transcriptional regulation of gene expression. mirna dysfunction is related to different types of cancer including hcc [91] . the mechanisms of hcc development caused by os can be summarized in fig. 3 [89]. 4.1.1. vitamin c and diallyl sulfide vitamin c, (l-ascorbate, l-ascorbic acid), is known by its antioxidant activity and the ability to capture free radical with singlet oxygen such as oh − or hoo − and superoxide ion o 2 − and produce dehydroascorbate. vitamin c activity is related to its free electron that interacts with electron deficient ions. several in vitro studies reported the antioxidant activity of vitamin c [92] . in vivo studies also showed the antioxidant properties of vitamin c in a dose-dependent manner. vitamin c administration protects guinea pigs, that do not form vitamin c, and osteodystrophy syndrome rats from oxidative stress when given oxidizing agents as endotoxin [93] and carbon tetrachloride [94] . it also protects rats exposed to cigarette smoke [95] . parquet is highly toxic nitrogen herbicide and has strong oxidizing stress. the administration of vitamin c reduces oxidative stress before (not after) parquet administration [96] . vitamin c has a promising role in cancer treatment through its selective cytotoxic activity for diverse cancer cell lines [97, 98] . in 1970s, a clinical study was performed and proved that ascorbate has an important role in increasing the survival of cancer patients at late stages [99] . combination of methotrexate with vitamin c is promising in cancer treatment through the reduction of h 2 o 2 produced from methotrexate in hep3b cells [100] . it was shown that vitamin c helped in the treatment of hepatotoxicity induced by dena in smp30 ko mice [101] . garlic contains diallyl sulfide (das) as a major active constituent. das is characterized by its anti-inflammatory activity through the modulation of cytokines as it inhibits the activity nf-kb [102] . das' anti-inflammatory activity is related to the nuclear factor erythroid 2-related factor 2 (nrf2) transcription activation and it also has antioxidant activity. nrf2 is an emerging regulator of cellular resistance to oxidants [103] . a combination of vitamin c and das was shown to offer several benefits, as inhibition of circulatory tnf-α and il-6 in dena-induced hcc in rats [104] and increases the sensitivity to chemotherapy as cisplatin in the treatment of hcc [105] . ginger (zingiber officinale) is one of the predominant herbaceous plants. it is a perennial plant and the main active part is the rhizome. ginger not only used as a condiment but also it has antiemetic and anticancer activity [106] . ginger has strong antioxidant and cell protection activity. this action of ginger is due to its potent active constituents as sesquiterpenoids, tannin, gingerols, shogaols and anthocyanin. several in vitro and in vivo studies documented the antioxidant activity of ginger. the protective effect of ginger was showed against several toxic agents, like bromobenzene and cisplatin [107] . another study displayed the chemopreventive effect of ginger against cancer [108] . ginger has a great activity in the treatment of experimental cancer in a rat model. it decreased the level of growth factors and αfetoprotein (liver tumor marker) after giving rats a daily dose about 50 mg/kg of ginger extract [109] . the anticancer effect of ginger is due to it's proapoptotic and anti-inflammatory properties. the anti-inflammatory activity of ginger was confirmed by inhibiting nf-κb and tnf-α after administration of 100 mg/kg of ginger in hcc rat model [110] . ginger contains other different constituents as clavatol, pinostrobin, and geraniol. these active components were detected by gas chromatography and mass spectrometry. ginger was found to inhibit the proliferation of cells in hepg-2 cell line (ic50, 900 μg/ml) [111] . one of the most popular active components in ginger is 6-shogaol which showed an anticancer effect against hepatoma cell line through the activation of ros-mediated caspase-dependent apoptosis in a multidrug resistance [112] . one of the most popular cultivated plants is broccoli which is distinguished by its high content of the antioxidant content. the most active antioxidant components in this plant are vitamins, flavonoids and carotenoids. isothiocyanates, the hydrolytic product of glucosinolate, is considered one of the antioxidant components, which motivates dna protection from damage through its antioxidant activity [113, 114] . the antioxidant property of broccoli may be direct by contributing in biochemical, cellular and physiological steps that inhibit free radical production, or indirect by inducing phase ii enzymes that have a protective effect against os [115] . the antioxidant activity of broccoli was observed in the human colon mucosa that exposed to oxidative stress [115] . broccoli showed a potential anticancer activity due to its high content of glucosinolates [116] . broccoli also contains a distinct component, sulforaphane, which is characterized by its activity as antioxidant and its ability to protect dna from breaking down by highly reactive electrophiles through increasing the antioxidant system activity and inhibition of inflammation [117] . antioxidant activity of sulforaphane is related to certain pathways, including the reduction of inflammation through inhibiting nf-κb and overexpression of transcription of nrf2, which has a very important role in keeping healthy cells and protect them from toxic chemicals and lifestyle-related factors [118] . previous in vivo studies registered that sulforaphane has abroad activity against different types of liver diseases related to toxic chemicals [119, 120] , consumption of alcohol [121] and using high calorie food [122] . broccoli has a major role in the suppression of different types of cancers, including liver hep-g2 and colon cancer. sulforaphane has different pathways related to its anticancer effect as it has anti-inflammatory, proapoptotic and cell cycle arresting action [123] . hbv and hcv infections are considered the chief reasons for hcc. usually, there are no symptoms for people with chronic infection but lately; cirrhosis and hcc are developed [124] . treating and overcoming hcv and hbv infection can help in the prevention of hcc development as they are oncogenic viruses [125] . the association between hcv infection and hcc varies worldwide. in western countries and africa, hcv is considered as the main cause of hcc, it also contributes to 80% to 90% of hcc cases in japan [126, 127] . as well as,80% of patients infected with hcv can progress to chronic hepatitis, with about 20% developing cirrhosis [128] . the hcv -related liver cirrhosis can increase the risk of liver cancer, with17-fold higher risk of developing hcc than in chronic hepatitis c infection alone, although this risk differs and depends on the degree of liver fibrosis caused by hcv [129] . the elevation of the risk of hcc development in patients infected with hcv arises from chronic inflammation, which results from the progression of liver fibrosis and cirrhosis. these inflammations cause alteration of the architecture of hepatocyte and defects of both cellular functions and the microcirculation of liver. hcv rna does not integrate into the host genome. alternatively, hcv viral proteins like hcv core protein and their induced host response have been involved in reactive oxygen species (ros) production, apoptosis, activation of transcription and modulation of immunity through up-regulation of tnf-α, il-6, and il-1, which participate in the transformation into malignancy [130] . hepatitis b virus (hbv) is a circular genome. chronic hbv infection can be confirmed by the presence of serum hbsag for a period not less than 6 months [131] . about 10%-25% of hepatitis b patients have a high risk of hcc development during their life. chronic hepatitis differs than other causes of hcc, as hcc occurs in the absence of cirrhosis [132] . after tobacco smoking, hbv is considered the second environmental carcinogen that affects individuals, resulting in about 55% of all hcc cases around the world [133] . the association of chronic hbv infection and hcc that now widely recognized was first explained by beasley and colleagues in 1981 [134] in taiwanese patients with positive serum hb surface antigen (hbsag). serum hbsag can be detected in 24%-27% of patients with hcc in japan, 41% of patients with hcc in the united states and 70% of patients with hcc in china in the absence of other risk factors [135] . once hbv arrives at the liver cell, transcription of messenger viral rnas occurs, followed by a translation into viral proteins and then, synthesis of dna of the virus. dna of the virus is then capable of integrating into the host genome in infected hepatocytes. cancer can be facilitated via this process through numerous ways, like rapid cell cycling of hepatocytes and viral dna integration into the host genome which causes instability and it may insert into, or adjacent to, genes that code proteins required for cancer development. it also leads to a chronic inflammation with fibrosis and proliferation of hepatocyte, which ultimately result in cirrhosis and cancer development [136] [137] [138] . andrographis paniculata nees (a. paniculata) is a medicinal plant, which belongs to family acanthaceae. it is used in japan, india, korea, china and other asian countries for a long time in the treatment of several diseases like inflammations, viral and bacterial infections and high blood pressure [139] . the most abundant di-terpene lactone found in the leaves and stems of a. paniculata is andrographolide. the andrographolide treatment was found to reduce the replication of hcv markedly and have a synergistic effect with the clinical trial drug psi-7977 or current antiviral drugs like telaprevir and ifn-α when used in combination to treat hcv. the mechanism of action of andrographolide fig. 3 . the mechanism of hcc development due to oxidative stress [89] . may be due to its ability to induce the p38 mapk/nrf2/ho-1 pathway, where, mapk stands for mitogen activated protein kinase and ho-1 is heme oxygenase-1. it was shown that andrographolide can be used as natural product or potential drug that is helpful in the treatment of hcv [140] . the treatment with a. paniculata aqueous extract was found to enhance the activity of hepatic enzymes and normalizes histopathological changes of malignant hepatic tissue induced by hexachlorocyclohexane [141] . it showed indirect and direct effects on tumor cells by inducing apoptosis and cancer cell necrosis, improving body's own immune system against tumor cells and inhibiting cell-cycle arrests and cancer cells proliferation [142] . the ethanolic extract of a. paniculata showed a cytotoxic effect against diverse human cancer cell lines like pc-3 (prostate), hepg2 (hepatoma), colon 205 (colonic) cancer cells and jurkat (lymphocytic) [143] . the inhibitory effect of andrographolide and its analogs on tumor cells may be due to their ability to depress cyclin-dependent kinase and induce the expression of inhibitory proteins of the cell cycle that result in blocking the cell cycle progression at g0/g1 [144] . andrographolide causes induction of apoptosis by several mechanisms including, the activation of caspase cascade, the release of cytochrome c from mitochondria and the activation of proapoptotic bcl-2 family members bax conformational change [145] . it also causes activation of ros-dependent c-jun nh 2 -terminal kinase (jnk) resulting in the activation of tumor suppressor p53 and thereby increasing p53 phosphorylation and protein stabilization [146] . silymarin is extracted from milk thistle seeds, silybum marianum l. gaertn., which belongs to asteraceae family [147] . the extract of silymarin contains silibinin, which consists of a mixture of two flavonolignans called silybin a (sa) and silybin b (sb). it has diverse pharmacological activities including, antioxidant, antiproliferative, immunomodulatory, antiviral activities and antifibrotic in different tissues and organs [148] [149] [150] . silymarin and its component silibinin possess antiviral activity against hcv infection in cell culture. their antiviral activity is due to their ability to block the entry of the virus, the synthesis of viral rna and protein, viral fusion, virus transmission and the activity of hcv n5sb rna dependent rna polymerase [148, 149, 151, 152] . one study showed that the treatment with a soluble form of silibinin in the form of daily intravenous injection causes a significant inhibition of hcv viral loads by 3-4 logs in 1-2 weeks in previous ifn non-responder patients [153] . several studies reported that silymarin has a potential anticancer effect against hcc. in a dose-dependent manner, silymarin inhibits the population growth of the human hepatocellular cancer cells (hepg2) as it elevates the percentage of apoptotic cells [154] . the antiproliferative activity of silymarin was also reported by another study without affecting the nontumor hepatic cells. in the g0/g1 phase, silymarin caused an increase in the percentage of cells, while in the s-phase it decreased the cell percentage associated with down-regulation of cyclin e, cyclin d1, phospho-rb and cdk4 and up-regulation of p53, retinoblastoma protein (rb), p27kip1, and p21cip1 [155] . silymarin also showed in vivo preventive and therapeutic efficiency against liver cancer. ramakrishnan et al. [156] reported that silymarin has a protective effect against dena-induced hcc in rats. silymarin also showed a potent preventive effect against spontaneous hcc in hbv x protein transgenic mouse model. oral silymarin in a dose-responsive manner causes a restoration of the early stage hepatic damage and fatty changes that lead to the recovery of hepatic tissue [157] . the oral administration of silibinin showed a significant reduction of hcc xenograft growth by inducing histone acetylation, apoptosis and expression of sod1 and inhibiting cell cycle progression, cell proliferation (ki-67 expression), erk and pten/p-akt signaling [158] . glycyrrhiza glabra, a perennial herb, originates from south-western and central asia and the mediterranean region. it showed numerous pharmacological activities like antioxidant, anti-inflammatory and immunomodulatory activities. the main constituent of glycyrrhiza glabra root is glycyrrhizin 1-9%, w/w [159] . glycyrrhizin has anti-viral, antiinflammatory, hepatoprotective and anti-tumor activities [160] . the antiviral activity was reported for glycyrrhizin and other components that isolated from glycyrrhiza species against different viruses, such as herpes simplex, hiv, severe acute respiratory syndrome, influenza virus, coronavirus, enteroviruses and hepatic viruses [161] [162] [163] . glycyrrhizin has been reported to be used in the treatment of hepatic diseases like chronic hepatitis c and b [164] . a preparation that contains glycyrrhizin was reported to reduce hepatic steatosis in transgenic mice expressing the full-length hcv poly-protein [165] . it was shown to have an inhibitory effect on hcv core gene expression and hcv fulllength viral particle both at protein and rna level and have a synergistic effect with interferon [166] . glycyrrhizin and other components of glycyrrhiza glabra showed antitumor activity in different kinds of cancers such as skin, liver and breast cancer, through inhibition of cellular proliferation, development and growth of cancer cells [167] . glycyrrhizic acid, a major bioactive component of the extract of glycyrrhiza glabra, has the ability to inhibit hcc occurrence in dena-treated mice [168] . another study showed that glycyrrhiza glabra extract has a potent effect in the treatment of hcc induced by dena/ccl4 in rats and this effect is more potent than the effect of cisplatin alone or cisplatin combined with glycyrrhiza glabra, so that cisplatin has several side effects and glycyrrhiza glabra is not associated with that side effects [169] . apoptosis, or programmed cell death, has a great interest in the field of oncology [170] . the recognition of each pathway of apoptosis is very important not only in understanding cancer development but also in the prevention and treatment of the disease. the normal tissue homeostasis maintained by keeping the balance between the proliferation of cells and their death. the imbalance between these two processes may lead to dysregulated clonal expansion, the cause of all neoplastic diseases [171, 172] . the mechanism of action of numerous cytotoxic agents includes apoptosis. several experimental approaches aimed to stimulate apoptosis that leads to the improvement of therapeutic response. numerous natural products play a vital role in the regulation of cellular proliferation and differentiation. the chemopreventive and chemotherapeutic activities of natural products may be due to their role in mediating different pathways involved in cancer development and progression [173] . 6.1. herbals with cytotoxic activity 6.1.1. nigella sativa nigella sativa (n. sativa), an annual flowering plant, originates from south and southwest asia and northern africa is grown almost all over the world [174] . n. sativa and its main constituent thymoquinone (tq) possess numerous therapeutic and pharmacological activities like antiinflammatory [175] , anticancer [176] , antioxidant [177] and immunomodulatory activities [178] . it has cytotoxic activity, as the ethyl acetate column chromatographic fraction of the ethanolic extract of n. sativa showed a cytotoxic effect against diverse cell lines such as molt4, hep g2 and ll/2 [179] . another in vitro studies showed about 50% cytotoxicity of a crude methanolic extract of n. sativa against dalton's lymphoma ascites (dla), ehrlich ascites carcinoma (eac) and sarcoma-180 cells (s-180 cells) [180] . its anti-cancer activity is due to its ability to exhibit powerful pro-apoptotic, anti-proliferative, anti-mutagenic, anti-metastatic and anti-oxidant effects. it also can inhibit tumor initiation and progression and has an anti-inflammatory and immunomodulatory effect. n. sativa can regulate signaling pathways like p53, inos and caspases [181] . the anti-tumor activity of n. sativa was reported in several in vivo and in vitro studies. a decoction that consists of seeds of n. sativa, smilax glabra rhizome, and hemidesmus indicus roots showed a significant improvement in the hepatocarcinogenesis induced by dena(4-6 g/kg/day) in rats [182] . the ethanolic extract of n. sativa showed a marked enhancement of dena-induced histopathological variations of the hepatic tissue [183] . another in vivo study showed that the administration of a methanolic extract of n. sativa in hcc albino rat model showed modulation of glucoregulatory enzymes [184] . aqueous extract of n. sativa showed in vitro antiproliferative activity and morphological changes like membrane damage and cell shrinkage in hepg2 cells, which lead to dna damage, cell death and a decrease in cell proliferation [185] . the mechanisms that explain the pharmacological effects of nigella sativa can be summarized in fig. 4 [186]. illicium verum (i. verum) hook belongs to family illiciaceae. it is commonly known as chinese star anise or star anise. it is an aromatic evergreen tree that originates from pakistan, china and other asian countries. due to its low toxic effects to humans, it was classified as "food and medicine" in 2002 by the ministry of health, people's republic of china [187] . the main active constituents that present in i. verum are sesquiterpenoids, monoterpenoids, lignans, phenylpropanoids, volatile compounds, and flavonoids. it also contains tannins, bitter principles and essential oils. these essential oils include transanethole, limone, α-pinene, β-phellandrene, farnesol, safrol and α-terpineol [188] . it also possesses antimicrobial, antioxidant, antifungal, analgesic, anti-inflammatory, sedative, insecticidal and anticonvulsive activities [189] . its cytotoxic activity was also reported in several studies [190] [191] [192] . it showed numerous mechanisms that involved in cell death such as apoptosis induction, scavenging of free radicals and tumor metastasis inhibition [191] . it was reported that alcoholic extract of i. verum showed a significant in vitro antiproliferative activities [190] . similar in vitro study also showed a marked inhibition of cell proliferation by its alcoholic extract by promoting apoptosis, growth inhibition and modulating the pro-apoptotic gene expression like bax and p53 [193] . separately, the cytotoxic effect of i. verum extract was studied in liver cancer model,it exhibited a significant anticancer outcome in hepatic tissue of rats with a significant improvement of tumor burden (decrease of nodule incidence, multiplicity, size, volume and liver weight). it also up-regulated phase ii detoxifying enzymes (glutathione-s-transferase) and decreased oxidative stress by restoration of superoxide dismutase activity [194] . sex has a vital role in hcc development as males are more diagnosed for hcc than females, with a ratio of 2:1-4:1. this may be due to the higher susceptibility of males to be infected with viral hepatitis, smoking, consuming higher amounts of alcohol and have a higher body mass index than females. the higher level of testosterone is associated with advanced hepatic fibrosis in males infected with hcv and hcc in hepatitis b carriers [195, 196] . another potent hepatocarcinogen is aflatoxin, which produced by aspergillus species found on corn, grains, soybeans or peanuts that stored in warm humid conditions [197] . several genetic and metabolic diseases are associated with hcc development such as wilson's disease, hemochromatosis, α-1 antitrypsin disease, glycogen-storage disease types i and ii, porphyrias and tyrosinemia [198] . other factors are also reported to be associated with the marked elevation of hcc development such as cigarette smoking and prolonged use of contraceptive pills [199] . diabetes mellitus is now considered as an independent risk factor for hcc [200, 201] . it causes liver cell damage through hyperinsulinemia and insulin resistance [202] . hyperinsulinemia induces hcc through inflammation, cellular proliferation and apoptosis inhibition. as well, the increase in insulin levels can lead to a decline in the synthesis of insulin growth factor binding protein 1 by the liver, which is supposed to cause an increase in the bioavailability of insulin-like growth factor 1, in addition to the fig. 4 . the cellular and molecular mechanisms of n. sativa that explain its pro-apoptotic, antiproliferative, cytotoxic, anti-oxidant, anti-metastatic, anti-mutagenic and nk-mediated effects. (lsa: lipid-bound sialic acid, tsa: total sialic acid, tnfα: tumor necrosis factor α, mda: malondialdehyde, afp: α-fetoprotein, il-6: interleukin-6, ros: reactive oxygen species, no: nitric oxide, gsh: glutathione, t-pa: tissue-type plasminogen activator, ifn-γ: interferon-γ, u-pa: urokinase-type plasminogen activator, pai-1: plasminogen activator inhibitor type 1) [186] . increase in apoptosis inhibition and cellular proliferation [203] . insulin has also been related to increased oxidative stress and the ros production, participating in dna mutation [204] . efficient storage of cereals liable to aspergillus attack, continuous inspection of both male and female sex hormones in suspected individuals, managing storage diseases as wilson's and hemochromatosis and controlling diabetes mellitus and other leading diseases will certainly contribute to a decline in hcc liability among risky individuals. the plants and their activities mentioned in this review could be summarized in table 1 : hcc is a prevalent disease in many countries around the world. it is highly related to the increase in deaths rate. the development of hcc passes through several intermediate steps such as molecular and transcriptional events that end finally to malignant transformation of hepatocytes. several factors are involved in these steps including; nafld, hcv, hbv, oxidative stress, chronic inflammation, some inborn metabolic errors, environmental toxins and some drugs, etc. accumulating evidence suggested that many dietary and natural products could be potential sources for prevention and treatment of liver cancer. these natural products (summarized in table 1 ) and their active ingredients can inhibit the liver cancer development and progression, through cutting the roads in front of the known leading risk factors for hcc. we here call urge the ministry of health in each country to establish records of liver patients, especially liver tumors. we recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid hcc progression and decrease its global incidence. we also draw attention of specialists in the food industry to add some of these natural products in different recipes to reduce the probabilities of liver diseases infections. none. none. the anti-inflammatory, anti-oxidant, cytotoxic and immunomodulatory [175, 176, 177, 178, 179] illicium verum cytotoxic activity, antioxidant, anti-inflammatory, induction of apoptosis, and inhibition of tumor metastasis [189, 190, 191, 192] therapeutic applications of herbal medicines for cancer patients molecular pathogenesis of hepatic fibrosis and current therapeutic approaches traditional herbal medicine: a review of potential of inhibitory hepatocellular carcinoma in basic research and clinical trial current status of herbal medicines in chronic liver disease therapy: the biological effects, molecular targets and future prospects prevalence of nonalcoholic fatty liver disease in the united states: the third national health and nutrition examination survey fatty liver hepatitis (steatohepatitis) and obesity: an autopsy study with analysis of risk factors prevalence of and risk factors for hepatic steatosis in northern italy frequency of abnormalities detected by abdominal ultrasound among japanese adults prevalence of fatty liver in children and adolescents prevalence of non-alcoholic fatty liver disease and advanced fibrosis in hong kong chinese: a population study using proton-magnetic resonance spectroscopy and transient elastography epidemiology of non-alcoholic fatty liver disease in china risk of cancer in patients hospitalized with fatty liver: a danish cohort study non-alcoholic fatty liver disease as a risk factor for hepatocellular carcinoma: mechanisms and implications nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace obesity, inflammation, and insulin resistance inflammation and metabolic disorders lipid homeostasis, lipotoxicity and the metabolic syndrome dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing il-6 and tnf expression liver transplantation in patients with nonalcoholic steatohepatitis-related hepatocellular carcinoma hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis hepatocellular carcinoma in japanese patients with nonalcoholic fatty liver disease, alcoholic liver disease, and chronic liver disease of unknown etiology: report of the nationwide survey recent advances in the herbal treatment of non-alcoholic fatty liver disease use of anti-aging herbal medicine, lycium barbarum, against aging-associated diseases. what do we know so far? lycium barbarum (goji) juice improves in vivo antioxidant biomarkers in serum of healthy adults immunomodulatory effects of a standardized lycium barbarum fruit juice in chinese older healthy human subjects hot water-extracted lycium barbarum and rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells effect of lycium barbarum polysaccharide on human hepatoma qgy7703 cells: inhibition of proliferation and induction of apoptosis chemical characterization of lycium barbarum polysaccharides and its inhibition against liver oxidative injury of high-fat mice the effect of lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats lycium barbarum polysaccharides protect mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation green tea polyphenols prevent toxin-induced hepatotoxicity in mice by down-regulating inducible nitric oxide-derived prooxidants depression by a green tea extract of alcohol-induced oxidative stress and lipogenesis in rat liver green tea extract protects against nonalcoholic steatohepatitis in ob/ob mice by decreasing oxidative and nitrative stress responses induced by proinflammatory enzymes therapeutic benefits of green tea extract on various parameters in non-alcoholic fatty liver disease patients, pak the effect of green tea extract supplementation on liver enzymes in patients with nonalcoholic fatty liver disease chemopreventive and therapeutic potential of tea polyphenols in hepatocellular cancer green tea constituent (-)-epigallocatechin-3-gallate inhibits hep g2 cell proliferation and induces apoptosis through p53-dependent and fasmediated pathways egcg inhibits activation of the insulin-like growth factor (igf)/igf-1 receptor axis in human hepatocellular carcinoma cells chemopreventive potential of green tea catechins in hepatocellular carcinoma epigallocatechin-3-gallate suppresses hepatic preneoplastic lesions developed in a novel rat model of non-alcoholic steatohepatitis therapeutic potential of resveratrol: the in vivo evidence resveratrol improves non-alcoholic fatty liver disease by activating amp-activated protein kinase resveratrol inhibits nonalcoholic fatty liver disease in rats resveratrol inhibits the expression of srebp1 in cell model of steatosis via sirt1-foxo1 signaling pathway alleviative effects of resveratrol on nonalcoholic fatty liver disease are associated with up regulation of hepatic low density lipoprotein receptor and scavenger receptor class b type i gene expressions in rats by reducing hexokinase 2, resveratrol induces apoptosis in hcc cells addicted to aerobic glycolysis and inhibits tumor growth in mice resveratrol down-regulates myosin light chain kinase, induces apoptosis and inhibits diethylnitrosamine-induced liver tumorigenesis in rats inflammation and cancer: back to virchow? inflammation and cancer stats in cancer inflammation and immunity: a leading role for stat3 infections as a major preventable cause of human cancer new functions for the matrix metalloproteinases in cancer progression role of nonresolving inflammation in hepatocellular carcinoma development and progression, npj precis suppressive effect of natural sesquiterpenoids on inducible cyclooxygenase (cox-2) and nitric oxide synthase (inos) activity in mouse macrophage cells xanthorrhizol inhibits 12-o-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor-kappa b evaluation of the antinociceptive activity and acute oral toxicity of standardized ethanolic extract of the rhizome of curcuma xanthorrhiza roxb xanthorrhizol: a review of its pharmacological activities and anticancer properties antiproliferative property and apoptotic effect of xanthorrhizol on mda-mb-231 breast cancer cells xanthorrhizol induced dna fragmentation in hepg2 cells involving bcl-2 family proteins advances in the study of berberine and its derivatives: a focus on anti-inflammatory and anti-tumor effects in the digestive system berberine suppresses proinflammatory responses through ampk activation in macrophages berberine mitigates cyclophosphamide-induced hepatotoxicity by modulating antioxidant status and inflammatory cytokines berberine inhibits inflammatory response and ameliorates insulin resistance in hepatocytes berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: the cellular mechanism berberine induces cell death in human hepatoma cells in vitro by downregulating cd147 berberine induces selective apoptosis through the ampkmediated mitochondrial/caspase pathway in hepatocellular carcinoma berberine inhibits p53-dependent cell growth through induction of apoptosis of prostate cancer cells the combinational effect of vincristine and berberine on growth inhibition and apoptosis induction in hepatoma cells alpinia: the gold mine of future therapeutics a review on the pharmacological activities and phytochemicals of alpinia officinarum (galangal) extracts derived from bioassayguided fractionation and isolation isolates of alpinia officinarum hance as cox-2 inhibitors: evidence from anti-inflammatory, antioxidant and molecular docking studies isolates from alpinia officinarum hance attenuate lps-induced inflammation in hepg2: evidence from in silico and in vitro studies diarylheptanoids from the rhizomes of alpinia officinarum and their anticancer activity diarylheptanoids derived from alpinia officinarum induce apoptosis, s-phase arrest and differentiation in human neuroblastoma cells galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-bid mitochondrial pathway chemosensitizing effect of alpinia officinarum rhizome extract in cisplatin-treated rats with hepatocellular carcinoma autophagy, mitochondria and oxidative stress: crosstalk and redox signalling survival rate in patients with hepatocellular carcinoma: a retrospective analysis of 389 patients hepatitis, cirrhosis, and hepatoma radical causes of cancer bagging survival trees viralinduced human carcinogenesis: an oxidative stress perspective role of oxidative stress and dna damage in human carcinogenesis hesperidin alleviates acetaminophen induced toxicity in wistar rats by abrogation of oxidative stress, apoptosis and inflammation is mitochondrial dna content a potential biomarker of mitochondrial dysfunction? mitochondrion oxidative stress and liver cancer: etiology and therapeutic targets circulating tumor cells and circulating tumor dna oxidative dna damage correlates with cell immortalization and mir-92 expression in hepatocellular carcinoma vitamin c as an antioxidant: evaluation of its role in disease prevention endotoxin increases oxidative injury to proteins in guinea pig liver: protection by dietary vitamin c the effect of vitamin c on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production vitamin c supplementation on hepatic oxidative stress induced by cigarette smoke in vivo dual effects of vitamin c on paraquat-induced lung damage: dependence on released metals from the damaged tissue pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer vitamin c enhances anticancer activity in methotrexatetreated hep3b hepatocellular carcinoma cells preventive effects of vitamin c on diethylnitrosamine-induced hepatotoxicity in smp30 knockout mice garlic (allium sativum l.) modulates cytokine expression in lipopolysaccharide-activated human blood thereby inhibiting nf-kappab activity diallyl sulfide enhances antioxidants and inhibits inflammation through the activation of nrf2 against gentamicin-induced 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hepatocellular carcinoma: a systematic review of epidemiologic evidence diabetes and cancer: a consensus report liver disease in patients with diabetes mellitus an evaluation of the role of insulin-like growth factors (igf) and of type-i igf receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against druginduced apoptosis the major lipid peroxidation product, trans-4-hydroxy-2-nonenal, preferentially forms dna adducts at codon 249 of human p53 gene, a unique mutational hotspot in hepatocellular carcinoma key: cord-309795-2kozsv4z authors: dewidar, bedair; kahl, sabine; pafili, kalliopi; roden, michael title: metabolic liver disease in diabetes – from mechanisms to clinical trials date: 2020-06-20 journal: metabolism doi: 10.1016/j.metabol.2020.154299 sha: doc_id: 309795 cord_uid: 2kozsv4z abstract non-alcoholic fatty liver disease (nafld) comprises fatty liver (steatosis), non-alcoholic steatohepatitis (nash) and fibrosis/cirrhosis and may lead to end-stage liver failure or hepatocellular carcinoma. nafld is tightly associated with the most frequent metabolic disorders, such as obesity, metabolic syndrome, and type 2 diabetes mellitus (t2dm). both multisystem diseases share several common mechanisms. alterations of tissue communications include excessive lipid and later cytokine release by dysfunctional adipose tissue, intestinal dysbiosis and ectopic fat deposition in skeletal muscle. on the hepatocellular level, this leads to insulin resistance due to abnormal lipid handling and mitochondrial function. over time, cellular oxidative stress and activation of inflammatory pathways, again supported by multiorgan crosstalk, determine nafld progression. recent studies show that particularly the severe insulin resistant diabetes (sird) subgroup (cluster) associates with nafld and its accelerated progression and increases the risk of diabetes-related cardiovascular and kidney diseases, underpinning the critical role of insulin resistance. consequently, lifestyle modification and certain drug classes used to treat t2dm have demonstrated effectiveness for treating nafld, but also some novel therapeutic concepts may be beneficial for both nafld and t2dm. this review addresses the bidirectional relationship between mechanisms underlying t2dm and nafld, the relevance of novel biomarkers for improving the diagnostic modalities and the identification of subgroups at specific risk of disease progression. also, the role of metabolism-related drugs in nafld is discussed in light of the recent clinical trials. finally, this review highlights some challenges to be addressed by future studies on nafld in the context of t2dm. nonalcoholic fatty liver diseases (nafld) is currently defined by lipid deposition that exceeds more than 5% of hepatocytes, as assessed from liver biopsy, and/or by more than 5 .6% hepatocellular fat content per liver weight, as assessed from magnetic resonance (mr) methods, in the absence of significant alcohol consumption and other causes of fatty liver [1, 2] . nafld, which affects about 25% of the population [3] , comprises a broad range of abnormalities ranging from simple fatty liver (steatosis) to non-alcoholic steatohepatitis (nash), characterized by inflammation, necrosis, and hepatocellular ballooning, and progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma (hcc) [2] . some gene variants promote risk of nafld by altering lipid droplet formation and de novo lipogenesis (dnl), such as variants of patatin-like phospholipase domain-containing protein 3 (pnpla3) and glucokinase regulatory protein [4] , or by decreasing very-low-density lipoproteins (vldl) export as shown for a missense mutation (e167k) in transmembrane 6 superfamily member 2 (tm6sf2) [5] . nevertheless, nafld is tightly associated with common acquired metabolic diseases such as obesity and type 2 diabetes (t2dm). the mutual relationship between both diseases is illustrated by several epidemiological data. the prevalence of steatosis and nash has been estimated to be 50 and 56%, respectively, in t2dm [6] . the age-adjusted relative risk of nafld is about 5.36fold higher in t2dm compared to healthy humans [7] . t2dm is also an emerging risk factor for nash progression to advanced fibrosis, cirrhosis and hcc [8, 9] . diabetes was even a better predictor for hcc development in people with nash and cirrhosis compared to other metabolic risk factors such as hyperlipidemia, body mass index (bmi) and hypertension [10] . recently, a consensus panel has proposed to rename nafld a metabolic-dysfunction-associated fatty liver disease (mafld) based on the presence of overweight/obesity, t2dm and evidence of so-called "metabolic dysregulation" [11] . future will tell, if this will help to better understand the multiple relationships between nafld and t2dm. in this context, nafld per se associates with more than double risk of incident diabetes pointing to specific liver-related mechanisms [12, 13] . moreover, multicenter studies in skandinavia and germany have recently found that diabetes can be stratified into subtypes j o u r n a l p r e -p r o o f the circulation. on the contrary, a recent study demonstrated that obesity-induced insulin resistance preceded inflammation in adipose tissues of mice [29] . indeed, adipose tissue inflammation might be a protective feedback mechanism as its local inhibition in mice induced ectopic lipid accumulation in liver, glucose intolerance, and systemic inflammation [30] . besides released inflammatory cytokines and ffa, adipose tissue could still communicate with the liver and muscle through secretion of different adipokines such as adiponectin and leptin. persons with nash have lower serum adiponectin compared to those with nafld with or without normal liver enzymes [31] . by contrast, the circulating levels of leptin were higher in people with nafld and t2dm, probably due to increased leptin resistance, and were associated with disease severity [32] . increased ffa influx to skeletal muscle promotes accumulation of intramyocellular lipid (imcl). reduced mitochondrial oxidation contributes as well to imcl as shown in aging and insulin-resistant humans [33] . consequently, skeletal muscle exhibits insulin resistance, which often precedes the onset of t2dm and insulin resistance in the liver [34] . lipid intermediate metabolites, in particular sn 1,2 diacylglycerols (dag), link imcl to skeletal muscle insulin resistance through activation of protein kinase c-theta (pkcθ) resulting in its translocation from cytoplasm to the plasma membrane [35] . muscles of insulin resistant humans with obesity and t2dm showed increased dag content and pkcθ activity as compared to healthy humans [35] . mutation studies highlighted serine amino acid residue (ser1101) of irs1 to be a substrate for activated pkcθ [36] . as a consequence of skeletal muscle insulin resistance, postprandial energy storage shifts from glycogen synthesis in the muscle into triacylglycerol (tag) in the liver, promoting nafld development [25] . gut microbiome is increasingly recognized as a modulator of liver pathogenesis through what is called the "gut-liver axis" [37] . distinctive alterations of gut microbiome were reported in humans having nash and t2dm [38] . the intestinal microbiome alters host metabolism by modulating the production of short-chain fatty acids (scfa) e.g. butyrate, acetate, and propionate, which have beneficial effects on insulin sensitivity, lipid and glucose metabolism [38] . also, intestinal dysbiosis could associate with increased intestinal permeability permitting translocation of bacterial lipopolysaccharide (lps) into the systemic circulation, j o u r n a l p r e -p r o o f 6 which could induce fat deposition in the liver, nash progression, weight gain, and diabetes [39] . moreover, intestinal microbiome could suppress the expression of fasting-induced adipocyte factor (fiaf) in intestinal epithelium, which functions as an inhibitor of circulating lipoprotein lipase, resulting in increased tag storage in the peripheral tissues [22] . also, ethanol-producing microbiome could increase blood alcohol concentration in nash, which is metabolized in the liver generating high levels of reactive oxygen species (ros). the last mechanism could explain the histological similarity between nash and alcoholic steatohepatitis [22] . furthermore, microbiota metabolize liver-derived primary bile acids into secondary bile acids. the latter are reabsorbed into bloodstream and may act as signaling molecules via a variety of receptors including farnesoid x receptor (fxr), which regulates the transcription of different metabolic genes involved in bile acid synthesis, transport, lipogenesis, and glucose homeostasis [40] . insulin resistance in both skeletal muscle and adipose tissues initiates liver steatosis by providing precursors and substrates for dnl and mitochondrial β-oxidation e.g. glucose, ffa and glycerol [25] . although reesterification of ffa derived from diet and adipose tissue is the dominant contributor to tag pool in the liver (59%), it did not increase in people with nafld. on the other side, dnl-derived ffa contribute by about 26%, which is severalfold higher as compared to individuals without nafld (10%) [41] . later, insulin resistance of the liver develops, resulting in increased gluconeogenesis and elevation of endogenous glucose production (egp) from the liver, which contributes to fasting hyperglycemia in individuals with t2dm [25] . insulin signaling inhibits typically hepatic gluconeogenesis through akt-induced phosphorylation of forkhead box (foxo1) and induces lipogenesis through activation of sterol regulatory element-binding proteins (srebp1c) and mammalian target of rapamycin complex (mtorc1) pathways. during hepatic insulin resistance, insulin does not suppress gluconeogenesis efficiently, while dnl is preserved or even increased. to explain this discrepancy, pathway-selective hepatic insulin resistance was postulated, which means that only one arm of insulin signaling is defective i.e. akt/foxo1 leading to reduced insulin-mediated suppression of gluconeogenesis, whereas insulin-activated srebp-1c/mtorc1 pathway remains intact and activates lipogenesis [28] . actually, dnl was reduced after induction of hepatic insulin resistance by feeding mice with j o u r n a l p r e -p r o o f 7 a high-fat diet (hfd) [42] , which challenges the concept of selective hepatic insulin resistance and suggest the existence of alternate mechanisms that contribute to increased dnl and gluconeogenesis in insulin-resistant liver, for example, hyperinsulinemia, insulinindependent re-esterification of adipose tissue-derived ffa, and increased acetyl coa generation from β-oxidation of ffa, which allosterically activates pyruvate carboxylase enzyme, leading to enhanced gluconeogenesis [25] . in addition, increased blood glucose level can activate carbohydrate response element-binding protein (chrebp) signaling pathway, thereby stimulating expression of several glycolytic genes, which provide additional metabolic precursors for dnl [43] . in line, chrebp overexpression induced stearoyl-coa desaturase 1 (scd1) expression, an enzyme responsible for the biosynthesis of monounsaturated fatty acids (mufa), resulting in increased liver fat content [44] . pkc epsilon (pkcε) is crucial in mediating hepatic insulin resistance and once activated by dag, it translocates to the cell membrane, and phosphorylates specific threonine residue (thr1160 in human and thr1150 in mouse) on insulin receptor leading to destabilization of the active configuration of insulin receptor kinase and inhibition of its tyrosine kinase activity [45] (figure 2 ). in general, both hyperglycemia and toxic lipids such as ceramides, dag, ffa, and cholesterol can induce deleterious effects on liver cells (glucolipotoxicity), which might initiate nafld progression from simple steatosis to nash and fibrosis via various mechanisms, including cell death, oxidative stress, endoplasmic reticulum (er) stress and mitochondrial disorders [46] . alterations in the activity and abundance of oxidative phosphorylation (oxphos) proteins and antioxidant enzymes were described in various animal models of nafld [47] . impaired hepatic mitochondrial function was evident as well in t2dm and nash [48, 49] . in a mouse model of choline-deficient diet-induced nafld, mitochondrial oxphos was increased at 12 weeks but lost at a later time point [50] . also, the higher maximal respiration rate of liver mitochondria was severalfold higher in insulin-resistant obese individuals with fatty liver as compared to lean individuals [51] . these studies highlight the flexibility of mitochondria to adapt to increased metabolic inputs to keep energy homeostasis and to protect against the harmful effects of increased ffa and tag in the liver. in nash, mitochondrial flexibility was lost, which was associated with increased ros production and exhaustion of protective antioxidant enzymes [51] (figure 2 ). whether loss of mitochondrial flexibility is a cause or consequence for nafld progression remains obscure. depletion of atp due to j o u r n a l p r e -p r o o f mitochondrial disorders, together with hyperglycemia and lipid overload could be inducers for another signaling pathway, termed unfolded protein response "upr", which is adaptive response to resolve unfolded or misfolded proteins and to restore er homeostasis [52, 53] . prolonged upr stress can activate jnk and nf-kb signaling pathways, which are involved in insulin resistance, liver steatosis, and inflammation [52] . furthermore, er stress could increase insulin resistance through induction of lipin-2 expression, which is a phosphatase enzyme that catalyzes biosynthesis of dag leading to activation of dag/pkcε axis [54] . interestingly, upr could also increase liver steatosis through activation of srebp-1c signaling pathway [53] . elevated ros and upr are well-identified pathways that could induce hepatocytes death in nash. hepatocytes apoptosis and necroptosis are the main forms of cell death in human steatohepatitis and diet-induced mouse models of nash [55] . the key fibrogenic liver cells, hepatic stellate cells (hscs), usually exist in a quiescent state and get activated by engulfment of apoptotic bodies, the inflammatory milieu, or damage-associated molecular patterns (damps) released from stressed and dying hepatocytes [56] . interestingly, ffa-mediated lipotoxic effects stimulate hepatocytes to release extracellular vesicles (evs) with distinctive micrornas (mirna) profile that increase the expression of fibrogenic genes in the surrounding hscs [57] (figure 3 ). free cholesterol could directly sensitize hscs to the action of tumor growth factor (tgf)-β, a potent fibrogenic cytokine [58] . treatment of human and rat immortalized hscs cell lines with saturated fatty acid increased the expression of various profibrogenic genes [59] . macrophages aggregate as well around dead hepatocytes forming a crown-like structure, a phenomenon that exists only in persons with nash but not in those with simple steatosis [60] . recruitment of more inflammatory cells from systemic circulation is facilitated by "find me" signals that are released from dead cells [61] . inhibition of inflammatory monocytes recruitment via inhibition of c-c chemokine receptors type 2 and type 5 (ccr2/ccr5) suppressed fibrogenesis and steatohepatitis in murine nash [62] . macrophages can modulate also hepatic insulin resistance and favor tag accumulation in hepatocytes through secretion of il-1β that downregulates peroxisome proliferator-activated receptor (ppar) α, a key transcriptional pathway involved in fatty acid oxidation [63] . liver sinusoidal cells (lsecs) are fenestrated cells that exist in close vicinity to hepatocytes, j o u r n a l p r e -p r o o f macrophages and hscs, which, under physiological conditions, regulate lipid transport, maintain the quiescence of kupffer cells, resident liver macrophages, and hscs [64] . at early stage of nafld, lsecs lose their fenestrae, a process termed capillarization, which could favor liver steatosis and initiate hscs activation [64] (figure 3 ). during nash, lsecs display a pro-inflammatory phenotype that promotes steatohepatitis [64] . autophagy is a self-degradative process which is used by the cells to remove misfolded proteins and damaged organelles [65] . singh et al. showed that the cells can use autophagic process as lipolytic mechanism to mobilize lipids from intracellular lipid store, which is termed in this case "macrolipophagy" [66] . various in vitro and in vivo studies showed that autophagy was decreased in fatty hepatocytes [67] . impairment of autophagy by palmitic acid in macrophages induces inflammatory il-1β production [68] . on the other side, selective loss of autophagic activity reduced liver fibrogenesis in cultured hscs [69] and protected against diet-induced insulin resistance [70] . the results highlight that the net effect of autophagy on nafld might depend on the tissue or type of the cells that show autophagic dysfunction and the stage of nafld. the liver biopsy is considered the gold standard for nafld diagnosis, especially for distinguishing steatosis from inflammation and fibrosis [2] . nevertheless, this technique has several limitations not only resulting from the invasive procedure and rare post-interventional complications, but also due to the small tissue volume, which might not be representative for the whole liver and may not take into account inhomogeneous distribution of nafldassociated alterations. novel imaging modalities such as ultrasound-or mr-based techniques are of increasing value, as recently reviewed [71] , but are still not generally available, so that there is an urgent need for noninvasive screening tools. non-invasive detection of nash and fibrosis remains challenging today. biomarker-based panels such as aspartate aminotransferase (ast)/platelets count ratio index, fibrosis-4 (fib-4) index, fibrotest, fibrospect ii, and nafld fibrosis score (nfs) offer variable diagnostic efficacy for assessing different stages of liver fibrosis [73, 74] . although combination of these panels could enhance their predictive values [73] , they still suffer from limited accuracy even compared to the alanine aminotransferase (alt) and ast, particularly in t2dm [75] . in this regard, transient elastography looks like a promising alternative in diabetes clinics for detection of liver fibrosis in nafld [74] . numerous biomarkers have been developed to specifically track features of nash progression and fibrosis. cytokeratin (ck) 18, an intermediate filament protein that is cleaved during cell death to ck18 m30 and ck18 m65, has been intensively investigated as a surrogate of nash-associated liver cell damage, but a recent meta-analysis of 25 studies reported a maximum sensitivity of 0.75 for nash diagnosis [76] and suboptimal diagnostic value was shown in t2dm [75] . the ecm turnover marker, type iii procollagen, can offer a diagnostic efficacy of 0.81 and 0.88 to detect moderate and advanced liver fibrosis in t2dm, respectively [77] . in 2016, the european associations for the study of the liver, obesity and diabetes (easl-easo-easd) jointly released recommendations for diagnosis and monitoring of disease severity in persons with suspected nafld and metabolic risk factors [78] . people with metabolic risk factors, such as t2dm, should undergo assessment by abdominal ultrasound, serum transaminases and fibrosis markers (e. g. fib-4, nfs). elevated transaminases or steatosis plus abnormal fibrosis test shall require referral to a specialist. this strategy has raised the question of a possible overreferral when adhering to these guidelines [79] . however, recent analyses show that a refined strategy of specific combining indices such as fli and fib-4 could reduce the number of people with t2dm for further diagnostic work up to a reasonable size [80] . this retrospective analysis also found that certain non-invasive biomarkers are consistently associated with different patterns of diabetes-related complications. analyses of the plasma metabolomic of insulin-resistant individuals with nafld suggested that bile salts, e. g. glycocholate, taurocholate, and glycochenodeoxycholate allow to detect nash in one study [81] , while another study reported an association with insulin resistance j o u r n a l p r e -p r o o f but not with liver necroinflammation [82] . also amino acids, specifically a glutamate-serineglycine index, was associated with hepatic insulin resistance and transaminases and discriminated individuals with fibrosis grade 3-4 from those with grade 0-2 independently of bmi [83] . the diagnostic performance of a serum-based lipidomic analysis was substantially lower for nafld detection in t2dm compared to healthy individuals [84] . nevertheless, certain sphingolipid species were recently found to be increased in insulin-resistant nash and to correlate with hepatic oxidative stress and inflammation [85] . one circulating small noncoding rna, mirna-122, was found to be more than 5.7fold in steatosis and further doubled in nash [86] . this mirna was also higher in t2dm with nafld than in those without nafld and provided better prediction of nafld when combined with ldl and waist-to-hip ratio [87] . moreover, extracellular vesicles (ev), which among other cargo also transport mirna, may be promising biomarkers for nafld as shown by higher serum levels in people with nafld, obesity and diabetes [107] . recently, metagenomics data derived from gut microbiota alterations allowed to detect advanced fibrosis in 86 nafld patients, of whom 23% had t2dm, with a robust diagnostic accuracy (auroc: 0.936) [88] . the current guidelines recommend only lifestyle modification and -for certain groupsbariatric or metabolic surgery to treat nafld [1, 2] . although the numerous activities in this field, no pharmacological treatment is currently approved or expecting approval for the use in nafld with or without concomitant t2dm (table 1 and 2), except for obeticholic acid (oca). marketing authorization application for oca has been submitted to the u.s. food and drug administration (fda) and european medicines agency (ema), awaiting fda decision by june 2020 [89] . against this background, current guidelines and recommendations primarily advise lifestyle modification (healthy nutrition and exercise) and weight loss in overweight/obese persons. the easl-easo-easd guidelines recommend to consider pharmacological treatment in people with nash when combined with fibrosis and in those with less severe disease, but high risk conditions for disease progression such as t2dm [78] . the american association for the study of liver diseases (aasld) recommends to limit pharmacological treatment to those with biopsy-proven nash and fibrosis [1] . j o u r n a l p r e -p r o o f weight management and physical exercise are key to the treatment of both nafld and t2dm. a proof-of-concept study showed that a hypocaloric diet with weight loss of ~8 kg within 12 weeks not only normalized fasting egp and thereby hyperglycemia, but decreased hepatic tag content down to normal concentrations in obese t2dm humans with nafld [90] . subsequent studies in larger cohorts extended these results by demonstrating that a very low-caloric diet with weight loss of about 15% rapidly normalized liver fat content in 90 in dividuals with t2dm, which persisted for one year if weight loss was maintained [91] . interestingly, mediterranean, low-saturated fat and high plant-based protein diets also improve steatosis in nafld combined with t2dm despite minor or no relevant weight loss [92, 93] . in addition, physical activity and exercise training interventions can also decrease liver fat content, which may not be exclusively depending on concomitant weight loss [94] . although the beneficial effects of structured behavioral treatment to improve histological endpoints, likely extend beyond reduction of hepatic fat content to ameliorating the grade and stage of inflammation and fibrosis [95] , only a minority of the people manages to adhere to dietary weight loss and exercise programs, which raises the issue of other therapeutic approaches [94] . weight-loss inducing drugs could be an attractive option for persons with nafld with a bmi > 30 kg/m 2 or >27 kg/m 2 in the presence of at least one metabolic comorbidity, as an adjunct treatment to lifestyle modifications [96, 97] . currently, the most-popular weight-loss inducing medications associated with at least 5% body weight decrease in one year as compared to placebo are orlistat, the fixed combination of phentermine and topiramate or naltrexone and bupropion, and the glucagon-like peptide-1 (glp-1) receptor agonist (glp-1ra), liraglutide [97] . of these drugs, orlistat treatment failed to improve liver histology when compared to placebo [98] , while no reports are available for topiramate, naltrexone, bupropion and phentermine regarding hepatic endpoints in humans with nafld [99] . only bariatric or metabolic surgery is a therapeutic option to induce sustained weight loss partricularly in people with combined nafld and t2dm. in obese humans, bariatric surgery resulted in 85% resolution of nash within one year [101] and in 77% complete remission of t2dm [102] . surgical weight loss improves glucose metabolism and insulin sensitivity by different mechanisms such as increased glp-1 secretion [103] and epigenetic modification [104, 105] . several antihyperglycemic drug classes were or are currently being investigated in clinical trials and preclinical models to evaluate their efficacy for people with nafld and with or without t2dm. metformin reduces body weight, hepatic gluconeogenesis -by yet unclear mechanisms [106] , and the risk of macrovascular complications, which is still controversially discussed due to lack of optimally designed clinical trials [116] . despite its action on hepatic metabolism, former small-scale studies reported conflicting results [109, 110] (table 1 ) and recent metaanalysis failed to demonstrate any effect of metformin on liver histology [111] . nevertheless, epidemiological, observational and preclinical studies suggest that metformin may reduce the risk of hcc and also in t2dm, possibly by promoting apoptosis, but controlled clinical trials are not available [112] . dpp4 degrades incretins such as glp-1 so that dpp-4i treatment increases the postprandial levels of endogenous glp-1, which leads to lower glucose peaks after meals [113, 114] . in individuals with nafld with prediabetes or recent onset diabetes, sitagliptin did not improve liver steatosis or liver fibrosis compared to placebo as assessed by mr-based techniques [115] . in line, another recent trial reported no benefits for sitagliptin versus placebo on liver fibrosis or steatohepatitis in t2dm [116] (table 1) . in contrast, a moderate reduction in liver fat content was observed with vildagliptin in individuals with t2dm [117] . j o u r n a l p r e -p r o o f 14 the actions of endogenous glp-1 are mimicked by glp-1 receptor agonists (glp-1ra), which effectively reduce blood glucose levels and also reduce the risk for cvd in t2dm [118] . in individuals with nafld and t2dm, liraglutide in combination with metformin reduced liver, subcutaneous, and visceral fat [119] . also, liraglutide improved hepatic steatosis measured by mr-based methods as well as resolved biopsy-proven nash without worsening of fibrosis [100, 120] (table 1) . however, a recent subanalysis did not detect an effect of liraglutide on liver fat content quantified by 1 h-mrs [121] (table 1) . respective trials with semaglutide and dulaglutide are still waiting for results (nct02970942, nct03648554). in an animal model of t2dm, exenatide also counteracted hcc development by suppression of stat3-regulated genes [122] . glucose-dependent insulinotropic polypeptide (gip) represents the second important incretin with proposed beneficial effects on peripheral energy metabolism [123] . tirzepatide, a novel dual agonist for gip and glp-1 receptors with probably greater efficacy than glp-1ra [124] , decreased transaminases and surrogate markers of liver injury paralleled by increased circulating adiponectin levels in people with t2dm [125] . a clinical trial on nash resolution is currently ongoing in persons with nash with or without t2dm (nct04166773). sglt2i inhibit reabsorption of glucose in the proximal renal tubule resulting in glucosuria and calory loss, thus effectively reducing blood glucose level and body weight in t2dm. moreover, sglt2is show beneficial effects on cardiovascular risk and progression of nephropathy [118, 126] . most, but not all recent randomized controlled trials (rcts) showed that sglt2i treatment resulted in reduction in liver fat content compared to placebo [127] [128] [129] [130] [131] (table 1) . in an open-label pilot study in liver-biopsy proven cohort of nash with t2dm, treatment with empagliflozin for 6 months reduced liver steatosis, ballooning and fibrosis and induced nash resolution in approximately half of those persons [132] . animal models further suggested anti-inflammatory, anti-oxidant and pro-apoptotic actions of sglt2i with cardio-protective effects as well as inhibition of nash progression and tumor growth of hcc [133, 134] . the dual sglt1/2i, licogliflozin, is expected to block both intestinal and renal glucose (re)absorption [135] and currently investigated to evaluate its efficacy on resolution of nash as monotherapy and as combination therapy with the fxr agonist, tropifexor, in people with nash and fibrosis (nct04065841). first results hint at improvement of transaminases and reduction of steatosis by licogliflozin in nash [136] . ppars is a family of nuclear receptors composed of multiple isoforms with wide tissue distribution [137] . the ppar-γ agonist pioglitazone had convincing efficacy on nash resolution in individuals with and without t2dm, but with conflicting results on fibrosis [138] [139] [140] (table 1) . of note, pioglitazone has been withdrawn in many european countries because of its disadvantageous safety profile [231] . however, pioglitazone exerts beneficial effects on cardiovascular outcomes in persons with t2dm and a history of cvd [141] . it has been proposed that non-ppar-γ dependent mechanisms, as the inhibition of the mitochondrial pyruvate carrier (mpc), might mediate the beneficial effects of pioglitazone on nafld [142] . however, a novel drug with ppar-γ sparing effects and mpc binding activity did not improve histological components of nash in individuals with t2dm compared to placebo [143] . elafibranor is a dual agonist for ppar-α and ppar-δ without ppar-γ stimulation [144] . based on a post-hoc analysis, nash was resolved without worsening of fibrosis in a higher percentage of people with and without t2dm in the elafibranor group as compared to the placebo [145] and a follow-up study on these findings has been initiated ( table 2) . recent announcements on an interim analysis state that elafibranor treatment failed to resolve nash and improve fibrosis when compared to placebo [146] . saroglitazar is another dual ppar-α/γ agonist with higher affinity for ppar-α. in a mouse model of nash, saroglitazar reduced liver steatosis, inflammation and prevented fibrosis development and a respective clinical trial is ongoing [147] ( table 2) . lanifibranor is a pan-ppar agonist for ppar-α, β, and γ receptors focused on treatment of liver fibrosis and nash (nct03008070). in an animal model of liver fibrosis, lanifibranor decreased hepatic collagen deposition [148] . several other strategies for pharmacological targeting of nash have emerged over the last few years; however, these strategies are not specifically designed for t2dm, but for the whole nafld collective. they comprise antiinflammatory drugs, but also modulators of other pathways, which are briefly summarized in the following sections. j o u r n a l p r e -p r o o f fxr can be activated by bile acids in a negative feedback mechanism to suppress bile acid synthesis [40] . oca is a potent semisynthetic and selective fxr agonist approved for treatment of primary biliary cholangitis [149] . oca treatment resulted in histologic improvement of nash in people with and without t2dm compared to placebo [150, 151] ; however, concerns were raised about oca-induced changes in plasma lipoprotein profile [152] . tropifexor, another potent fxr agonist [153] , is currently being tested in combinatorial approaches of nash treatment (cenicriviroc and licogliflozin; nct03517540). oltipraz is a synthetic dithiolethione with antisteatotic effects by inhibiting the activity of lxr-α. it activates adenosine monophosphate activated protein kinase (ampk) and inactivates s6k1, affecting lxr-α thus reducing lipogenesis and increasing lipid oxidation [154] . a recent 24-week phase 2 clinical trial found decreased steatosis measured by 1 h-mrs with oltipraz compared to placebo treatment [154] and a respective phase 3 clinical trial is ongoing ( table 2 ). chemokine receptors type 2 (ccr2) and type 5 (ccr5) are expressed on various inflammatory and fibrogenic cells [155] . cenicriviroc is a ccr2/crr5 dual antagonist that reduced insulin resistance, liver inflammation and fibrosis in diet-induced models of nash [62] . in recent rcts in a nash cohort with fibrosis, cenicriviroc treatment did not improve nas but may reduce liver fibrosis [156, 157] . currently, there is an ongoing clinical trial to evaluate the effects of cenicriviroc on fibrosis in nash ( table 2) . both people with obesity, metabolic syndrome or t2dm as well as those with nafld are at increased risk for dyslipidemia. statins, inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme a (hmg-coa) reductase, are generally safe and have unmet efficacy to decreased serum ldl and prevent cardiovascular outcomes, despite slightly increasing the risk of t2dm [2] . use of statins associated with a 46% lower relative risk of hepatic decompensation and mortality in cirrhosis and a trend towards lower fibrosis progression in non-cirrhotic liver diseases [158] . however, the data on liver histology ist limited [159] so that statins are not currently recommended for the management of nafld [2] . the inhibitor of intestinal j o u r n a l p r e -p r o o f cholesterol absorption, ezetimibe, decreased the histological nas, but not consistently liver fat content in an analysis of the few available studies [160] . fenofibrate, a ppar-α agonist, does not affect or even increase liver fat content or volume [161, 162] . nevertheless, the combination of statins with certain anti-nash drugs, such as oca, could be beneficial to counteract drug-related increases in ldl during long-term treatment. inhibition of dnl in nafld may be achieved by inhibition of acc as this enzyme catalyzes the conversion of acetyl coa into malonyl coa, which acts as a substrate for fatty acid synthesis and inhibitor for fatty acid β-oxidation [163] . in a recent clinical trial in individuals with nash with and without t2dm, treatment with the dual acc1 and acc2 inhibitor gs-0976 decreased liver steatosis without improvement of fibrosis [164] . the major concern of this strategy is that decreased dnl in nafld might channel lipids towards other harmful directions, e.g. increased blood lipids [163] . scd1 is a key enzyme for hepatic lipogenesis that catalyzes the conversion of saturated fatty acids to mufa and its downregulation protected mice from high carbohydrate-induced liver steatosis [165] . a clinical trial with aramchol, an inhibitor of scd1, showed a reduction in liver fat content, resolution of nash and improvement of liver fibrosis in persons with nafld with prediabetes or t2dm [166] . these results paved the way for a respective phase 3 clinical trial ( table 2) . mitochondrial uncoupling describes any process that uncouples the electron transport from atp synthesis in mitochondria [167] . 2,4-dinitrophenol (dnp) was widely used for the treatment of obesity before its discontinuation due to life-threatening serious adverse events [168] . to overcome the side effects of dnp, improved formulas of dnp were recently developed to decrease the toxic to effective dose ratio (dnp-methyl ether (dnpme) and controlled-release mitochondrial protonophore (crmp)) [169, 170] . oral crmp targets mainly the liver due to the first-pass effect and decreased hepatic insulin resistance, hepatic steatosis and liver fibrosis in a methionine-choline deficient rat model of nash [170] . crmp was also effective in nonhuman primates with diet-induced nafld for reduction of hepatic steatosis and egp [171] . thyroid hormone deficiency has been associated with nafld development [172] . thyroid hormone analogues decreased liver fat, liver transaminases, and inflammatory and fibrosis markers in animal models of nash [172] . resmetirom is a liver-targeted highly selective thr-β agonist which showed efficacy in reducing liver fat content in nash with and without t2dm [173] . currently, there is ongoing clinical trial for evaluation of long-term outcomes of resmetirom and its efficacy on nash resolution ( table 2 ). vitamin e is a potent antioxidant [174] . in nash without t2dm, vitamin e improved nas without worsening of fibrosis [139] . despite concerns regarding adverse effects of long term vitamin e usage, treatment with vitamin e for ≥2 years reduced the risk of liver failure in a nash cohort with advanced fibrosis and with or without t2dm [175] . therapeutic manipulation of intestinal microbiome is still in its infancy. rodent data showed efficacy for fecal microbiota transplantation in improving nafld in a diet-induced nash model [176] . also, modulation of the intestinal microbiota by antibiotic treatment reduced liver transaminases in nafld [177] . the use of prebiotics and probiotics in obese nafld/nash is currently not supported by high-quality clinical studies with mr-or biopsy-based endpoints [178]. the evidence for shared pathophysiological mechanisms between t2dm and nafld will help to develop strategies for detecting and treating both diseases and preventing their leading complication, cvd. altered lipid and energy metabolism, insulin resistance, low-grade inflammation and intestinal dysbiosis represent key targets. in addition to weight loss by lifestyle modification or bariatric surgery, glp-1ra and sglt2i are promising antihyperglycemic concepts with beneficial effects on nafld and cvd. in addition, many j o u r n a l p r e -p r o o f metabolism-based drugs are currently studied comprising ppar agonists, endocrine dual coagonists to modulators of hepatic metabolism or microbiota. nevertheless, several roadblocks need to be overcome to reduce the burden of nafld in t2dm. first, there is still lack of preclinical animal models that encapsulate essential features of human nash and diabetes. second, available biomarkers lack diagnostic efficacy to identify nafld progression. innovative strategies such as cluster analysis already enabled detection of a diabetes subtype (sird) with high risk of nafld [14] . combination with computational integration of multiomics data shall identify specific disease signatures and pave the way to precision medicine and targeted management of t2dm and nafld. finally, studies on so-called endpoints are scarce, which may be due to the need of long-term studies to evaluate liver-related mortality, to the current neglect to accept cvd morbidity and mortality as nafld outcome and to ongoing discussions on the relevance of surrogate markers. the research of the authors is supported in part by grants from the german federal ministry phase iii registered interventional trials on "clinicaltrials.gov" for nafld as accessed on 10 th april 2020. bl, baseline; ccr2/5, c-c chemokine receptors type 2 and type 5; fxr, farnesoid x receptor; hba 1c , glycated haemoglobin; lxr, liver x receptor; mpc, mitochondrial pyruvate carrier; na, data not available; nafld, non-alcoholic fatty liver disease; nfs, nafld fibrosis score; ppar, peroxisome proliferator-activated receptor; nash, non-alcoholic steatohepatitis; scd, stearoyl-coa desaturase; sglt, sodium-glucose cotransporter; thr, thyroid hormone receptor; t2dm, type 2 diabetes. *only placebo-controlled, randomized clinical trials are listed, except for saroglitazar, a 4-arm active-controlled study. the diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the american association for the study of liver diseases easl-easd-easo clinical practice guidelines for the management of 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fibrosis total fecal microbiota transplantation alleviates high-fat diet-induced steatohepatitis in mice via beneficial regulation of gut microbiota efficacy of rifaximin on circulating endotoxins and cytokines in patients with nonalcoholic fatty liver disease key: cord-300187-fr6tme32 authors: kearns, shawn title: infectious hepatopathies in dogs and cats date: 2009-11-26 journal: top companion anim med doi: 10.1053/j.tcam.2009.06.004 sha: doc_id: 300187 cord_uid: fr6tme32 this article serves to review the various infectious diseases that affect the liver primarily or as a part of systemic infection. although bacterial infections are probably the most common cause of infectious hepatitis, the clinician should be aware of other potential organisms and other commonly involved systems. therefore, this article includes a description of common bacterial, mycobacterial, viral, fungal, protozoal, parasitic, and rickettsial diseases in dogs and cats. alimentary flora circulates to the liver under various clinical conditions. these bacteria are extracted by kupffer cells, killed by neutrophils, or excreted in bile in healthy clinical states. a low-flow, low-pressure perfusion of hepatic sinusoids may allow superior removal of bacteria by phagocytes, and pressure differentials in the biliary system may limit retrograde access of enteric organisms. 2, 4, 5 changes in this sinusoidal flow may decrease the effectiveness of phagocytosis when portal flow is compromised. bowel disease, cholestasis, immunosuppression, and altered gut motility result in altered portal circulation, and the subsequently unchecked bacterial access to the liver may result in bacterial hepatitis or cholangiohepatitis. common isolates implicated in bacterial hepatitis and cholecystitis include escherichia coli, enterococcus spp, bacteroides spp, streptococcus spp, and clostridium spp. 6 cultures can be obtained by liver aspirate, liver biopsy, and cholecystocentesis. a combination of liver and gall bladder samples (fig 1) may increase the likelihood of identification of the offending organism(s). surgical or laparoscopic biopsies may be more rewarding for culture growth compared with aspirates. 6 in suspected cases, broad-spectrum antibiotics for common enteric isolates should be initiated pending specific culture results. focal micro-and macro-abscesses have also been documented in dogs and cats. 7, 8 predisposing causes include alterations in blood flow, trauma, ascending biliary infections, liver lobe torsions, 9 immunocompromised clinical states, 10 and neoplasia. 7, 11 microabscesses are often identified in association with extrahepatic infection and sepsis. 7, 12 ultrasound has greatly enhanced the early diagnosis of hepatic abscesses. 8 greater than 50% of solitary abscesses are polymicrobial. antimicrobial treatment should be directed at both anaerobes and aerobes regardless of whether anaerobic cultures are negative if a polymicrobial hepatic infection is documented. 2 bacterial isolates in hepatic abscesses are similar to those identified in diffuse bacterial hepatic disease. however, clinically rare isolates including klebsiella, listeria, salmonella, brucella, yersinia pseudotuberculosis, actinomyces, nocardia, and pasturella have also been documented. 13 focal abscesses may require surgical drainage and antibiotic therapy. treatment in all cases must be implemented for a minimum of 6 to 8 weeks. leptopirosis is an extremely common nonenteric bacterial infection in the canine liver. leptospires are thin, filamentous, spiral-shaped motile bacteria with a lipopolysaccharide outer envelope. direct transmission occurs via contact with infected urine, venereal and placental tissues, or fluids. indirect transmission can occur through contaminated water sources, soil, food, or bedding. the organism can stay stable for several months with the right environmental conditions. the organism initially penetrates the mucous membranes and rapidly multiplies after entry into the vascular space. dissemination and replication occur in many tissues, including the liver. however, the organism tends to persist in the kidney and can be shed for weeks to months after infection. certain serovars are more frequently associated with hepatic involvement and include leptospira icterohaemorrhagiae and l. pomona. young dogs (ͻ6 months of age) seem to develop signs of hepatic dysfunction more frequently in disease outbreaks. 14 profound hepatic dysfunction may occur without significant histologic changes because of subcellular damage produced by bacterial toxins. the endothelial damage, subsequent thrombosis, and possible disseminated intravascular coagulation seen in acute disease may contribute to hepatic damage. chronic hepatitis has been reported as a sequelae to leptospiral infection. 15, 16 diagnosis is usually made based on clinical signs and serologic titers. however, leptospirosis polymerase chain reaction (pcr) performed be-fore treatment may increase testing sensitivity given vaccinal interference and delayed seroconversion in the acute phase. 17 penicillins are the treatment of choice in the acute phase and must be followed by appropriate antibiotics to eliminate the carrier state. alternatively, doxycycline may be used for both the acute and carrier states. bartonella spp are gram-negative fastidious bacteria and are well adapted for the intracellular environment. a recent case report documented b. henselae and b. clarridgeiae dna in the liver of 2 dogs with granulomatous inflammation. both had a positive clinical response to azithromycin and demonstrated biochemical reduction in hepatocellular enzymes. 18 another dog with peliosis hepatitis (a rare vascular condition characterized by multiple, randomly distributed blood-filled cavities throughout the liver) had b. henselae dna amplified from multiple hepatic specimens by pcr. 19 helicobacter canis has been isolated from the liver of a single dog with hepatitis. 20 helicobacter spp have also been amplified from hepatic tissue in cats with cholangiohepatitis. further studies are required to determine whether these organisms are associated with inflammatory liver disease. these organisms are difficult to culture, and this failure may reflect the fastidious nature of these bacteria. pcr positivity may reflect the presence of intestinal helicobacter from the enterohepatic circulation or transient colonization rather than a true disease association. 21 francisella tularensis (tularemia) is a pleomorphic, gramnegative, nonspore-forming bacillus. this disease frequently occurs as a result of exposure to ticks or wildlife. macrophages are the primary host cells, and bacteremia with multiorgan involvement is common. lungs, spleen, liver, and skin are common sites for embolic spread, resulting in microabscesses and granulomatous disease. puppies and young cats appear more susceptible to infection, and dogs are generally more resistant to infection. clinical findings include depression, oral/lingual ulceration, regional or generalized lymphadenomegaly, hepatosplenomegaly, panleukopenia with severe toxic neutrophil changes, hyperbilirubinemia, and bilirubinuria. [22] [23] [24] examination for evidence of microscopic agglutinating antibody is most frequently used for diagnosis, although indirect fluorescent antibody testing may be useful as well. 24 aminoglycosides are the primary treatment in humans. however, tetracyclines (doxycycline), chloramphenicol, and quinolones are commonly used in dogs and cats. unfortunately, clinical relapse is common with these antibiotics. tyzzer's disease (clostridium piliforme) is caused by a flagellated, spore-forming, gram-negative intracellular parasite. although spores have been identified in rodent species, interspecies transmission via ingested feces has not been documented. however, spontaneous disease has been documented in dogs and cats. [25] [26] [27] [28] colonization of the liver results in multifocal, periportal hepatic necrosis and may result from a currently unidentified toxin. 29 minimal inflammation may be present despite extensive necrosis. 30 death usually occurs within 24 to 48 hours once the organism is in the liver. [31] [32] [33] [34] [35] [36] [37] rhodococcus is a soil-borne pleomorphic, gram-positive bacteria normally associated with suppurative infections in figure 1 . fine-needle aspirate and cytology from the gallbladder of a cat with cholangiohepatitis. the aspirate consists predominantly of bacteria of mixed type. the bacteria are frequently present in chains (black arrow). also, note dark brown-staining amorphous material (bile pigment: yellow arrow). the finding of bacteria in cytologic specimens of bile is considered abnormal. the following organisms were cultured from the bile: escherichia coli, streptococcus pneumoniae, an anaerobic bacterial rod, prevotella oralis, and a gram-positive rod that could not be classified. courtesy of the pathology department, angell animal medical center, boston, massachusetts. domestic livestock. inhalation from soil or wound inoculation are the suspected routes of transmission. disseminated infection and death have been reported in a single dog. 38 clinical reports are rare in cats. mycobacterium spp are aerobic, nonspore-forming, nonmotile bacteria with a wide host affinity and pathogenic potential. they are typically classified based on growth in culture and by the pathologic production of tubercles or granulomatous disease. mycobacterium tuberculosis and m. bovis are the most pathogenic, and humans are reservoirs for these species. aerosolized organisms in sputum are considered the primary mode of transmission. however, m. bovis can be acquired via uncooked meats and wildlife reservoirs. mycobacterial disease is often subclinical in dogs and cats, but signs may be associated with granuloma formation in various organs. 39, 40 nontuberculous mycobacterium, including those in the mycobacterium avium complex, are saprophytic opportunistic organisms primarily implicated in disseminated disease in cats [41] [42] [43] [44] [45] and occasionally in dogs. 46 -53 no clear associations have been identified with retroviral diseases. canine and feline breeds with potentially increased susceptibility include basset hounds, 51 miniature schnauzers, 53 siamese, 45 and abyssinians. 42 dogs with m. avium complex-induced disease will often demonstrate extensive granulomatous disease of the intestine, spleen, liver, and mesenteric lymph nodes. animals undergoing immunosuppressive drug therapy with inhibition of cell-mediated immunity may be at risk for disseminated disease, including renal transplant patients. 43 acidfast cytology can demonstrate bacilli, although false negatives can occur. negative bacterial images may be identified on routine stains (fig 2) . pcr may provide greater sensitivity and safety than culture. 52 combination therapies are often required, because organisms build resistance quickly, particularly with disseminated disease. although not a risk for immunocompetent individuals, dogs and cats infected with saprophytic mycobacterium pose a risk for immunodeficient people. mycobacterium lepraemurium was considered the main causative agent for feline leprosy until recently. however, m. visibilis has been associated with feline multisystemic granulomatous mycobacteriosis, resulting in diffuse cutaneous disease and widespread dissemination to multiple internal organs. 54 organisms responsible for disseminated fungal infections include histoplasma capsulatum, coccidioides immitis, blastomyces dermatitides, aspergillosis sp, cryptococcussp, and sporothrix schenckii. most are dimorphic, saprophytic, opportunistic fungi that exist in the mycelial stage in the environment. spores are produced in the mycelial stage and be-come pathogenic on inhalation, ingestion, or inoculation. dissemination occurs via the hemolymphatic system. specific environmental conditions are required for the individual organisms, and this dictates their geographic range. histoplasma capsulatum is located primarily in the temperate and subtropical areas of the world. organisms are phagocytized by mononuclear cells and replicate intracellularly once they are inhaled and converted to the yeast phase. the primary clinical signs in dogs are associated with the gastrointestinal system (diarrhea, tenesmus, mucous, fresh bloods in stools). clinical signs in cats are vague. dissemination to other visceral organs (including the liver) has been documented in both species. [55] [56] [57] clinically affected animals are usually young (1-4 years of age). diagnosis is usually achieved with fine-needle aspirate or exfoliative cytology of affected organs. aspergillosis is primarily associated with rhinitis. however, several reports have documented systemic infections in german shepherds and in non-shepherd breeds. aspergillus terreus 58 -62 and a. deflectus 63, 64 have been most frequently implicated in systemic infection. predisposing factors include optimal climatic conditions, access to a partial strain, or subtle defects in mucosal immunity. 65 disseminated aspergillosis has also been documented in cats. 66, 67 neurologic deficits, spinal column pain, urinary system disorders, and respiratory pathology are the primary presenting clinical signs. prototheca is a saprophytic, achlorophyllous alga found in the southeastern united states. three species of prototheca have been identified, but p. zopfii is the only one associated with disseminated disease. the organism is associated with sewage, slime flux of trees, and animal waste. transmission generally occurs through ingestion or penetration of injured skin or mucosa. disease can develop with diminished host resistance or concurrent diseases. 68 concomitant large intestinal diarrhea and ocular signs should prompt clinical consideration of prototheca infection. dissemination via blood or lymph to other organs including the liver is common. various stages of development of the organism may be identified on cytology or histopathology. urine culture and sediment are also useful in organism identification. 69 this disease is invariably fatal, although disease progression may be delayed with various antifungal and antibacterial agents. 70 -73 coccidioides immitus is a dimorphic fungus with preference for the alkaline sandy soil environment found in the lower sonoran life zone in the southwestern united states, western mexico, and central and south america. mycelia are produced during rainfall, but arthroconidia develop with soil drying and become airborne under dry and windy conditions. inhalation is the primary mode of infection in dogs and cats. the spherule (tissue parasitic form) undergoes division with eventual rupture. the severity and extent of clinical disease depend on immunocompetence and range from a mild, asymptomatic, pulmonic form to severe, life-threatening disseminated disease. dissemination most commonly involves the axial and appendicular skeleton and overlying skin. tissues from abdominal viscera, the central nervous system (cns), pericardium, myocardium, and prostate can also be involved. 74, 75 cytology or histology may reveal spherules, although diagnosis is often made based on history, clinical signs, and positive serology. antigens for sero-testing commonly use tube precipitin and complement fixation with agar gel immunodiffusion. sporothrix schenckii causes a chronic granulomatous disease of worldwide distribution. infection is usually the result of trauma and inoculation with infective conidiophores. the skin is the primary target organ. however, disseminated disease has been reported, particularly in the cat. no clear dissemination pattern has been identified because of low case numbers, but affected organs include the internal lymph nodes, liver, lungs, eyes, bone, muscles, and cns. 76, 77 diagnosis is frequently made by cytology. blastomyces dermatitidis is found primarily in mississippi, missouri, the ohio river valley, the mid-atlantic states, and some canadian provinces. growth of the organism requires sandy, acidic soil with some proximity to water. preferred sites for dissemination include the skin, eyes, bones, and lymph nodes, although dissemination to the liver has been reported. 78, 79 cryptococcus neoformans has a worldwide distribution. inhalation may be the primary mode of infection, and sites of infection tend to be areas of the body with cooler temperatures, including the respiratory passages and subcutaneous tissues. the fungus is occasionally disseminated to the kidneys and rarely to the liver. 80 treatment of most disseminated fungal infections involves the use of triazoles, including itraconazole and fluconazole, as well as amphoterocin b. 59,81-85 clinical signs may resolve in many cases, but relapses occur and patients with severe clinical illness generally have a poor prognosis. leishmania, transmitted by the sandfly (lutzomyia in the new world, phlebotymus in the old world), frequently causes cutaneous and visceral lesions in the dog. promastigotes transmitted by the female sandflies become amastigotes in the vertebrate and are phagocytized by mononuclear cells. the organism travels through hemolymph organs to remote dermal sites and other organs. clinical signs will not develop in all exposed animals, and the immune response at the time of infection appears important in determining development of disease. leishmania infection should be considered in dogs from endemic areas with marked hyperglobulinemia or in those with a travel history to endemic areas. mild increases in liver enzymes are often noted. however, unlike the kidneys, the liver is not a primary target organ. infection can be associated with chronic hepatitis. 86 definitive diagnosis is made by demonstration of organisms on cytology or histopathology, or by serology, culture, or pcr. amphotericin b in a soybean oil lipid emulsion has been intravenously administered for higher clinical cure success rates and greater numbers of negative posttreatment parasitologic tests compared with other treatments. other less successful treatment options include allopurinol and the pentavalent antimonials. 87 hepatozoon canis is a worldwide protozoal disease reported in domestic dogs and is most prevalent in subtropical and temperate climates. the primary vector is the rhipicephalus sanguineous tick, which is primarily located in warm and temperate regions. transmission occurs through ingestion of ticks containing mature protozoal oocyts. sporozoites are released in the intestine on tick ingestion and penetrate the gut wall, invade mononuclear cells, and disseminate. target organs include the bone marrow, spleen, and lymph nodes but can involve other internal organs such as the liver, kidney, and lungs. 88, 89 the most striking clinicopathologic abnormality is leukocytosis with evidence of parasitemia of the white cells on peripheral blood smears. clinical findings can range from incidental hematologic findings to severe life-threatening illness. hepatitis, glomerulonephritis, and pneumonitis have all resulted from h. canis infection. 90 coinfections with other protozoal diseases (toxoplasma, leishmania, and babesia spp) or other tick-borne diseases (ehrlichia spp) and immunosuppressive states can predispose animals to clinical illness. the hepatitis is associated with developing meronts within the liver and their associated neutrophilic and mononuclear inflammation. hepatozoon has also been documented in felines. [91] [92] [93] microscopic detection of gamonts in peripheral blood smears is the most frequently used diagnostic test. imidocarb is the treatment of choice in dogs. subcutaneous or intramuscular injections are administered every 14 days until gamonts are no longer visualized in the leukocytes. a new species, hepatozoon americanum, was identified in 1997, with the amblyomma maculatum tick as its definitive host. 94 this emerging disease has spread to the north and the east since its initial identification in the gulf coast region. clinical signs are often severe, even in the absence of other diseases or in the presence of immunosuppression. waxing and waning clinical signs are attributed to repeated cycles of asexual reproduction and pyogranulomatous inflammation. the primary site of infection for the merozoites is the cardiac and skeletal muscle. however, single zoites can enter circulation and reproduce asexually at distant locations. 95 diagnosis is most often made with muscle biopsy, although a recent study has identified promise in the use of pcr testing. 96 an enzyme-linked immunosorbent assay has been developed with sporozoites as the antigen. 97 no treatment effectively eliminates the tissue stages of h. americanum. however, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and an excellent quality of life. 98 the microsporidial parasite encephalitozoon cuniculi is an obligate intracellular protozoan. infection likely occurs by inhalation or ingestion of spores from contaminated urine or feces shed by infected hosts. the organism undergoes asexual reproduction or binary fission after infecting host cells and ruptures, leading to infection of new cells or shedding of resistant spores into the environment. typical organs of localized infection include the kidney, liver, lungs, and brain with resultant granulomatous inflammation. 99, 100 cats and older dogs are not commonly affected, and renal disease predominates in young dogs. cytological examination of fluids (particularly urine) is important in making a diagnosis in animals with disseminated disease as other tests are commercially unavailable. cytauxzoon felis is a tick-borne protozoal disease of domestic and wild cats. the bobcat is the natural reservoir in north america and is usually asymptomatic despite persistent erythroparasitemia. the tissue phase of infection consists of the development of large schizonts in mononuclear phagocytes. the schizonts line the lumens of vessels in most organs, eventually leading to vessel occlusion. merozoites are released into blood or tissue fluid once the host cells rupture and infect red blood cells. late-stage parasitemia can often be detected on blood films at about 1 to 3 days before death. most clinical signs, including those associated with liver abnormalities, are due to schizont-associated mechanical obstruction. however, parasite by-products may also be toxic, pyogenic, and vasoactive. the anemia is regenerative but mild in comparison with clinical icterus. this may be useful in differentiating this infection from hemotropic mycoplasmas. demonstration of piroplasms in wright's-stained or giemsa-stained blood films most frequently provides a definitive diagnosis. histopathology reveals schizont-laden mononuclear phagocytes in the veins of the lungs, liver, and spleen. the prognosis is generally considered poor, but different geographic strains may have varying virulence. 101 treatment with diminazene or imidocarb has been somewhat successful. 102 toxoplasma gondii is an obligate intracellular coccidian parasite that infects almost all warm-blooded animals. do-mestic cats are the definitive hosts and excrete the infective oocyts. three stages of the life cycle are considered infectious, including oocyst sporozoites, tissue cyst tachyzoites, and tissue cyst bradyzoites. transmission can occur through ingestion of oocysts or tissue cysts and via congenital transmission. other reported modes include lactation, transfusions, and transplantation. 103 a higher frequency of disease is reported in dogs and cats fed raw meat or those in a rural/ feral environment. the extra-intestinal life cycle is the same in all hosts, and sporozoites encyst in the intestinal lumen, penetrate cells, and divide into tachyzoites. the tachyzoites can form cysts in the cns, muscle, and visceral organs, and may persist for the life of the host. clinical signs were diverse in 100 cats with histologically confirmed toxoplasmosis, and more than 90% had pulmonary, cns, and liver manifestations. 104, 105 in dogs, disseminated infection is most often associated with canine distemper, other infections including ehrlichiosis and immunosuppression, or vaccination with live attenuated vaccines. 106 clinical cases in cats have been seen with steroids, cyclosporine use, hemotropic mycoplasms, and viral disease. [107] [108] [109] liver and lung involvement is associated with quicker mortality than other organ involvement. tachyzoites may be detected on cytology of various organs and body fluids. however, diagnosis is most frequently based on clinical signs, serology (immunoglobulin g, immunoglobulin m), and response to treatment. clindamycin is the treatment of choice. neospora caninum is a protozoan similar to toxoplasma. dogs and coyotes are considered definitive hosts, and deer and cattle are intermediate hosts. the predominant mode of transmission is transplacental in the dog, and clinical signs are usually secondary to exacerbation of a congenital infection. acute phases of infection include widespread dissemination to many organs, including the liver, whereas chronically infected animals are restricted to muscular and neuronal sites. 103 serology and muscle biopsy often provide a diagnosis, although tachyzoites may be detected in other parasitized tissue or body fluid. 110 sarcocystis canis is an apicomplexan protozoan with no particular geographic distribution. infection results in disseminated disease, including protozoal hepatitis. 111, 112 many reports involve puppies, suggesting the presence of congenital infection. however, the life cycle is still unknown. sarcocystis canis is the only sarcocystis species known to form schizonts in canine tissue. infectious canine hepatitis (ich) is caused by adenovirus type 1. this is the only virus with primary tropism for the liver. 113 infection leads to severe hepatic necrosis and can also cause ocular and renal changes. the virus localizes in the tonsils after oronasal exposure, spreads to regional lymph nodes, and disseminates via the thoracic duct. hepatic parenchymal cells and vascular endothelial cells are the prime targets of viral localization, and injury leading to centrilobular to panlobular hepatic necrosis ranges from self-limiting to fatal. most affected dogs are less than 1 year of age and unvaccinated. severely affected dogs can become moribund and die within hours of disease onset and with few predictive clinical signs. if patients survive the acute phase, they may develop clinical signs including vomiting, diarrhea, and abdominal pain. 114, 115 those that survive may go on to develop chronic hepatitis and fibrosis, likely secondary to self-perpetuating liver inflammation rather than chronic infection. 116 diagnosis is frequently made based on clinical signs and serology, although the virus can be isolated in cell cultures. this disease is rarely encountered because of the high efficacy of vaccination. canine acidophil hepatitis is believed to be caused by a viral agent. however, the specific agent is not yet identified. disease has been reproduced via injections of sterile liver homongenates from spontaneously affected animals. acute infections can lead to acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. diagnosis is made on histology because acidophils are scattered throughout lesions. this disease has only been reported in great britain. 117, 118 canine herpesvirus causes tissue necrosis and localized mucosal or generalized systemic infections in young or immunocompromised animals. the virus only infects dogs because of specific cell-surface receptors. replication occurs via viral dna synthesis within the host nucleus. transmission occurs through direct contact with mucosal secretions from the respiratory or genital tract of animals. factors predisposing to infection in puppies include hypothermia and a poorly developed immune system. newborns can acquire disease in utero, during passage through the birth canal, during contact with infected littermates, from oronasal secretions of the dam, and from fomites. puppies less than 1 week of age are more susceptible to generalized fatal infections. dissemination leads to hemorrhagic necrosis in several organs including the adrenal glands, kidney, liver, lungs, and spleen. clinical signs include loss of interest in nursing, loss of body weight, soft yellow-green feces, abdominal discomfort, and dullness. a marked increase in alanine aminotransferase is often noted on biochemistry profile. definitive diagnosis is by viral isolation. 119 feline leukemia virus is a single-stranded retrovirus that replicates in many tissues. clinical illness is generally related to the hematopoietic system and the immune system. feline leukemia virus has also been associated with icterus and various inflammatory and degenerative liver diseases including focal liver necrosis. 120 feline infectious peritonitis (fip) is a feline coronavirus that has undergone frequent rna mutations, resulting in an ability to enter and replicate in macrophages. an immunemediated vasculitis occurs if the virus is not eliminated. affected cats develop signs related to target organ lesions (kidney, liver, cns, intestine) or due to fluid redistribution. abnormal liver enzymes can occur because of hepatitis, hepatic lipidosis, or prehepatic sequalae of vasculitis, erythrocyte destruction, and hypoxia. hyperbilirubinemia is common and usually secondary to vasculitis in the liver. 121 histopathology is required for definitive diagnosis but is sup-ported by history, physical examination, and laboratory findings. a new pcr test may also prove useful in the diagnosis of fip. 122 treatment is generally unrewarding. conflicting information exists on the usefulness of feline recombinant interferon, although it may be beneficial for a subpopulation of fip-infected cats. 123, 124 rickettsial diseases the most common agents encountered in dogs with clinical evidence of liver involvement include the ehrlichia sp, rickettsia rickettsii, and borrelia burgdorferi. these organisms can infect either hepatocytes or endothelial cells. hepatic involvement in erhlichia infections occurs in more than 80% of human patients, leading to mild transient increases in transaminases. 125 liver injury may be related to organism proliferation in hepatocytes and stimulation of immunologic and nonspecific inflammatory mechanisms. rocky mountain spotted fever is vasculotropic in nature and can cause moderate increases in transaminases. experimental evidence with borrelia suggests direct hepatic invasion by the spirochetes in conjunction with cellular and humoral immunologic mechanisms. 126 an association with borrelia was observed and confirmed with liver biopsy in 2 dogs. lesions were consistent with lobular dissecting hepatitis and mixed multifocal inflammation leading to focal pyogranulomas in the other. 2 chronic cholangitis associated with liver fluke infestation in endemic areas is primarily observed in cats and less frequently in dogs. most infections are due to opistorchus and metorchis, which require 2 intermediate hosts. the first hosts are water snails, and the second hosts include a wide variety of fish with encysted metacercariae. the final host acquires infection by ingestion of fish, and the young liver flukes migrate to the liver through the bile ducts. this results in bile duct thickening and dilation. rarely, cysts may be formed as well. 127 a slight to moderate inflammation may be seen both within the ducts and in the portal areas. although eosinophils may be present, they are usually limited in numbers. the number of liver flukes and eggs within the dilated bile ducts varies markedly, and often only limited evidence of liver flukes or eggs is identified. platynosomum concinnum is a trematode of the feline biliary system. terrestrial snails, lizards, toads, and terrestrial isopods act as intermediate hosts based on geographic location. disease is most prevalent in the tropical and subtropical climates. clinical cases involve adult indoor or indoor-outdoor cats. the severity of clinical signs is proportional to the number of adult flukes as well as to the duration of parasitemia. early diagnosis can be difficult. however, diagnosis is easier when eggs have been identified in the bile. 128 treatment of p. concinnum and liver fluke infections is best accomplished with praziquantel. there are many infectious diseases that ultimately affect the liver. few, however, have primary tropism for hepatic tissue. testing should be directed based on signalment, geographic locale, and primary presenting complaint. cytology and/or histopathology of the liver will most frequently provide a definitive diagnosis in clinical situations with liver involvement. the prognosis is guarded with many disseminated infections. toxic, metabolic, infectious, and neoplastic liver diseases infectious diseases of the dog and cat detection of portal and systemic bacteremia in dogs with severe induced hepatic disease and multiple portosystemic shunts diseases of the gallbladder and biliary tree cholangitis/cholangiohepatitis complex in the cat bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease: 1998-2003 hepatic abscesses in cats: 14 cases hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options liver lobe torsion and liver abscess in a dog hepatic abscesses associated with diabetes mellitus in two dogs hepatocellular carcinoma with secondary abscessation in a cat hepatic abscesses in dogs hepatic abscesses in dogs: 14 cases (1982-1994) infectious diseases of the dog and cat chronic active hepatitis in dogs associated with leptospires chronic hepatitis associated with leptospiral infection in vaccinated beagles clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease peliosis hepatis in a dog infected with bartonella henselae helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis association of helicobacter with cholangiohepatitis in cats feline tularemia on tularemia in a cat acute tularemia in three domestic cats bacillus piliformis infection in an adult dog tyzzer's disease in kittens with familial primary hyperlipoproteinaemia tyzzer's disease in a puppy tyzzer's disease in puppies infectious diseases of the dog and cat the liver in systemic disease. an innocent bystander naturally occurring tyzzer's disease as a complication of distemper and mycotic pneumonia in a dog tyzzer's disease in a dog tyzzer's disease in an adult cat naturally occurring tyzzer's disease in a cat naturally occurring tyzzer's disease in a puppy tyzzer's disease complicated with distemper in a puppy tyzzer's disease in puppies vapa-negative rhodococcus equi in a dog with necrotizing pyogranulomatous hepatitis, osteomyelitis, and myositis mycobacterium tuberculosis complex infection in a dog putative transmission of mycobacterium tuberculosis infection from a human to a dog disseminated mycobacterium avium infection in a cat disseminated mycobacterium avium infection in young cats: overrepresentation of abyssinian cats disseminated mycobacterium avium complex infection following renal transplantation in a cat tuberculosis in cats disseminated mycobacterium avium complex infection in three siamese cats fatal mycobacteriosis with hepatosplenomegaly in a young dog due to mycobacterium avium a case of disseminated tuberculosis in a dog caused by mycobacterium avium-intracellulare systemic mycobacterium smegmatis infection in a dog disseminated mycobacterium avium infection in a dog disseminated mycobacterium avium complex infection in a dog tuberculosis in five basset hounds systemic mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat disseminated mycobacterium avium infection in three miniature schnauzer litter mates histologic and genotypic characterization of a novel mycobacterium species found in three cats atypical histoplasma capsulatum infection in a dog disseminated histoplasmosis in dogs: 12 cases (1981-1986) disseminated histoplasmosis in cats: 12 cases (1981-1986) disseminated aspergillosis in two dogs in israel long-term survival of four dogs with disseminated aspergillus terreus infection treated with itraconazole disseminated aspergillus terreus infection in a dog disseminated aspergillosis in a dog with diskospondylitis and neurologic deficits disseminated aspergillosis in a dog systemic mycosis due to aspergillus deflectus in a dog disseminated aspergillosis attributable to aspergillus deflectus in a springer spaniel canine disseminated aspergillosis systemic aspergillosis and mucormycosis in 23 cats feline disseminated aspergillosis altered immune function in a dog with disseminated protothecosis urinary tract manifestations of protothecosis in dogs more than meets the eye: subretinal aspirate from an acutely blind dog disseminated protothecosis causing acute blindness and deafness in a dog disseminated protothecosis in a dog infectious diseases of the dog and cat deep mycotic infections in cats disseminated coccidioidomycosis in a dog pathology of sporotrichosis in 10 cats in rio de janeiro disseminated sporotrichosis in a cat ocular changes in a cat with disseminated blastomycosis legendre am: blastomycosis, in greene ce fatal disseminated cryptococcosis and concurrent ehrlichiosis in a dog disseminated opportunistic fungal disease in dogs: 10 cases infectious diseases of the dog and cat cryptococcosis coccidioidomycosis and paracoccidioidomycosis infectious diseases of the dog and cat chronic hepatitis associated with canine leishmaniosis (leishmania infantum): a clinicopathological study of 26 cases initial and long term efficacy of a lipid emulsion of amphotericin b desoxycholate in the management of canine leishmaniasis hepatozoon canis infection in two dogs canine hepatozoonosis in oklahoma hepatozoon canis infection feline hepatozoonosis granulomatous cholangiohepatitis in a cat due to a protozoan parasite resembling hepatozoon canis hepatozoon species infection in domestic cats: a retrospective study a new hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in north america characterization of stages of hepatozoon americanum and of parasitized canine host cells diagnosis of canine hepatozoon spp. infection by quantitative pcr an indirect enzymelinked immunosorbent assay for diagnosis of american canine hepatozoonosis treatment of dogs infected with hepatozoon americanum: 53 cases (1989-1998) mammalian microsporidiosis experimental encephalitozoonosis in neonatal dogs cats surviving natural infection with cytauxzoon felis: 18 cases (1997-1998) administration of diminazene aceturate or imidocarb dipropionate for treatment of cytauxzoonosis in cats toxoplasmosis and neosporosis infectious diseases of the dog and cat acute primary toxoplasmic hepatitis in an adult cat shedding toxoplasma gondii oocysts fatal toxoplasmosis in five cats acute toxoplasmosis following renal transplantation in three cats and a dog feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis neonatal toxoplasmosis in littermate cats histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952-1990) neospora caninum associated with septic peritonitis in an adult dog fatal cutaneous and visceral infection in a rottweiler dog associated with a sarcocystis-like protozoan fatal hepatic sarcocystosis in a puppy with eosinophilia and eosinophilic peritoneal effusion canine viral diseases viral hepatitis of dogs (rubarth's disease) infectious canine hepatitis (hepatitis contagiosa canis use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis a new transmissible agent causing acute hepatitis, chronic hepatitis, and cirrhosis in dogs persistent hepatitis and chronic fibrosis induced by canine acidophil cell hepatitis virus canine herpesvirus frequency and significance of feline leukemia virus infection in necropsied cats feline infectious peritonitis and feline enteric coronavirus feline infectious peritonitis: typical findings and a new pcr test use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis effect of feline interferonomega on the survival time and quality of life of cats with feline infectious peritonitis ehrlichial diseases of humans: emerging tickborne infections host-pathogen interactions in the immunopathogenesis of lyme disease severe cholestatic liver disease secondary to liver fluke key: cord-016130-5q9ufu28 authors: linday, linda a. title: nutritional supplements and upper respiratory tract illnesses in young children in the united states date: 2010-12-17 journal: preventive nutrition doi: 10.1007/978-1-59259-880-9_21 sha: doc_id: 16130 cord_uid: 5q9ufu28 key points: in the united states, children have lower blood levels than adults of eicosapentaenoic acid (epa), an important ω-3 fatty acid that helps decrease inflammation; vitamin a, the “anti-infective” vitamin; and selenium (se), a trace metal that is an intrinsic part of glutathione peroxidase, an important free-radical scavenging enzyme. epa, vitamin a, and se are important in controlling inflammation and can be supplied by oral nutritional supplements. cod liver oil contains epa (and other important ω-3 fatty acids), and vitamin a as well as vitamin d. fish oil contains ω-3 fatty acids (including epa) but no vitamins. our clinical research demonstrates that daily supplementation with a flavored cod liver oil (which meets european purity standards) and a children’s multivitamin-mineral with trace metals, including se, can decrease morbidity from upper respiratory tract illnesses, otitis media, and sinusitis in young children living in the united states. these supplements can be used by practitioners on an individual basis, when clinically indicated; the supplements can be purchased in the united states without a prescription. socioeconomically disadvantaged children are at risk for micronutrient deficiencies. however, their families may not be able to afford to purchase these supplements, which are not available through medicaid, the special supplemental nutrition program for women, infants and children, or the food stamp program. if our results are confirmed in larger studies, a system change will be needed to provide these supplements to nutritionally vulnerable, socioeconomically disadvantaged children living in the united states. key element in the pathophysiology of these disorders. this chapter discusses the role of essential fatty acids, vitamins, and trace metals in the pathophysiology of inflammation; reviews our clinical research on the use of a lemon-flavored cod liver oil (which meets european purity standards) and a children's chewable multivitamin-mineral with se for the prevention and adjunctive treatment of these disorders; reviews the history of cod liver oil, including its importance in the discovery of vitamin d and the anti-infective properties of vitamin a; and discusses the current clinical use of these supplements. if additional research confirms the utility of these supplements in improving the health of young children, the problem of access to these supplements by socioeconomically disadvantaged children in the united states will need to be addressed. children under age 5 yr had an average of 20.7 million ambulatory care visits per year for upper respiratory conditions in the united states from 1993 to 1995, with another 14.5 million visits per year for om during the same time period (1) . in addition to their cost, unnecessary health care visits for the treatment of colds generate a significant number of inappropriate antibiotic prescriptions (2, 3) , although the situation is now improving (4, 5) . bacterial antibiotic resistance is considered to be a major public health problem in the united states, and an interagency federal action plan has identified the decrease of unnecessary antibiotic prescriptions as critical to combating antimicrobial resistance (6) . in 1996, gates (7) estimated that the total annual cost of treating om with effusion in the united states was $5 billion, including both direct and indirect costs. this estimate included the cost of surgical placement of tympanostomy tubes (ventilation tubes placed in the tympanic membrane of the ear), a procedure that is commonly performed for the treatment of this disorder (8) . frequent om with effusion in early childhood may be associated with later speech and language problems, although causative relationships have not been definitively established (9, 10) . sinusitis also is a common and costly condition (11) ; in 1996, overall health care expenditures for sinusitis in the united states were estimated to be $5.8 billion, of which $1.8 billion was for children age 12 yr or younger. the primary treatment of sinusitis in children is medical management. adenoidectomy may be helpful (12) , although endoscopic sinus surgery is reserved for chronic, refractory cases (13, 14) . chronic sinusitis has a major negative impact on the quality of life of children whose disease is sufficiently severe to require endoscopic sinus surgery (15). viral illnesses usually are brief and self-limited conditions. however, viral infections produce inflammation, enhance nasopharyngeal bacterial colonization and adherence, alter the host's immune defenses, and are associated with bacterial complications, including acute om (16) , sinusitis, and pneumonia (17, 18) . acute om occurs in about 20% of children with viral upper respiratory infections (17) . although viral vaccines are in development, the use of vaccines to prevent upper respiratory tract infections is hampered by the large number of different viruses causing these infections (17) . the us advisory committee on immunization practices voted to recommend influenza vaccination for children ages 6 to 23 mo for the coming influenza season of 2004 to 2005; the impact of this recommendation remains to be seen (19) . despite the fact that streptococcus pneumoniae is the most common cause of bacterial acute om, the heptavalent pneumococcal polysaccharide conjugate vaccine only produced a 6% reduction in the overall number of episodes of acute om from any cause (20) . of inflammatory eicosanoids (29) . the level of ω-3 fatty acids and other pufas is largely determined by diet, unlike proteins, whose structure is genetically determined (26, 30) . changes in dietary habits in the united states in the last 20 to 30 yr have markedly increased the amount of ω-6 efa consumed (as in vegetable oils), whereas the amount of consumed ω-3 efa (as in cod liver oil and fish oil) has decreased (26, 31, 32) . the optimal ratio of ω-6/ω-3 efa in the diet is 3 to 4:1; in the united states, it is currently 10 to 20:1. this abnormal ratio has been linked with numerous disease states, especially those associated with inflammation. ω-3 fatty acid levels can be increased by eating more fatty fish and by consuming nutritional supplements such as cod liver oil, algalderived long-chain fatty acids, or fish oils (26, 31, 32) . free radical-induced lipid peroxidation may play a role in the acute (33) (34) (35) and chronic inflammation (36) of a guinea pig model of acute, unilateral om caused by infection with s. pneumoniae. inflammatory mediators have been demonstrated in both experimental and human middle ear effusions; these mediators include leukotrienes and prostaglandins, which are metabolites of aa and are derived from phospholipids in cell membranes. treatment with specific inhibitors of these mediators has prevented the development of om in some animal models of om (37) . free radicals are significant components of the inflammatory response, which is part of the pathophysiology of pneumococcal infections (38) and the influenza a virus (39) ; the latter are important causes of om. reactive oxygen species (ros) (40) have also been implicated in sinusitis. the association between respiratory virus infections and acute om in children is well-established (41, 42) . oral supplementation with efas (43) and zinc (44) has been shown to decrease the incidence of respiratory infections in children. trace elements (including zinc and se) have been shown to have important effects on the regulation of immune responses (45) . supplementation with ω-3 fatty acids has been reported to be beneficial in preventing infection in surgical patients (46) . the importance of ω-3 fatty acids and trace metals (including zinc and se) is already recognized in the relatively new field of "immunonutrition" (47) (48) (49) . the clinical efficacy of antioxidants in the treatment of om has been reported by two groups of russian investigators (50, 51) . of interest is the work of ginsburg (52) , who proposed that the main cause of tissue damage in infectious and inflammatory conditions is synergistic interactions among ros, microbial hemolysins, enzymes, and cytokines. antibiotics may have anti-inflammatory actions in additional to their antibacterial effects (53, 54) . macrolide antibiotics, effective in the treatment of adults with chronic rhinosinusitis (55) , have anti-inflammatory properties that contribute to this effect. however, the overuse of antibiotics is associated with the development of bacterial antibiotic resistance. in finland, the prevalence of macrolide-resistant group a streptococci diminished after the heavy use of macrolide antibiotics decreased (56) . in this age of antibiotic resistance, it is preferable to use antibiotics for their antibacterial effects rather than as anti-inflammatory agents. in our first study (57) , we obtained blood samples from 44 children undergoing clinically indicated ambulatory surgery at the new york eye & ear infirmary (nyee). there were 39 subjects in the tympanostomy tube group (tt); these children were undergoing placement of tympanostomy tubes for frequent ear infections and/or persistent middle ear effusion, with or without concomitant adenoidectomy and/or tonsillectomy. their mean (± standard deviation [sd]) age was 3.8 ± 1.8 yr; 72% were male; approximately half were hispanic and half were white; approximately half were private patients; and almost half were taking vitamin supplements. the comparison group (comp) was composed of children undergoing eye-muscle surgery as well as those undergoing ear, nose, and throat procedures such as bronchoscopy or laryngoscopy that did not involve the ears, adenoids, or tonsils. these subjects were slightly older, with a mean age of 5.7 ± 1.8 yr, and there was a lower percentage (20%) of private patients. no demographical information was available for the six adults in the adult control group (ac), supplied by ann moser of the peroxisomal disease section of the kennedy krieger institute genetics laboratory (baltimore, maryland). data regarding red blood cell (rbc) fatty acids, trace metals, and vitamin a were available for subsets of these subjects. the rbc fatty acid data are summarized in table 1 . the mean values for epa were lower in both groups of nyee children than in the acs supplied by kennedy krieger. the mean rbc epa values were: (a) tt = 0.31% ± 0.02% (standard error [se]), (n = 16); (b) comp = 0.31% ± 0.04% (se), (n = 5); and (c) ac = 0.48% ± 0.4% (se), (n = 6). these differences were statistically significant when analyzed by both parametric (anova, p < 0.002; f[2,24] = 8.336) and nonparametric (kruskal-wallis anova by ranks, p = 0.007; h[2, n = 27] = 9.924) tests. no other significant differences in rbc fatty acids were noted among the three groups, although additional differences might become apparent with larger sample sizes. in 1986, japanese investigators reported lower plasma levels of ω-3 fatty acids in young normal and atopic children than in adults (58) . they ascribed these findings to dietary changes in japan in the 30 yr prior to the study. data for plasma se, zinc, and copper for the children in the tt group are summarized in table 2 ; data from the published literature for children (59) and adults (25) for these parameters is also included, as are international system units. there was no statistically significant difference between the mean plasma se for study subjects (tt = 110 ng/ml ± 16.3 sd; n = 39) and the published values for children. both groups of children had lower se levels than published values for adults (p < 0.005, anova; f = 20.442; f[0.005] 3140 = 4.47) (60). statistical analyses were performed only for se because only the variances for se were homogeneous (bartlett's test for homogeneity of variances (60). the mean plasma vitamin a (retinol) level for the tt group was 39.1 μg/dl ± 10.8 (sd); (range: 23.6-72.1 μg/dl; n = 39). (multiply conventional units by 0.0349 to convert to international units [61] ). ballew (62) defined an inadequate vitamin a level as less than 20 μg/dl and a suboptimal vitamin a level as less than 30 μg/dl. the upper limit of the pediatric reference range for vitamin a is 80 μg/dl (61) . therefore, overall, the values for our subjects were within the reference range. russell (63) stated that a vitamin a level of 40 μg/dl predicted normal dark adaptation 95% of the time; 69% (27/39) of our subjects had vitamin a levels less than or equal to 40 μg/dl, although parents denied symptoms of night blindness for all children. our finding is consistent with ballew's (62) report that the 75th percentile for serum retinol levels in children ages 4 to 8 yr was 39.0 μg/dl. in addition, 15% (6/39) of our sample had suboptimal levels (<30 μg/dl), although none were inadequate (62) . in this suboptimal group, five of six children were not taking vitamin supplements, five of six children were hispanic, five of six children were general service patients, and four of six children were female (all of whom were hispanic). the overrepresentation of hispanic children in the subgroup of children with suboptimal vitamin a levels was also consistent with previous reports (62) . on a group basis, there was no statistically significant difference in the mean vitamin a levels between the subgroup of children whose families reported that they were taking vitamin supplements (39.7 μg/dl ± 11.6 sd; n = 17) and the subgroup of children whose families reported that they were not taking vitamin supplements (38.7 μg/dl ± 10.4 sd; n = 22). our subjects took a variety of prescription and over-the-counter vitamin preparations; no children were receiving or had taken cod liver oil; only one child in the fatty acid subgroup had a history of fish oil ingestion. of the eight different children's vitamin preparations examined, all contained vitamin a palmitate, vitamin a acetate, and/or β-carotene. the blood-level data (57) revealed that (a) study children had lower levels of rbc epa than adult controls; (b) 69% of study children had plasma vitamin a (retinol) levels in the lower reference range, with 15% in the suboptimal range; and (c) study subjects, like other children, had lower levels of plasma se than adults. therefore, for our clinical studies, we chose cod liver oil as a source of both vitamin a and epa and used it in conjunction with a marketed children's chewable multivitamin/mineral preparation containing se. we specifically chose cod liver oil as a source of vitamin a on the basis of our blood-level data, which revealed no significant difference in the vitamin a levels between the subgroups of children who were taking vitamin supplements and those who were not. although we could have used fish oil as the source of epa, fish oil does not contain vitamin a or d. the detailed contents of these supplements are shown in table 3 . the vitamin a content of cod liver oil used in our initial pilot study on om was 2000 to 2500 iu/teaspoon (5 ml); in the subsequent two studies, the vitamin a content was decreased to 1000 to 1250 iu/5 ml. to explore the clinical utility of these supplements, we then performed an open-label secondary prevention study, in which each child served as his or her own control (64) . we studied one om season, from september 1, 2000 to march 31, 2001 (57) . all children were patients of the soho pediatrics group, a private group practice in lower manhattan, new york. children were required to have had at least one episode of om from september 1 to november 30, 2000 (the early portion of om season under consideration). children with a known allergy to fish were excluded. eight children were enrolled, ranging in age from 0.8 to 4.4 yr; seven were caucasian, half were female, and all families were english-speaking. after enrollment, subjects received 1 teaspoon of lemon-flavored norwegian cod liver oil and one-half of a tablet of carlson's scooter rabbit chewable multivitamin-mineral (mvm) tablet per day (see table 3 ). mothers were instructed to crush the mvm tablet, measure the cod liver oil, and mix both in a small amount of food (such as applesauce, yogurt, or rice cereal) to administer the supplements to their children. parents were informed verbally and in writing that supplements were to be given only in the amounts required by the study and that study supplements were to be kept out of reach of children. of the eight children who entered the study, one could not tolerate the taste of cod liver oil. the remaining seven children received antibiotics for om for 12.3 ± 13.4 (p < 0.05; mean ± sd) fewer days during supplementation than before supplementation during the om season under study (see fig. 1 ). five of seven subjects had no additional episodes of om during supplementation, although it had no apparent effect on established serous middle ear effusions in two children. however, because our study lacked a randomized, parallel control group, we could not exclude the possibility that the decreased antibiotic usage we found might have occurred without the use of study supplements. based on our prior research (discussed in ref. 57 ) and the historical studies on cod liver oil and upper respiratory illnesses (65), we hypothesized that use of the study supplements by young children would decrease their doctor visits for upper respiratory illnesses during the late fall, winter, and early spring. we studied the effect of daily use of these supplements on the number of pediatric visits by young, inner-city, latino children from late autumn 2002 to early spring 2003 (83). we did not have a matched placebo for liquid cod liver oil. although adults and older children can swallow capsules, infants and toddlers cannot. furthermore, if capsules were cut open to administer the contents, the distinctive odor and taste of cod liver oil would immediately become apparent (57) . the absence of a matching placebo for liquid cod liver oil precluded our performing a classical double-blind, placebo-controlled study. lack of a placebo coupled with the fact that cod liver oil can be purchased without a prescription by interested parties (57, 66) led us to choose a study design in which we randomized pediatric sites, rather than individual patients. this type of design has been used in the worldwide studies of vitamin a supplementation, which are discussed in subheading section 5.3, that have included randomization by ward, household, village, or district (67) . it was also used in a food-consumption study to avoid changes in food habits resulting from knowledge of the other treatment (68) . randomized site design is commonly used in behavioral and educational studies where no placebo is possible (69) (70) (71) (72) ; a recent study of herd immunity and the pediatric heptavalent pneumococcal vaccine also used a randomized site design (73) . to minimize the influence of the study on the behavior of the participating families (69), we used a "no-contact" control group (74, 75) , which has also been used in behavioral and educational studies. the study was performed at pediatrics 2000, a multisite, private, pediatric group practice in new york city. two of the offices with similar demographics (low-income latino families), located 1.1 miles apart in upper manhattan, were randomized to a supplementation site and a medical records control site. study participants were children ages 6 mo to 5 yr of either gender and any race, religion, or nationality who were patients enrolled at the two offices where the study was being performed. study participants were required to be in new york city from enrollment through april 2003 (with the exception of brief vacations), and to have some type of medical insurance. patients who routinely received additional health care at other practices or medical centers were excluded; children with known fish allergy, a chronic, life-threatening condition (such as hiv/aids or cancer), feeding disorders, and epilepsy were also excluded. study materials were available in both english and spanish. per practice routine, two professional coders reviewed all charts from both sites, coded the visits, and entered the data into a computer with ndc medisoft™ network professional 7.02 software (76) . participants in the medical records control group were enrolled from october 21 to november 10, 2002; those in the supplementation group were enrolled from november 11 to december 12, 2002. we were unable to randomize enrollment at the two sites because the lemon-flavored cod liver oil used in the study (which was manufactured in norway) had been reformulated with less vitamin a (77) and was delayed in the us customs office. the study follow-up/supplementation period ended on may 1, 2003. a total of 94 children (47 at each site) were enrolled in the study. the mean age of the supplementation group was 2.03 yr (±1.04 sd), and the mean age of the control group was 2.08 yr (±1.10 sd). there were no statistically significant differences in the demographical characteristics of the study participants in the two groups: most were latino children from low-income families (as indicated by health insurance), and their mothers were predominantly unmarried immigrants from the dominican republic whose first language was spanish. children of at least 1 yr of age received 1 teaspoon of carlson's lemon-flavored cod liver oil per day and one-half tablet of carlson's scooter rabbit chewable mvm, the same doses used in our previous research administered in the same manner (57) . however, the vitamin a content of the cod liver oil was approximately half that used in our first pilot study of om (see table 3 ). thus, the full dose of supplements provided a total of 3750 iu of vitamin a and 700 iu of vitamin d per day. however, in the current study, the starting dose of supplements was halved for children ages 6 mo to 1 yr .visits were classified as upper respiratory visits, other illness visits, or visits not analyzed on the basis of the icd-9 visit code (78) . the primary outcome measure was upper respiratory visits during the follow-up/supplementation period; other illness visits during the same time period were considered as secondary outcome measures. as shown in figs. 2 and 3 and table 4 , the supplementation group had a statistically significant decrease in the mean number of upper respiratory visits over the course of the follow-up/supplementation period (p = 0.042; r = 0.893; r 2 = 0.797; y = 0.602 −0.002x), whereas the medical records control group had no change in this parameter (p = 0.999; r = 0.0006; r 2 = 0.0000; y = 0.259 + 1.43 × 10 -6 x). there was no statistically significant change in the mean number of other illness visits for either study group during the same time period. although there was a significant difference in the pattern of decreasing upper respiratory visits over time in the supplementation group, there was no difference in the total number of visits made by the two groups. data were analyzed on an intention-to-treat basis. as reported by their parents, 70% of our subjects completed a 5-to 6-mo course of lemon-flavored cod liver oil. by comparison, only 47% of families reported compliance with antibiotic prophylaxis for om in a study of latino children who attended an otolaryngology clinic (79) . our favorable compliance rates may partly result from the fact that young children in the dominican republic are often given cod liver oil or similar supplements, although families rarely continue this practice after moving to the united states. inflammation and edema of the sinonasal mucosa are important in the pathophysiology of sinusitis. based on our previous research and the similarities between om and sinusitis (80), we hypothesized that these nutritional supplements would also be effective adjunctive therapy for the treatment of children with chronic and/or recurrent sinusitis. therefore, we performed a 4-mo, open-label, dose-titration study in which each patient served as his or her own control (64, 81) . study participants were private pediatric otolaryngology outpatients of jay n. dolitsky, md, who resided in the new york metropolitan area and had a clinical diagnosis of chronic and/or recurrent sinusitis as well as symptoms of at least 3 mo of duration that were refractory to treatment with antibiotics. subjects were between ages 2 and 18 yr, of either gender and any race, religion, or nationality. children with known allergy to fish; chronic, life-threatening condition (such as hiv/aids or cancer); feeding disorder; seizure disorder; known cystic fibrosis; aspirin-intolerant asthma; and family plans to move outside the metropolitan area during the course of the study were excluded. subjects were enrolled from late january to early march 2003 and received supplements for 4 mo from the time of enrollment. primary endpoints were the number of doctor visits for acute respiratory illnesses and the child's sinus symptoms, which were quantified using a pediatric sinusitis symptom questionnaire (82) . the starting dose of supplements in the current study was the same as in our previous research (57): 1 teaspoon (5 ml) of carlson's lemon-flavored cod liver oil and one-half of a tablet of carlson's scooter rabbit chewable multivitamin-mineral per day (providing a total of 3750 iu of vitamin a and 700 iu of vitamin d per day). the vitamin a content of the cod liver oil was lower than in our first pilot study of om (57) but was the same as that used in our study of latino children (83; see table 3 ). supplement doses could be doubled to an intermediate dose (providing 7500 iu of vitamin a and 1400 iu of vitamin d per day) within 2 to 3 wk . if higher doses were needed, cod liver oil was discontinued and fish oil was administered instead (fish oil does not contain vitamin a or d). the maximum dose of fish oil was 3 g/d, and the maximum dose of multivitamin-minerals was four half-tablets per day (providing 10,000 iu of vitamin a and 800 iu of vitamin d per day). the titrated doses of vitamins a and d were higher than those used in our previous study (57) but were well-below the lowest daily toxic doses of these vitamins (61) (19,860 iu/d for vitamin a and 2800 iu/d for vitamin d). the us food and drug administration (fda) considers fish oil at dosages up to 3 g per day as safe for adults and children (84) . our four subjects were caucasian males, ranging in age from 4.2 to 9.8 yr, with chronic/recurrent sinusitis for at least 3 yr prior to entry in the study. three subjects had a positive response; one subject dropped out for administrative reasons. the responders had decreased sinus symptoms, fewer episodes of acute sinusitis, and fewer doctor visits for acute illnesses at 4, 6, and 8 wk after beginning study supplements. their parents reported that they had begun to recover from upper respiratory illnesses without complications, which was unusual for these children, as was improvement in springtime; their improvement had previously been limited to the summer months or periods of home-schooling. our findings are consistent with prior work by other clinical investigators. in a study of upper respiratory tract infections in young children, wald and colleagues (85) noted that an inflamed respiratory mucosa may not completely recover between episodes of infection. parsons (86) hypothesized that inflammation and edema of the sinonasal mucosa was the primary event in sinusitis, with bacterial infection as a secondary phenomenon. chronic/recurrent sinusitis is a debilitating disorder that may require treatment with intravenous antibiotics and/or endoscopic surgery. use of these supplements as adjunctive therapy for children with chronic/recurrent sinusitis is an inexpensive, noninvasive intervention that clinicians can use for selected patients, pending the outcomes of definitive, large, well-controlled studies. in the 1930s, during the pre-antibiotic era, lipoid aspiration pneumonia was reported with cod liver oil, mineral oil, and egg yolk, which were used at that time to treat sick and debilitated infants (87) . in 1950, caffey (88) reported vitamin a toxicity in children who were mistakenly treated with high-dose, long-term vitamin a administered in highly concentrated fish liver oil preparations that were available at that time. however, none of caffey's patients had received cod liver oil, and the highly concentrated fish liver oil preparations they received are no longer available in the united states. in our clinical studies, parents were instructed to crush the half-tablet of mvm, measure the cod liver oil, and mix both with a small amount of food (such as applesauce, yogurt, or rice cereal) before administering the supplements to their child. additionally, parents were informed both verbally and in writing that supplements were to be given only in the amounts required by the study and that study supplements were to be kept out of reach of children. the principal investigator spoke spanish, and all parental study materials were available in both spanish and english. to date, we have not encountered problems with aspiration or overdose in our studies. there is a clear association between viral respiratory infections and acute exacerbations of asthma in both children and adults (17) . there is also a link between sinusitis and asthma (89, 90) , with rhinovirus infections linked to both sinusitis and exacerbations of asthma (90) . additionally, latino children have a high incidence of asthma (91) . in view of the results of our studies (57, 81, 83) , we believe that these supplements could be clinically useful for young children (particularly latino children) with asthma, and we are currently beginning to organize research in this area. similarly to other countries worldwide (92) , socioeconomically disadvantaged children in the united states are at risk for micronutrient deficiencies (57, 62, 93) . although the supplements used in our research can be purchased in the united states without a prescription, their cost may pose an excessive financial burden to lowincome families. cod liver oil does not have a national drug code number, it is not available through medicaid in new york, and the children's vitamins we have located that are available through this system do not contain se or other trace metals. additionally, cod liver oil is not available through the united states department of agriculture (usda) special supplemental nutrition program for women, infants and children (wic); our request for such availability can be found online at http://www.fns.usda.gov/wic/anprmcomments/ ihp-06.pdf. furthermore, purchase of vitamins with us food stamps is not permitted (see http://www.fns.usda.gov/fsp/faqs.htm#9). if our results are confirmed in larger studies, a system change will be required to provide these supplements to nutritionally vulnerable, socioeconomically disadvantaged children living in the united states. egyptian and greek physicians may have understood the value of liver (high in vitamin a) for the treatment of night blindness, an early ocular manifestation of vitamin a deficiency (94, 95) . the use of fish oils in medicine was mentioned by hippocrates, and pliny discussed the use of dophin liver oil for the treatment of chronic skin eruptions (96) . however, these classical physicians did not appear to know about the use cod liver oil. the coastal fishermen of northern europe apparently used cod liver oil for many years for the treatment of aches and pains (96, 97) . however, the first recorded use of cod liver oil by physicians was from the manchester infirmary in england during the 1780s (96, 97) , where it was found to be very effective for "old pains" and "rheumatism," which were probably cases of osteomalacia (a bone disease of adults) (96) . the pattern of discovery was that the use of cod liver oil by fishing folk and peasants was accidentally observed by a physician, who then tried it and made it known to the medical profession (96) . guy (96) states that there was no further mention of cod liver oil in the english medical literature until its revival in 1841 by bennett, who had observed its use in germany. bennett reported that in holland, cod liver oil had obtained a wide reputation as a cure for rickets (a bone disease of children) "long before its remedial properties were acknowledged by physicians" (see ref. 97, p. 67) . in the 1820s, schenk and schuette published independent reports in the german literature regarding the value of cod liver oil for curing rickets (97) , and schuette reported that he used cod liver oil successfully for 25 yr. cod liver oil for the treatment of rickets was introduced in france by trousseau in the 1830s (97) . the demand for cod liver oil was so great that all types of substitutes were used, and reports of failure, contamination, and substitutes for cod liver oil began to appear in the literature before the middle of the 19th century. vitamin a was discovered as the result of a long, incremental process with contributions by numerous investigators (95, 98) . at the end of the 19th century and the beginning of the 20th century, nutritional theories were tested under well-controlled laboratory conditions through the administration of experimental diets to animals, and specific factors necessary for their growth and survival began to be identified. during this time, frederick hopkins, at cambridge university, proposed that there were "accessory factors" in foods that were necessary for life but that had not been previously identified; casimir funk named these factors "vital amines" or "vitamines" (65) . in 1913, in the same issue of the journal of biological chemistry, two groups independently reported the existence of a fat-soluble factor that was essential for the growth of rats (65, (99) (100) (101) . mccollum and davis of the university of wisconsin (99) demonstrated that after a certain age, the growth of rats was dependent on an ether extract from eggs or butter. using a different experimental diet, osborne and mendel (100) , of yale university, found that there was an "essential accessory factor" in butter needed for the normal growth of rats. this fat-soluble growth factor, originally termed "fat-soluble a," soon became known as "vitamine a" (65). the discovery of vitamin d was closely tied to work on the prevention and treatment of rickets. during the industrial revolution, rickets spread rapidly throughout europe, particularly among the urban poor, who lived in the sunless alleys of factory towns and urban slums (97) . in 1918, mellanby (102) , an english physician and professor of pharmacology, reported the first animal model of rickets, which he developed in puppies. in a simple, two-page report to the physiological society, he noted that the daily administration of foods such as butter, cod liver oil, or 500 cc of milk (among others) was effective in preventing rickets in his model, whereas casein and linseed oil were among the substances that were ineffective. mellanby felt that rickets was a deficiency disease and stated that "the anti-rachitic accessory factor has characters related to the growth accessory factor [vitamin a], although it is not identical with the latter …" (ref. 102, p. xi) . however, mellanby was not able to distinguish these two factors; this was accomplished by mccollum and his new collaborators at johns hopkins university. in the 1920s, mccollum and his colleagues developed a rat model of rickets that could also be cured with cod liver oil. they were then faced with the same question that perplexed mellanby: was the anti-rachitic factor vitamin a, or was it another substance with a similar distribution as fat-soluble vitamin a (103)? it was known that the vitamin a-deficient animals in these studies often developed ocular abnormalities, including dryness of the eyes, corneal ulceration, and blindness, similar to xeropthalmia in humans (65) . additionally, hopkins demonstrated that oxidation destroyed fat-soluble a (103) . using these facts, in 1922, mccollum and his colleagues (104) reported that when cod liver oil was oxidized for 12 or 20 h, it could no longer cure xerophthalmia, although it could prevent rickets. therefore, they concluded that the anti-xerophthalmic and the anti-rachitic properties were a result of two distinct substances, and that the antirachitic factor, which specifically regulated bone metabolism, was the more heat-stable factor. because this was the fourth vitamin to be discovered, mccollum's group named it vitamin d in 1925 (97) . the fact that both exposure to sunlight and cod liver oil could prevent or cure rickets was perplexing and controversial (105) . careful experiments by chick and coworkers (103) , working in vienna from 1919 to 1922, confirmed the value of both cod liver oil and sunlight in the prevention and treatment of rickets in young infants. in 1919, huldschinsky (97), a pediatrician in berlin, used light from a mercuryvapor quartz lamp (which includes ultraviolet [uv] wavelengths) to cure four cases of advanced rickets in children with up to 2 mo of treatment. when huldschinsky exposed one arm of a rachitic child to the uv irradiation, he found that the rickets in the child's other arm was cured to the same degree as in the exposed arm. therefore, he concluded that phototherapy was not a local effect and speculated that as a result of exposure to uv light, something was formed in the skin that was then carried to other sites, where it had its anti-rachitic effect (105) . in 1925, hess and weinstock (97) reached similar conclusions based on experimental work in animals. these theories were confirmed in 1936, when windaus, working in germany, demonstrated that skin contains the natural prehormone of vitamin d, which is converted to vitamin d 3 when the skin is exposed to uv irradiation (including light from a mercury-vapor lamp) (97). historical investigators were well-aware of an association between rickets and respiratory diseases. in their 1917 paper on rickets, hess and unger stated that "rickets is a predisposing cause of these respiratory diseases (pulmonary tuberculosis, pneumonia, and whooping cough)" (ref. 106, p. 1583 ). in her 1927 paper on community control of rickets, eliot stated that "susceptibility to upper respiratory infections, such as colds, bronchitis and pneumonia, is greatly increased in infancy and early childhood by rickets" (ref. 107, p. 114 ). based on prior animal studies and clinical work by german investigators, ellison discounted the contribution of vitamin d in the efficacy of cod liver oil for measles. nonetheless, he acknowledged that "it is possible that some adjuvant effect was obtained from the co-operation of the two factors [vitamins a and d]" (ref. 108, p. 710 ). in a 1936 study of vitamins a and d (individually or combined) for children hospitalized with measles, mackay noted "there is much to indicate that resistance to infections is reduced in children suffering form an overt deficiency of either of these vitamins [vitamin a or d]" (ref. 109, p. 127 ). semba noted that cod liver oil, a rich source of vitamins a and d, was used as a treatment for tuberculosis for more than 100 yr (110) . in the 1840s, charlotte brontë, the author of jane eyre, suffered from tuberculosis, and her treatment included cod liver oil (111) . a 1917 textbook on tuberculosis, although recognizing that there was no specific treatment for tuberculosis at that time, stated that "one of the oldest and best established remedies for the treatment of tuberculosis is cod liver oil" (ref. 112, p. 467); however, the mechanism of action of cod liver oil was unknown. the situation had changed little by 1946, when goldberg's textbook stated that cod liver oil "has been used empirically for many centuries in the treatment of pulmonary tuberculosis without any definite knowledge of its action" (ref. 113 , p. c-81). however, the use of cod liver oil for tuberculosis faded as specific treatments were developed, and "cod liver oil" is not listed in the index of a modern textbook on tuberculosis (114). mellanby (see section 4.3.) had a large colony of dogs that were maintained on experimental diets. in 1926, mellanby reported, "at one period in the course of my experimental investigations on dogs, the work was greatly hampered by the development of an inflammatory condition of the lungs" (ref. 115, p. 518), which was bronchopneumonia. on postmortem examination, the pneumonia was largely restricted to the vitamin adeficient dogs, and he speculated that this might be relevant to respiratory illness in children (65, 115) . in 1928, green and mellanby reported that a deficiency of vitamin a, but not vitamin d, caused increased infections in a rat model, leading them to call vitamin a an "anti-infective" agent; they speculated that this was related to the epithelial changes caused by vitamin a deficiency (116) . in 1932, ellison (65, 108) reported the results of a study of concentrated cod liver oil for children who were hospitalized with measles. ellison was aware of mellanby's work on the anti-infective properties of vitamin a and also knew that vitamin a deficiency damaged epithelial cells in the respiratory tract (117, 118) . ellison specifically chose to study measles because it was "a disease which attacks epithelial defences and whose incidence is greatest in those members of the community who are most likely to be suffering from various grades of vitamin deficiency…the children of the poorest classes" (ref. 108, p. 709). he studied 600 children under age 5 yr who were admitted to the grove hospital (london) with measles. the cases were randomized by ward to treatment with a highly concentrated cod liver oil preparation or a control treatment of standard treatment (no placebo was used). treatment with cod liver oil reduced measles mortality by approximately one-half, from 8.7% in the control group to 3.7% in the treated group (65, 108) . based on animal studies and german clinical work, ellison attributed the efficacy of cod liver oil to vitamin a, although he did concede that some adjuvant effect could have been obtained from the cooperation of the two factors (108) . a subsequent study published in 1936 (65, 109) reported that neither vitamins a and d together nor vitamin d alone had an effect on reducing the mortality rate from measles. however, the control mortality rate in this later study decreased to 2.6%, making it difficult to demonstrate an improvement. by 1940, numerous studies had been conducted to evaluate the ability of vitamin a (usually given as cod liver oil) to decrease the incidence of respiratory infections. the results were mixed, with about half showing a positive impact and the rest demonstrating no effect (65) . however, cod liver oil did have a significant impact on decreasing industrial absenteeism (65) . in a 1935 study of cod liver oil for the prevention of the common cold in school children, the investigator was not able maintain a control group given no supplements because enthusiastic families purchased cod liver oil for their children outside of the study (65, 66) ; this finding is relevant to our current work. with the introduction of sulfa antibiotics and penicillin in the 1930s to 1940s (65), as well as the improvements in diet in industrialized countries in the late 1930s, interest in anti-infective therapy shifted to antibiotics and away from vitamin a (98). in the mid-1920s, uv radiation of food and a variety of other substances was demonstrated to produce anti-rachitic properties (105) . steenbock patented the addition of provitamin d to foods followed by uv irradiation to produce anti-rachitic activity. in the 1930s, the addition of provitamin d 2 to milk followed by uv irradiation was widely practiced in the united states and europe. rickets was eradicated as a significant public health problem in the countries that used this vitamin d fortification process (105) . in the late 1940s, otto isler and his collaborators in basel reported the synthesis of all-trans-vitamin a from the inexpensive precursor β-ionone (95) . in the same time period, arens and van dorp (94) reported the synthesis of retinoic acid. within a few years, the price of vitamin a fell 10-fold, and it became economically feasible to add vitamin a more generally to foods. during the latter part of the 19th century, cod liver oil was rarely used in america, although the reason for this lack of use is not clear (96, 97) . however, there was a resurgence in interest, and in 1917, hess and unger wrote, "for many years cod liver oil has been regarded as the sovereign remedy for rickets" (ref. 106, p. 1583) . they successfully prevented rickets with cod liver oil in susceptible african-american babies in a lowincome neighborhood in new york city (106, 119) . hess urged officials to dispense cod liver oil at the baby health stations at cost, but they declined because it would be too expensive, and they thought that additional milk would be preferable to cod liver oil (106) . cod liver oil and sunlight were highly valued for the prevention of rickets, and nurses taught mothers of infants how to use these remedies for their infants (107) . from the 1920s to the 1940s, many children in the united states were given cod liver oil each day (26, 65) with orange juice (which was know to prevent scurvy). however, older preparations of cod liver oil had an unpleasant taste, the quality of different preparations was erratic (119) , and medical professionals became concerned about lipoid aspiration pneumonia (87) and vitamin a toxicity (88) . by the 1950s, cod liver oil had been largely replaced by synthetic vitamins in the united states; however, the latter do not contain ω-3 fatty acids, which have anti-inflammatory properties (26) and important effects on immune function (47, 120) . in norway, the norwegian nutrition council continues to recommend supplementation with cod liver oil beginning at age 4 wk, because it provides ω-3 fatty acids in addition to vitamin d (121). before columbus made his first voyage to america, basque fisherman were secretly fishing the massive stocks of cod and other groundfish off the new england coast (122, 123) . their salt cod was a staple in mediterranean markets, and cod was a staple of the european diet for more than 400 yr (122) . although fishermen exploited cod for centuries, the technological innovations of the 20th century led to the collapse of cod stocks in north america. motorized boats dragged the ocean floor with massive trawl nets, destroying both cod fish and their habitat. factory ships with refrigeration have almost erased the limit to the amount of cod that can be caught and sold internationally without spoiling (123) ; increasingly powerful and accurate sonar produces detailed readouts of nooks where schools of fish may lurk; and shipping fleets can position themselves precisely through use of the satellites of the global positioning system (124) . despite growing regulations on allowable catches and fishing equipment, cod stocks have continued to decrease across the north atlantic. in 1992, the canadian government declared a temporary moratorium on cod fishing; the moratorium was extended in 1994. in 2003, with cod stocks showing no sign of recovery, the canadian government banned all cod fishing off its eastern provinces and identified some cod populations as endangered. the us government also imposed restrictions on cod fishing (123) . however, it is unclear whether north atlantic cod stocks will recover. the level of polychlorinated biphenyls (pcbs) and dioxins in fish and fish oils has become a concern as oceans have become progressively contaminated with industrial waste. this issue was addressed in the united kingdom and europe by purity standards (125) , which were revised and made more strict in 2002 (126) . in the same year, the uk food standards agency reported that exposure to dioxins had decreased by 75% over the previous 20 yr and that the levels of dioxins and pcbs found in most of the samples in their most recent fish oil survey were lower than in previous surveys that were performed in 1994 and 1996 (126) . mercury contamination of fish is also a concern, and the fda advises that young children and women of childbearing age should avoid tilefish, swordfish, shark, and king mackerel because of their elevated levels of mercury (127) . however, an analysis of us fish oil supplements revealed no detectable mercury, with a limit of detection of 0.1 μg of mercury per gram (128). after a 40-yr hiatus, interest in the anti-infective properties of vitamin a was rekindled in the 1980s by the observation of increased mortality in indonesian children who had vitamin a deficiency and xerophthalmia (65, 129) . the first symptom of eye disease from vitamin a deficiency is night blindness; at this stage, bitot's spots (superficial, foamy gray, triangular spots) may be present on the conjunctiva (129, 130) . this is followed in later stages by xerophthalmia (dryness of the conjunctiva), keratomalacia (corneal ulceration), and blindness (77) . since the 1980s, numerous studies have been performed regarding the effect of vitamin a supplementation on the health of children in developing countries. for a complete review of this subject, the reader is referred to chapter 23, as well as reviews (110, 131, 132) , and meta-analyses (67, 133, 134) . for the purpose of this chapter, the findings are summarized to provide a basis of comparison to the status of vitamin a in the developed world as well as to provide a perspective on the results of our research. vitamin a supplementation of children in developing countries decreased overall childhood mortality by about 30% (67, 132) . community-based studies of vitamin a supplementation have indicated that it may decrease the severity, but not the incidence, of diarrhea (131) . in children hospitalized with measles in the developing world, vitamin a supplementation decreased mortality by an average of 60% (67, 132, 135) ; the decrease in mortality from measles-related pneumonia was particularly noteable (67) . the modern studies are consistent with the results of ellison's historical study of cod liver oil for children who are hospitalized with measles (see section 4.6.). the role of vitamin a supplementation in measles is also consistent with the fact that infectious diseases that induce the acute-phase response transiently depress serum retinol concentrations, that vitamin a deficiency impedes the normal regeneration of mucosal barriers damaged by infection, and that it also diminishes the immune function of white blood cells (136, 137) . however, several placebo-controlled trials have demonstrated that high-dose vitamin a supplementation is not effective in decreasing the severity of pneumonia in hospitalized children in developing countries and that large doses of vitamin a may be harmful when given to well-nourished children in these areas (132, 134) . additionally, vitamin a supplementation is not effective for children who are hospitalized with pneumonia caused by respiratory syncytial virus, which is a paramyxovirus similar to measles and an important cause of infantile bronchiolitis and pneumonia (138, 139) . in a multicenter study performed in the united states, patients who received vitamin a actually had longer hospital stays than those who received placebo (138) . infection with hiv has become increasingly prevalent in many developing countries. vitamin a supplementation of children younger than age 5 yr who are hiv-positive decreases aids-related deaths as well as total mortality and morbidity from diarrhea (140) . small, frequent doses of vitamin a may be more protective than large, periodic doses. additionally, adequate dietary vitamin a intake is associated with a significant decrease in mortality (141) , diarrheal and respiratory infections (142) , and stunting (143) . new strategies in vitamin a supplementation in developing countries include targeting at-risk populations, improving dietary sources of vitamin a, using horticultural approaches, fortifying food, and addressing multinutrient deficiencies (140). modern studies of vitamin d indicate that calcitriol (1,25 dihydroxyvitamin d), the active form of vitamin d, has important nonclassical effects beyond the regulation of calcium metabolism. these include the modulation of hormone and cytokine production and secretion as well as the regulation of proliferation and differentiation (144) . calcitriol, a potent inhibitor of human t-lymphocyte proliferation (145, 146) , and vitamin d analogs have been shown to be effective in the prevention and treatment of some models of autoimmune disease in rodents-particularly autoimmune diabetes in mice (144, 147, 148) . in 1997, muhe and colleagues (110, 149) reported the importance of nutritional rickets in the development of pneumonia in developing countries. this is consistent with the work of historical authors discussed earlier, who were also aware of this association. vitamin a deficiency that is severe enough to cause blindness is uncommon in the developed world (62) . however, some segments of the us population, particularly socioeconomically disadvantaged children (93) as well as african-and mexican-american children (62), may have suboptimal levels of vitamin a. in 1932, ellison recognized that children from lowincome households were the most likely to have vitamin deficiencies (see section 4.6.) (108) . consistent with these reports, in our original study, five of six children with suboptimal levels of vitamin a were hispanic general-service patients (57) . additionally, young children in the united states-particularly those in the toddler and preschool age groups-may not have adequate dietary intakes of vitamin a (150) . in developed countries, high intakes of vitamin a (but not β-carotene) by pregnant women have been associated with teratogenesis (151), leading to recommendations that prenatal vitamins should contain no more than 8000 iu of preformed vitamin a (152) . additionally, high intakes of vitamin a by postmenopausal women in the united states (153) and 49-to 51-yr-old men in sweden (154) have been associated with a higher risk of hip fractures. as a result, vitamin a supplementation and fortification of food with vitamin a in western countries has been questioned (77) . as discussed under section 3.3., the amount of vitamin a in norwegian cod liver oil has been reduced. nonetheless, the norwegian nutrition council continues to recommend supplementation with cod liver oil beginning at age 4 wk, because it provides ω-3 fatty acids in addition to vitamin d (121). numerous investigators have stated that vitamin a deficiency rarely exists alone and that it is usually accompanied by variety of other nutritional deficiencies (92, 131, 132, 134, (155) (156) (157) (158) . in a 1986 review, mejía (92) noted that vitamin a deficiency primarily affects the world's most underprivileged populations, which, because of their limited socioeconomic condition, also lack a variety of other essential nutrients. he emphasized the importance of the interaction between nutrients and reviewed the established relationships of vitamin a status to protein, dietary fat, vitamin e, zinc, and iron (92) . mejía also mentioned the more controversial links of vitamin a to iodine metabolism; vitamins c, k, and d; calcium, and copper. realizing that the relationships might be direct or indirect, he emphasized the importance of considering these interactions when "treating or preventing vitamin a deficiency both at the clinical and at the population levels" (ref. 92, p. 95) . olson (155) reported that deficiencies of various other nutrients, including protein, α-tocopherol (vitamin e), iron, and zinc, adversely affects the transportation, storage, and utilization of vitamin a. he also noted that the absorption of vitamin a and carotenoids is markedly reduced when diets contain very little fat (<5 g/d). more recently, villamor and fawzi (132) stated that supplementation with vitamins and minerals in addition to vitamin a is likely to "reduce the burden of adverse health outcomes," because of the physiological interactions between nutrients and overlapping micronutrient deficiencies, including iron and zinc. semba (98) noted that antenatal supplementation with multivitamins reduced fetal deaths and low birthweight in pregnant women who were infected with hiv, but vitamin a alone had no significant effect. semba (110) also discussed the role that other deficiencies of vitamin d (149) and zinc (159) may have in susceptibility to respiratory infections. we agree with semba, who stated that "further studies are needed to address the use of vitamin a in multi-micronutrient supplements, as there is increasing evidence that other coexisting micronutrient deficiencies may limit the efficacy of vitamin a" (ref. 131, p. 105). our work is consistent with the historical uses of cod liver oil, vitamin a as the "antiinfective" vitamin, the link between rickets and respiratory tract infections, the modern understanding of immunomodulatory effects of vitamin d, the importance of ω-3 fatty acids and trace metals in decreasing inflammation, the clinical observation that inflamed respiratory mucosa may not completely recover between episodes of infection, and the current concept of the importance of multiple micronutrient deficiencies. we have demonstrated that use of flavored cod liver oil (which meets european purity standards) and a chewable children's multivitamin-mineral with trace metals, including se, can decrease morbidity from upper respiratory tract illnesses, om, and sinusitis in young children living in the united states. these supplements were particularly wellaccepted by latino families from the caribbean, where use of cod liver oil is a cultural tradition. currently, there is adequate information for practitioners to recommend the use of these supplements, when indicated, to their individual patients; information for practitioners and families is available online at http://www.drlinday.com. the supplements can be purchased in the united states without a prescription. further research is needed to evaluate the effect of the supplements on antibiotic prescription for these illnesses and to explore their role as adjunctive therapy in asthma. additionally, our findings need to be confirmed in larger studies to facilitate large-scale, policy decision making. use of these supplements has the potential to improve children's health and decrease the cost of their health care. however, cod liver oil does not have a national drug code number and is not available through medicaid in new york, and the children's vitamins we have located that are available through this system do not contain se or other trace metals. also, cod liver oil is not available through the usda wic program; our request for such availability can be found online at http://www.fns.usda.gov/wic/anprmcomments/ ihp-06.pdf. furthermore, purchase of vitamins with us food stamps is not permitted (see http://www.fns.usda.gov/fsp/faqs.htm#9). socioeconomically disadvantaged children living in the united states are at risk for micronutrient deficiencies. although the supplements used in our research can be purchased in the united states without a prescription, their cost may pose an excessive financial burden to low-income families. if our results are confirmed in larger studies, a system change will be needed to provide these supplements to nutritionally vulnerable, socioeconomically disadvantaged children living in the united states. this work was supported in part by the department of otolaryngology of the new york eye and ear infirmary (new york, ny). j. r. carlson laboratories, (arlington ambulatory health care visits by children: principal diagnosis and place of visit antibiotic prescribing for children with colds, upper respiratory tract infections, and bronchitis misconceptions about colds and predictors of health service utilization trends in antimicrobial prescribing rates for children and adolescents reduction in antibiotic use among us children interagency task force on antimicrobial resistance: a public health action plan to combat antimicrobial resistance. part 1: domestic issues cost-effectiveness considerations in otitis media treatment the role of adjuvant adenoidectomy and tonsillectomy in the outcome of the 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among sudanese children dietary vitamin a intake in relation to child growth vitamin d analogues in insulin-dependent diabetes mellitus and other autoimmune diseases: a therapeutic perspective comparison of the effects of 1,25-dihydroxyvitamin d3 on t lymphocyte subpopulations the immunobiology of vitamin d 1,25-dihydroxyvitamin d3 prevents insulitis in nod mice prevention of autoimmune diabetes in nod mice by 1,25 dihydroxyvitamin d3 case-control study of the role of nutritional rickets in the risk of developing pneumonia in ethiopian children subclinical vitamin a deficiency: a potentially unrecognized problem in the united states teratogenicity of high vitamin a intake continuing caution is needed vitamin a intake and hip fractures among postmenopausal women serum retinol levels and the risk of fracture recommended dietary intakes (rdi) of vitamin a in humans nutrition and development: other micronutrients' effect on growth and cognition iron and zinc supplementation improves indicators of vitamin a status of mexican preschoolers impact of zinc supplementation on morbidity from diarrhea and respiratory infections among rural guatemalan children heights, il) donated the nutritional supplements used in this research but had no other role in the design or conduct of the study. information for practitioners and families regarding this research is available at http://www.drlinday.com. key: cord-001567-3bw7jbzq authors: borlak, jürgen; singh, prashant; gazzana, giuseppe title: proteome mapping of epidermal growth factor induced hepatocellular carcinomas identifies novel cell metabolism targets and mitogen activated protein kinase signalling events date: 2015-02-25 journal: bmc genomics doi: 10.1186/s12864-015-1312-z sha: doc_id: 1567 cord_uid: 3bw7jbzq background: hepatocellular carcinoma (hcc) is on the rise and the sixth most common cancer worldwide. to combat hcc effectively research is directed towards its early detection and the development of targeted therapies. given the fact that epidermal growth factor (egf) is an important mitogen for hepatocytes we searched for disease regulated proteins to improve an understanding of the molecular pathogenesis of egf induced hcc. disease regulated proteins were studied by 2de maldi-tof/tof and a transcriptomic approach, by immunohistochemistry and advanced bioinformatics. results: mapping of egf induced liver cancer in a transgenic mouse model identified n = 96 (p < 0.05) significantly regulated proteins of which n = 54 were tumour-specific. to unravel molecular circuits linked to aberrant egfr signalling diverse computational approaches were employed and this defined n = 7 key nodes using n = 82 disease regulated proteins for network construction. string analysis revealed protein-protein interactions of > 70% disease regulated proteins with individual proteins being validated by immunohistochemistry. the disease regulated network proteins were mapped to distinct pathways and bioinformatics provided novel insight into molecular circuits associated with significant changes in either glycolysis and gluconeogenesis, argine and proline metabolism, protein processing in endoplasmic reticulum, hifand mapk signalling, lipoprotein metabolism, platelet activation and hemostatic control as a result of aberrant egf signalling. the biological significance of the findings was corroborated with gene expression data derived from tumour tissues to evntually define a rationale by which tumours embark on intriguing changes in metabolism that is of utility for an understanding of tumour growth. moreover, among the egf tumour specific proteins n = 11 were likewise uniquely expressed in human hcc and for n = 49 proteins regulation in human hcc was confirmed using the publically available human protein atlas depository, therefore demonstrating clinical significance. conclusion: novel insight into the molecular pathogenesis of egf induced liver cancer was obtained and among the 37 newly identified proteins several are likely candidates for the development of molecularly targeted therapies and include the nucleoside diphosphate kinase a, bifunctional atp-dependent dihydroyacetone kinase and phosphatidylethanolamine-binding protein1, the latter being an inhibitor of the raf-1 kinase. electronic supplementary material: the online version of this article (doi:10.1186/s12864-015-1312-z) contains supplementary material, which is available to authorized users. liver malignancies are common cancers worldwide and are responsible for approximately one million deaths each year with most hcc patients having poor prognosis as a result of rapid disease progression. the relative 5-year survival rate is about 15% and can be attributed to an advanced stage of disease at the time of diagnosis, the occurrence of cirrhosis and of other co-morbidites [1] . detection of early stages of disease is essential for an improved prognosis and overall survival. however, apart from alpha-fetoprotein (afp) only a few serological markers are available in clinical practice (such as glypican-3, mir-21, fucosylated gp73, α-fucosidase) with afp diagnostics remaining unsatisfactory because of its low sensitivity and the non-specific correlation between the clinical behavior of hcc and afp blood levels. for this reason, new biomarkers are in strong demand [2, 3] and more selective markers, such as soluble interleukin-2 receptor levels, are evaluated [4] . importantly, research into the molecular pathogenesis of hcc identified several signalling pathways as deregulated. this inspired the development of molecularly targeted therapies such as the multikinase inhibitor sorafenib that inhibits signalling of c-raf-1, mek, erk, vegfr, pdgfr and other kinases, effectively [5] . given that epidermal growth factor is an important mitogen for hepatocytes we were particularly interested in an understanding of the consequences of its targeted overexpression in liver. in our initial study we reported the oncogenomics and pathology of egf induced hepatocarcinogenesis and an identification of molecular circuitries linked to exaggerated egfr signalling [6] . furthermore, we investigated the serum proteome of egf-tumourbearing mice to obtain information on disease-regulated proteins and to search for novel biomarkers at different stages of disease [7] . note, a regulatory loop was proposed whereby egf induces transcriptional activation of hdac2 by ck2α/akt activation in liver cancer cells [8] . additionally, inhibition of egfr in different animal models by erlotinib was shown to attenuate liver fibrosis and the development of hepatocellular carcinoma [9] thus suggesting new therapeutic intervention strategies in the prevention of hcc. indeed, the egf receptor tyrosin kinase plays a much wider role in the immortalization of different cell types as originally anticipated [10] , and is highly expressed in a number of solid tumours and egfr over-expression correlates well with tumour progression, resistance to chemotherapy and poor prognosis. the present study aimed at an identification of disease regulated proteins to facilitate an improved understanding of its complex signalling networks and to search for cross-talk amongst other pathways while an identification of disease regulated proteins would aid the development of molecularly targeted therapies. for this purpose, a two-dimensional electrophoresis and maldi-tof/tof ms strategy was employed to identify disease regulated proteins in an egf transgenic mouse model of hcc. this resulted in an identification of 96 statistically significant regulated proteins of which 54 are uniquely expressed in liver cancer. importantly, 11 out of 54 mouse tumour specific proteins were likewise uniquely expressed in human hcc and 49 disease regulated proteins identified in egf induced liver cancer were similarly regulated in human hcc, as determined by immunhistochemistry using different antibodies and the information given in the publically available human protein atlas depository. clinical significance of the identified proteins could be demonstrated and a total of 37 so far unkown proteins could now be related to egf induced liver cancer, several of which are likely candidates for the development of molecularly targeted therapies. this includes the nucleoside diphosphate kinase a, bifunctional atp-dependent dihydroxyacetone kinase, phosphatidylethanolamine binding protein1, i.e. an inhibitor of the raf-1 kinase as well as aldo-keto reductase family 1 proteins, members c14 and c6, interleukin 25 and the v-crk sarcoma virus ct10 oncogene homolog. finally, to gain insight into the molecular circuitries of egfr induced hepatocarcinogenesis diverse computational approaches were employed. this revealed master regulatory proteins and permitted network constructions of 82 disease regulated proteins with protein-protein interactions being confirmed for > 70% of regulated proteins in string analysis. their regulations were also studied by immunohistochemistry in egf transgenic hcc. we also compared the serum and liver proteomes of hcc bearing mice and found 10 proteins to be similarly regulated, thus evidencing leakage of tumour proteins that can be detected in serum. obviously, these are highly interesting biomarker candidates, 6 of which were also regulated in human hcc as determined by immunohistochemistry. all animal work followed strictly the public health service (phs) policy on humane care and use of laboratory animals of the national institutes of health, usa. formal approval to carry out animal studies was granted by the animal welfare ethics committee of the state of lower saxony, germany ('lower saxony state office for consumer protection and food safety' (laves)). the approval id is az: 33.9-42502-04-06/1204. a total of n = 12 c57/bl6 non-transgenic and n = 12 egf transgenic mice (aged 6-8 months), weighing 25-33 g, were housed in makrolon® type iii cages. water and food (v1124-000, ssniff, the netherlands) was given ad libitum. the temperature and relative humidity was set to 22 ± 2°c and 40-70%, respectively and a 12-h day and night cycle was used. tris, urea, thiourea, chaps, dithiothreitol, bromophenol blue, glycerin, sodium dodecyl sulfate, glycine, temed, ammoniumperoxodisulfate, ammonium sulfate, ammonium bicarbonate, colloidal coomassie blue, and acrylamide were purchased from roth (karlsruhe, germany). iodacetamide was obtained from serva (heidelberg, germany) and benzonase was purchased from novagen (darmstadt, germany). ampholytes (biolyte 3-10) were purchased from bio-rad laboratories (münchen, germany) and destreak was obtained from amersham bioscience (freiburg, germany). mice were anesthetized with ketamine 10% 100 μl/100 g and xylazine 2% 50 μl/100 g, and after surgical removal the liver was perfused and rinsed with ice cold ringer solution until free of blood. approximately 0.1 g of the liver sample was ground in a mortar under liquid nitrogen flow. then, the samples were processed with 0.5 ml of a buffer containing 40 mm tris base, 7 m urea, 4% chaps, 100 mm dtt, and 0.5% (v/v) biolyte 3-10 first (lb2). the suspensions were homogenized by sonication (3 × 20 s) and after addition of 3 μl of benzonase (endonuclease that degrades dna and rna) were left at room temperature for 20 min. the samples were then centrifuged at 12,000 g for 20 min. the pellets were washed and sonicated for 5 min with a further 0.5 ml of lb2 and centrifuged at 12,000 g for another 20 min, and the resulting two fractions of supernatant were collected (extract a). finally, the pellets were redissolved with 0.5 ml of buffer containing 40 mm tris base, 5 m urea, 2 m thiourea, 4% chaps, 100 mm dtt, and 0.5% (v/v) biolyte 3-10 (lb3), sonicated, and centrifuged at 12,000 g for 20 min. the pellet was collected, and the supernatant was marked as extract b. from the same animals, a further 0.1-g portion was ground in a mortar, but was now treated with 0.5 ml of lb3. the suspensions were sonicated, incubated with benzonase, and centrifuged. the pellets were then washed with another 0.5 ml of lb3, sonicated and centrifuged, and the supernatants were collected (extract c). proteome mapping was done under a variety of conditions, e.g. extraction with lysis buffers 2 and 3. in addition, proteins were separated at two different ph ranges [5] [6] [7] [8] [9] [10] . a total of 4 independent experiments were carried out, and duplicate measurements were run for each experiment. the protein concentration of all extracts was determined using the bradford assay. liquid-phase ief pre-fractionation was performed in the rotofor cell system (bio-rad) following the supplier's instructions. ion exchange membranes were equilibrated overnight in the appropriate electrolyte (anion exchange membranes in naoh 0.1 m and cation exchange membranes in h 3 po 4 0.1 m). after four runs ion exchange membranes were always discarded and new membranes were replaced for the other samples. for each run, the electrode chambers were filled with appropriate fresh electrolytes (30 ml) . initially, the cell was filled with pure water and run for 5 min at 5 watts constant power to remove residual ionic contaminants from the membrane core and ion exchange membranes. approximately 32 ml of lb2 were used to fill the cell. a total of 60 mg of total proteins in approximately 2 ml of lb2 were added to the cell to reach the maximum loadable volume (40 ml) . focusing started at 12 watts constant power. after approximately 4 hours the voltage increased to 3000 v and the wattage decreased to 3 w. the focused proteins were harvested in 20~1.5 ml fractions, and ph values were checked. fractions having ph values between 3 and 7.0 were collected and denoted "a-a" (acid). fractions having ph values > 7.0 were collected and denoted "a-b" (basic). again, the protein concentration was determined for both fractions (a-a and a-b) by the bradford method. approximately 30 mg of protein were recovered at the end of the liquid-phase ief pre-fractionation from an initial 60-mg load. the losses are accounted for by the multi-step pre-fractionation procedure, but are not the result of a precipitate that could not be dissolved in our lysis buffer. after each run the membrane core was cleaned with naoh 0.1 m overnight and sonicated for 5 min in water before the new focusing. isoelectric focusing (ief) -first dimension ief was performed using precast linear ipg strips. the 17-cm ipg strips 7-10 and 5-8 were loaded with 1.5 mg of proteins by active rehydration (12 h, 50 v) . samples destined to be separated by ipg strips 7-10 received an excess of hydroxyethyldisulphide (hed) (destreak™) prior to the focusing run. focusing began at 250 v for 20 min in rapid mode, 10,000 v for 5 h in linear mode and 10,000 v for 50,000 vh in rapid mode (for the ipg strips 5-8). ief for the strips 7-10 was carried out at 250 v for 60 min in rapid mode, 10,000 v for 3 h in linear mode and 10,000 v for 50,000 vh in rapid mode. each sample was analysed in duplicate. control and hcc samples were run always at the same time (6 control and 6 hcc samples). after ief, the ipg strips were either stored at −80°c or transferred to 10 ml of equilibration buffer (6 m urea, 30% w/v glycerin, 2% w/v sds, 50 mm tris-hcl ph 8.8) with 2% w/v dtt and 0.5% v/v bromophenol blue solution (0.25% w/v bromophenol blue, 1.5 m tris-hcl ph 8.8, 0.4% w/v sds) and incubated for 20 min at room temperature. strips were removed and incubated in equilibration buffer with 4% w/v iodoacetamide and 0.5% v/v bromophenol blue solution for further 20 min at room temperature. finally, the strips and 10 μl sds-page molecular weight standard on filter paper were placed on top of the 20 cm x 20.5 cm 12% second-dimension gel (12% v/ v acrylamide/bis solution, 375 mm tris, ph 8.8, 0.1% v/v sds, 1/2000 temed, 0.05% v/v aps). both were fixed in place with a 0.5% w/v agarose overlay. gels were run in protean plus dodeca cell (bio-rad) at 70 v for approximately 14 h, followed by 200 v until the bromophenol blue dye reached the bottom of the gel. the running buffer (25 mm tris, 0.2 m glycin, 0.1% sds) was cooled externally to 16°c. gels/proteins were fixed overnight in 30% ethanol, 2% phosphoric acid, and washed 3 x 20 min with 2% phosphoric acid. the gels were equilibrated with 15% ammoniumsulfate, 18% ethanol, 2% phosphoric acid for 15 min and finally stained with colloidal coomassie blue for 48 h. after staining, gels were washed 10 min with pure water and scanned on a molecular fx scanner (bio-rad) at 100 μm resolution. protein spots were imaged first automatically and then manually and analysed using the pdquest™ software (bio-rad). the normalization was carried out in total density in gel mode according to the manufacturer's recommendation. gels were excised using the spot cutter of bio-rad and placed into 96-well microtiter plates. excised gel spots were washed manually with 20 μl of water for 10 min and destained twice, first with 15 μl ammonium bicarbonate 50 mm for 5 min and then with 15 μl 50% ammonium bicarbonate 50 mm -50% acetonitrile for 5 min. finally, the gel particles were covered by acetonitrile until gel pieces shrunk and left to dry for 10 min. all gels/proteins were digested manually in situ with 4 μl of ammonium bicarbonate 50 mm containing 20 ng trypsin (sequencing grade modified trypsin, promega, germany). after 15 min each gel piece was re-swelled with 10 μl of ammonium bicarbonate 50 mm and incubated for 4 h at 37°c. after 4 h the reaction was stopped by adding 10 μl of trifluoroacetic acid 1% containing 1.5% (w/v) n-octyl-beta-d-glucopyranoside (ogp) (applichem). for the application of the samples, 4 μl of peptide solution were loaded onto an mtp anchor chip target 600/384 (bruker daltonics) previously prepared with a saturated solution of matrix, alpha-cyano-4-hydroxy-cinnamic acid (alpha-hcca) (bruker daltonics, bremen, germany). maldi-ms was performed on an ultraflex ii maldi-tof/tof (bruker daltonics) mass spectrometer equipped with a smartbeam™ laser and a lift-ms/ms facility. the instrument was operated in positive ion reflectron mode and an acceleration voltage of 25 kev for the peptide mass fingerprint (pmf) mode. typically, 600 spectra, acquired at 100 hz, were summed and externally calibrated. in the case of ms/ms-cid the lift device was used for selection and fragmentation of the ions; the acceleration voltage in the ion source 8 kv, the timed ion selector was set to 0.4% (relative to parent mass), and argon was used as collision gas (~4-6 × 10-6 mbar). resulting fragments were further accelerated in a second source by 19 kv and analysed by a two-stage gridless reflectron. typically, 400 shots were accumulated for the parent ion signal and 1000 shots for the fragments. flexcontrol™ 3.0, and flexanalysis™ 3.0 were used as instrument control and processing software (bruker daltonics, bremen, germany). a calibration standard was used for the external calibration of spectra (peptide calibration standard for mass spectrometry, which covered the mass range~1000-4000 da internal calibration was achieved using trypsin autolysis products (m/z's 1045.564, 2211.108 and 2225.119) resulting in a mass accuracy of ≤ 50 ppm. spectra were collected by the flexcontrol software without smoothing or baseline subtraction and a peak resolution higher than 6000 or 7000 a.u. in case of dhb and chca matrixsample preparation, respectively. the spectra were sent to the flexanalysis software which labeled the peaks for protein identification by proteinscape 1.3 or biotools 3.1 (bruker daltonics). trypsin autolysis products, tryptic peptides of human keratin and matrix ions were automatically discarded by proteinscape (mass control list). proteinscape score booster feature was used to improve database search results by automatic iterative recalibrations and background eliminations. protein scores greater than 53 were considered significant (p <0.05, mascot) and an annotation as mouse protein as the top candidates was requested in the search when no restriction was applied to the species of origin. identified proteins were checked individually for further considerations. for pmf peak picking the snap peak detection algorithm, a signal to noise threshold of 6, maximal number of peaks 100, a quality factor threshold 50 and baseline subtraction tophat was applied. peptide masses were searched against the swiss-prot database (download 2005-197 228 sequences, 71 501 181 residues) employing the mascot server (in-house mascot-server, matrix sciences ltd., http://www.matrixscience.com/, revision 2.0.0), taking into account carbamidomethyl of cysteines -carbamidomethyl (c)-as fixed modification and possible oxidation of methionine -oxidation (m)-as a variable modification but allowing one missed cleavage. based on initial data, ion precursors were selected by proteinscape for tandem ms data acquisition (by lift-tof/tof, bruker daltonics, bremen, germany). in the mascot ms/ms ions search, the restriction mammalia was applied with peptide tolerance of (70 ppm and ms/ms tolerance of (0.9 da (fixed and variable modifications as pmf). the acceptance criteria for pmf-based identification were an individual ions score >27, at least five matching peptides and 10% peptide coverage of the theoretical sequences. livers, dissected from egf-overexpressing mice aged between 7-9 months, were fixed in 4% buffered paraformaldehyd and embedded in paraffin. 5 μm thick sections were deparaffinized and rehydrated through a descending alcohol series followed by a 4 min washing step in destilled h 2 o. subsequently, antigen retrieval was performed in citrate buffer (ph 6) by autoclaving the sections 15 min at 121°c. the envision kit (dako, hamburg, germany) was used for immunohistochemistry. the slides were rinsed with destilled h 2 o and after a 5 min incubation step in tris-buffered saline (washing buffer), endogenous peroxidase activity was blocked with dako peroxidase blocking reagent for 5 min followed by a second washing step. thereafter, the sections were blocked for 10 min with protein-block serum free (dako) and incubated with primary antibodies for 45 min. details of antibody dilutions with washing buffer are given in additional file 1: table s1. in the case of goat primary antibodies a rabbit-anti-goat bridging antibody (dako) was employed. specifically, the bound primary antibodies or bridging antibodies were detected by use of labelled polymer hrp anti-rabbit secondary antibody (envision kit; dako) and the immunoreactivity was visualized by dako liquid dab substrate chromogen system in a 5 min incubation. finally, the sections were counterstained with harris haematoxylin for 2 min, dehydrated in an ascending alcohol series, coverslipped and examined under a light microscope (leica, jülich, germany). a total of n = 122 disease regulated proteins were filtered for statistical significance at p < 0.05 (table 1 ). this yielded n = 96 statistically significantly regulated proteins two of which had identical accession number, i.e. aah81431 = atp synthase h+ transporting mitochondrial f0 complex, subunit d and bac36241 = apoa1 but differed in their spot ids as a result of posttranslational modifications. the statistically significantly regulated proteins were grouped into four different categories to yield 54 tumour specific (to), 9 up-regulated (ur), 19 down-regulated (dr) and 14 proteins only expressed in healthy non-transgenic control livers (co). categorization of tumour regulated proteins based on ontology terms 82 non-redundant tumour proteins covering to, ur and dr categories were considered and analysed for ontologies using the genexplain software (v.2.4.1), the biological pathways tools reactome (http://www.reactome.org) and kegg (http://www.genome.jp/kegg) and wikipathways (http://wikipathways.org). the tumour regulated proteins (to + ur + dr) were subjected to functional classification based on ontology terms and a p-value of <0.01 was considered to be significant. moreover, disease regulated proteins were analysed with the cytoscape software version 3.0.2 using the function go-tree levels and number or % of proteins for a given term (see additional file 2: table s2 ). master regulatory proteins were searched based on the designated workflow of the genexplain software. it is designed to find master regulatory molecules upstream of an input list of regulated tumour proteins. after annotation of the input datasets the tool for master regulator finding over geneways network (http://www.genexplain.com) was applied. specifically, the geneways software is used to automatically extract, analyse, visualise and integrate molecular pathway data from the published peer reviewed literature. it is based on document sorting, term identification, term meaning disambiguation, information extraction, ontology, visualization and system integration [61] . the following filtering threshold was used, i.e. score cutoff (0.2), search collection (geneways hub), maximum radius [4, 10] , fdr cutoff (0.05), z-score cutoff (1.0), penalty (0.1) and decay factor (0.1) (additional file 3: table s3 ). protein network for disease regulated proteins were also constructed using the string software (http:// string-db.org/). the underlying database informs on known and predicted protein-protein interaction and the constructed networks are based on active prediction methods of neighborhood, gene fusion, co-occurrence, co-expression, databases and textmining. eventually, confidence scores were calculated for each interaction pair and only those above default cutoff scores (0.4) were selected. finally, mapping of pathways information from reac-tome, kegg and wikipathways have been implemented the proteins are sorted in alphabetical order, and the ncbi annotation is given in the accession number column. molecular weight, pi, and mascot scores are also given. the column "gels", "c" (c = control) and "t" (t = tumour) indicate the frequency of positive identification of proteins in a total of 48 independent gels, whereas "lb2" and "lb3" (lb = lysis buffer) refers to the different lysis buffers employed. furthermore, references are given for those proteins which have already been described as hcc-associated whereas those marked with a star (*) are so far unknown as egfr disease regulated in hepatocellular carcinoma. over protein networks using information of known pathways and sustained proteins connecting these pathways in a given network. the histopathology and oncogenomics of egf induced liver cancers was previously reported [6] and an important finding of the study was the 100% incidence of malignant tumour formation in less than one year after birth. notably, a sequence of events was observed that initially consisted of diffuse large cell dysplasia followed by multiple dysplastic foci and nodules and growth of hcc. figure 1 a and b depict the histopathology of healthy non-transgenic control liver and egf induced tumours, respectively. after protein extraction 2de was performed. subsequently, the gels were scanned on a bio-rad molecular fx scanner at a 100 μm resolution. image analysis was done with the pdquesttm software and spots were detected automatically. a total of 122 proteins differed in expression or were de novo expressed when 2de gels of non-transgenic controls and hcc mice were compared (see table 1 for detailed information on the proteins identified and figure 1e -g depicting examples of zoomin-gels of some regulated proteins). among them are 96 statistically significantly regulated proteins (p ≤ 0.05) of which 63 were significantly up-regulated (ratio hcc/control ≥ 2) and included fibrinogen and subunits of it, vimentin, cu/zn superoxide dismutase, and apolipoprotein e ( figure 1f (i-iv), while 33 proteins were repressed in expression (ratio hcc/control ≤ 0.6) and included arginase 1, dhdh protein, glutathione peroxidase 1 and agmatine ureohydrolase ( figure 1g (i-iv) and table 1 ). a reference 2-de map of mouse liver and serum proteins was constructed that consists of more than n = 500 proteins [2, 7] . note, in our previous efforts we identified n = 25 serum proteins as regulated in the egf transgenic disease model. among them were alpha-fetoprotein, clusterin, fibrinogen-α and -γ, serum amyloid component p and several apolipoproteins all of which were significantly up-regulated. based on the combined use of 2de and maldi-ms a total of n = 122 differentially expressed proteins were identified (table 1 ) and included isoforms as well as post translational modifications of albumin (5 up-regulated spots), alpha enolase (4 down-regulated spots), apoliproptein a-i (2 up-regulated spots), atp synthase h+ transporting mitochondrial (2 down-regulated spots), fibrinogen beta (2 up-regulated spots), glycine n-methyltransferase (3 spots, in controls only), hsp60 (2 down-regulated spots), nit protein 2 (2 down-regulated spots), peroxiredoxin 6 (1 up-regulated spot and 1 down-regulated spot), and 4931406c07rik (2 up-regulated spots) (see table 1 ). importantly, a total of n = 37 so far unknown disease regulated proteins were identified that can now be related to egf induced liver cancer. these are marked with an asterisk in table 1 . furthermore, a comparison of serum and liver proteoms revealed n = 10 proteins to be regulated in common, thus evidencing leakage of tumour proteins into systemic circulation (table 2) . among them was serum afp; it's up-regulation and that of others was confirmed by western blot analysis (figure 2a-e) . likewise, apolipoprotein e was up-regulated both in serum and tumour samples, the ratio hcc/control being 2.2 and 3.9, respectively. in a previous study on human hcc increased expression of apoe was observed in 88% of study cases; however, gene apoe expression and serum levels were unchanged to suggest its accumulation and impaired secretion [21] . two isoforms of alpha-2-macroglobulin were up-regulated in serum of hcc-bearing mice (spot 1: ratio hcc/control = 1.8; spot 2: ratio hcc/control = 3.2). its expression was exclusively associated with tumours. finally, serum amyloid component p was up to 10-fold up-regulated in serum and its tissue expression was tumour specific ( table 2) . to further evidence disease regulated proteins and to provide information on their subcellular localization a total of n = 8 proteins were studied by immunohistochemistry. five of them were selected for their novelty (see table 1 ) while amphiregulin and epiregulin were chosen for their importance in the egf-signalling pathway. furthermore, hnf4α was studied for its pivotal role in liver cancer [62] . depicted in figure 3 are immunohistochemistry stainings performed with egf transgenic livers to confirm regulation and predominant cytoplasmic expression of arginase ii. note, arg2 is only expressed in hcc and recent evidence suggest modulation of arginine levels in the extracellular milieu to be part of an immune escape mechanism whereby lack of local arginine weakens tumour-infiltrating lymphocytes as t cells require adequate argine levels [63] . likewise, the tumour specific and cytoplasmic expression of the f-actin capping protein α1 subunit (capza1) and the predominant nuclear expression of tubulin β that was particularly visible beneath the liver capsule may possible promote microtubule stability and interactions of microtubules with endogenous proteins. furthermore, the induced and predominat cytoplasmic expression of the gdp dissociation inhibitor 2 (gdi2) protein is part of the control of vesicular trafficking. this protein is known to regulate gdp-gtp exchange amongst members of the rab family of proteins. the tumour specific and cytoplasmic expression of amphiregulin supports the notion of a switch in autocrine signalling and it has been reported that amphiregulin is a prognostic marker for poor outcome of a variety of malignancies including colorectal liver metastasis [64] . finally, the repressed nuclear expression of hnf4a was not unexpected and confirms earlier findings [62] . based on the information given in table 1 the human protein atlas depository (www.proteinatlas.org, version 12) was interrogated. as shown in additional file 4: table s4 48 out of 96 mouse liver cancer regulated proteins were likewise regulated in human hcc. it should be noted that for some proteins several antibodies were used to study their expression; only representative data were considered. importantly, out of the 54 proteins uniquely expressed in mouse liver tumours n = 11 were likewise uniquely expressed in human hcc thus evidencing clinical significance of our findings. we compared our previously published transcriptomic data of egf induced liver cancers with the proteomic data obtained in the present study. such comparisons revealed n = 22 genes to be significantly regulated of which n = 17 are in common regulated whereas for n = 5 genes transcript expression was opposite to that of the coded proteins (see additional file 5: table s5 ). 82 of the 96 significantly regulated proteins were mapped to 40 different biological processes (see figure 4a ) of which prominent examples are regulation of arginine metabolism and amino acid import, regulation of cdc42 protein signal transduction', cellular response to oxidative stress, hydrogen peroxide and superoxide, glycolysis and gluconeogenesis, regulation of cholesterol transport, protein-lipid complex and plasma lipoprotein particle remodeling, positive regulation of steroid metabolic process, negative regulation of calcium ion transmembrane transporter activity and release of sequestered calcium ion into cytosol by sarcoplasmic reticulum, (see additional file 6: table s6 ). in figure 4a -c the go biological process, cellular components and molecular functions are depicted. note, some of the ontology terms could be grouped, i.e. chaperone-mediated protein complex assembly and folding, endoplasmic reticulum unfolded protein response, er-nucleus signalling pathway and response to er oxidative stress as well as hypoxia, blood coagulation, developmental growth and regulation of programmed cell death. as depicted in figure 4b 76 significantly regulated proteins were mapped to 21 cellular components (see additional file 7: table s7 ), i.e. mitochondrial crista, matrix and inner membrane, endoplasmic reticulum lumen, early endosome and cytoplasmic membrane-bounded vesicle, chylomicron, very-low and high density lipoprotein particle, proteasome accessory complex, peroxisome, extracellular vesicular exosome and extracellular membrane-bounded organelle. furthermore, 75 significantly regulated proteins were mapped to 21 molecular functions (see figure 4c ) and included arginase activity, fructose-bisphosphate aldolase activity, hydrolase and oxidoreductase activity, acting on carbon-nitrogen (but not peptide) bonds, acting on aldehyde, ch-oh group or oxo group of donors, nad or nadp as acceptor as well as steroid dehydrogenase activity. in addition, phosphatidylcholine-sterol o-acyltransferase activator activity, cholesterol transporter activity, sterol transporter, antioxidant and lipid transporter activity as well as electron carrier and serine-type endopeptidase inhibitor activity were prominent functions. finally, proteins functioning in metal ion and purine ribonuleoside triphosphate binding, lipoprotein particle receptor binding, chaperone and oxygen binding, binding of magnesium ion and nad, protease and single-stranded dna binding were observed as disease regulated (additional file 8: table s8 ). in all, 96 significantly regulated proteins were classified by the reactome, kegg and wikipathway databases, respectively. the different databases provided similar information with the majority of tumour proteins acting in 4 major metabolic pathways (see figure 5 and information derived from cluego and cluepedia). for example, the proteins aldoa, aldoc, fbp1 and pkm function in glycolysis and gluconeogenesis whereas akr1c6, aldoa, aldoc and fbp1 are part of the fructose and mannose metabolic pathway. likewise, atp5h and ndufv1 are part of the oxidative phosphorylation pathway and mdh1 and pkm contribute to pyruvate metabolism. similarly, the proteins akr1c14, akr1c18, akr1c6, alb, apoa1, apoa4, apoe, fdps, gpx1, hacl1 and plg take part in the metabolism of lipids, arachidonic acid and lipoproteins whereas the proteins agmat, arg1, arg2, bckdha, ckb, cps1, haao and phgdh are specified for arginine and proline metabolism. in the same manner the proteins gpx1, itpa and nme1 contribute to the metabolism of nucleotides and related to this are the proteins itpa, pkm and psmc5 which are part of the purine metabolic pathway. apart from these pathways a highly significant regulation of the blood coagulation cascade, figure 2 western blotting of serum proteins in control and egf transgenic mice. for the commonly regulated proteins in serum and tumours their regulation in liver tissue was confirmed by 2de and maldi-tof/ms (see table 1 ). depicted are western blots for serum proteins. note, with the exception of egf the regulated serum proteins were already reported in our earlier publication [7] . platelet activation and fibrinolysis was observed as defined by the proteins crk, fga, fgb, fgg, plg and sod1 all of which were highly significantly regulated. furthermore, trna aminoacylation (aars, gars and sars), advanced glycosylation endproduct receptor signalling (alb, capza1 and lgals3), peroxisome (ech1, hacl1 and sod1), protein processing in endoplasmic reticulum (ganab, hyou1 and pdia4), proteasome (psmc5 and psmd11) and activation of chaperone genes by xbp1(s) and 'unfolded protein response' (hyou1 and lmna) are pathways significantly perturbed in liver cancer induced by egf (see additional file 9: table s9 and additional file 10: table s10 ). using the designated workflow of the genexplain platform (see methods section) we searched for master regulatory proteins. the software is designed to identify molecules upstream of regulated tumour proteins to assist in the construction of molecular circuitries. after annotation of the input datasets the tool "find master regulators in networks (geneways)" was used to identify key nodes amongst 54 proteins exclusively expressed in tumours (to). this revealed 24 upstream regulatory molecules. among them five were selected for their link to the egfr signalling pathway, i.e. plaur, fgfr1, ptbp1 and agtrap while the protein s100a1 was chosen for its importance in the plaur/egfr network, (see additional file 11: figure s1a -e). in additional file 3: table s3 and additional file 12: table s11 , the tumour regulated proteins distributed amongst the selected master regulatory molecules are summarized. in support of its biological significance the constructed networks were enriched with gene expression data from transgenic non-tumour and tumour tissues. thus, the gene and protein data were merged and hybrid networks for each master regulatory protein were constructed. subsequently, these were merged into one (see figure 6 ) and the integrated hybrid network consisted of n = 82 network proteins of which n = 20 were tumour specific. in support, the genes coding for lmna, i.e. a component of the nuclear lamina that is frequently up-regulated in cancers and mvp that codes for multidrug resistance were up-regulated (ur-t) whereas nme, a suppressor of metastasis was repressed in expression (dr-t). likewise, the genes coding for igals3, i.e. a beta-galactoside-binding protein frequently overexpressed in cancers and pcbp1 that is involved in transcription and functions as an inhibitor of invasion [65] were up-regulated in transgenic nontumour livers (ur-tr-nt) whereas transcript expression of aars, a member of trna synthases and anaxa6, a calcium-dependent, phospholipid-binding protein with important roles in the tumour microenvironment and metastasis were repressed (dr-tr-nt). finally, the entire network was enriched with expression data of 16 and 17 genes, respectively that were significantly regulated in tumour and non-tumour transgenic livers. next, we searched for master regulatory molecules by considering 82 regulated tumour proteins obtained from the comparison tumour specific or up-and down regulated as compared to healthy non-transgenic controls (to + ur + dr). this revealed 29 filtered (threshold radius of 10) upstream regulators. among these 7 were selected as candidates because of their regulation in liver tumours and their link to egfr signalling. notably, in the constructed network all master regulators were significantly up-regulated and included pdia4, apeh, pebp1 and apoe while the protein expression of arg1, fbp1 and haao was repressed (see additional file 13: figure s2a-g) . note, in the case of arg1 transcript expression was equally repressed. in additional file 3: table s3 and additional file 12: table s11 the tumour regulated proteins distributed amongst the selected master regulatory molecules are summarized. in support of its biological significance the fused hybrid network was enriched for gene expression data derived from transgenic non-tumour and tumour tissues. thus, the integrated hybrid network consisted of 34 out of 82 regulated proteins and gene expression calls evidenced 6 of the 27 up-regulated tumour (to + ur) proteins to be regulated at the transcript level as well whereas among the 7 down-regulated tumour proteins (dr) the gene arg1 was repressed in expression. likewise, gene expression data from non-tumour transgenic livers evidenced 5 genes out of 27 networks partners to be increased in expression (ur-tr-nt) and among the 7 down-regulated networks proteins the gene phb was repressed (dr-tr-nt). thus, when the tumour gene expression data of the entire network was considered a total of 22 genes were regulated, of which 13 were up-regulated and 9 were repressed in expression, (see figure 7) . based on the information of the hybrid master regulatory network and in addition to other disease regulated proteins summarized in table 1 (note, some of the proteins were not part of the networks) a total of n = 122 disease regulated proteins were considered for network construction. after filtering for non-connected proteins the string database informed on n = 151 protein-interactions of which n = 76 were disease regulated as identified in the present study. among these 45, 24 and 7 were either up-, down-or not statistically significantly regulated. furthermore, gene expression calls for 45 up-regulated proteins were supported by 5 up-and 4 down-regulated genes identified in tumours and 4 up-and 6 down-regulated genes in transgenic non-tumour livers. likewise, gene expression calls for 24 down-regulated proteins were supported by 8 and 5 downregulated genes in tumours and transgenic non-tumour livers, respectively. therefore, the entire network was supported by 14 induced and 17 repressed tumour specific gene expression changes and 16 up-regulated and 13 downregulated genes observed in transgenic non-tumour livers. as depicted in figure 8 the proteins of the fusion network displayed functional associations via the egf/egfr network and included 69 out of 96 (72%) significantly regulated tumour proteins with 6 out of 7 master regulators being connected to egfr through the network's proteins (see additional file 14: table s12 for possible protein-protein interactions and related scores). of the 151 network proteins 109 could be mapped to distinct pathways. after removal of non-relevant terms such as alzheimer disease a total of 94 proteins were mapped to 6 pathways with meaningful associations (see figure 9 ) and consisted of 'platelet activation, signalling and aggregation (platelet degranulation)' , 'lipoprotein metabolism' , 'mapk signalling pathway' , 'glycolysis and gluconeogenesis', 'metabolism of amino acids and derivatives (arginine and proline metabolism)', 'apoptosis' and 'egfr1 signalling pathway'. additionally, a total of 2 and 3 tumour regulated proteins were mapped to the hif-1 signalling and protein processing in endoplasmic reticulum pathways, respectively. the pathway mapping was also supported by gene expression data with 10 up-and 9 down-regulated genes in tumours and 9 up-and 6 downregulated genes in transgenic non-tumour livers. note, two of the significantly regulated tumour proteins, i.e. crk and pebp1 are members of the egfr1 signalling pathway with pebp1 also functioning as a master regulator while the other regulated proteins are connected to egfr signalling through cross-talk among the pathways (see additional file 15: table s13 ). figure 6 integrated master regulatory network for proteins uniquely expressed in tumours. based on network information obtained for the 5 different master regulators an integrated hybrid network was constructed. the network contained 82 proteins including 20 with connectivity to egfr signalling (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partitioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression. recent research into the molecular pathogenesis of hcc evidenced significant alterations in signalling pathways. given the fact that the epidermal growth factor is an important mitogen for hepatocytes we were particularly interested in investigating the consequences of its targeted overexpression in the liver. in our previous study we employed chromatin immunoprecipitation followed by cloning and sequencing of dna to search for tumour associated gene regulations targeted by novel hnf4alpha p1 and p2 promoter-driven isoforms. this identified egf-receptor substrate (eps15r) and eps15 as regulated by the p2 promoter-driven hnf4alpha splice variant in mouse and human hcc. a molecular circuitry was proposed whereby eps15 and eps15r mediate internalization of activated egfr to stimulate receptor recycling, therefore responding to mitogenic signalling of egf [66] . in the present study disease proteomics was performed to further investigate the role of egf in liver cancer. this identified 122 regulated proteins of which 37 are novel and have not been reported so far. a total of 63 proteins were significantly up-regulated (table 1) . among these 18 were extra-cellular or secreted proteins and included albumin and isoforms of it, apolipoproteins (apoe, apoa4 and apoai), α-, β-, γfibrinogen, plasminogen as well as interleukin 1 receptor antagonist (il-1ra). note, an isoform of apoa1 was already proposed as serum marker of hcc [67] and based on ihc staining il-1ra expression was confirmed in about 70% of mouse liver adenoma and carcinoma cases; however preneoplastic foci as well as normal hepatocytes surrounding the lesions were negative. furthermore, rt-pcr analysis confirmed mouse hepatic tumours to contain both secreted and intracellular forms of il-1ra [51] and figure 7 integrated master regulatory network for hcc regulated proteins. based on network information obtained for 7 different master regulators an integrated hybrid network was constructed. the network contained 114 proteins including 34 with connectivity to egf/egfr signalling (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partitioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression. serum levels of il-1ra were monitored to assess therapeutic efficacy of radiofrequency ablation in hcc patients [68] . an important finding of the present study is the statistically significant regulation of 20 mitochondria associated proteins of which 13 were repressed while 7 were upregulated. similar results were reported by chignard and wei sun with mitochondrial proteins being the second largest proportion of regulated proteins in human viral hcc [15, 69] . among the repressed proteins were nadh dehydrogenase (ubiquinone) 1 alpha subcomplex 8 and prohibitin, a mitochondrial chaperone. this protein, when deleted (prohibitin ko mice) induced fibrosis, bile duct metaplasia, liver dysplasia and eventually multifocal hcc. however, its overexpression in tumour cell lines inhibited cell proliferation to demonstrate tumour suppressor function [70] . likewise, glutathione peroxidase 1 (response to oxidative stress) and argininosuccinate synthetase 1 (ass, urea cycle) were repressed. note, ass is the first of two enzymes to convert citrulline to arginine and this pathway allows cells to synthesize arginine from citrulline to function in no production, ammonia detoxification and synthesis of polyamines. several reports suggest ass deficiency to be common in tumour cell lines [25] [26] [27] [28] [29] [30] , and the present study confirms ass expression to be confined to healthy non-transgenic control liver, but ass was absent in tumour tissue extracts (see table 1 ). ablation of ass in diverse tumours suggests a tumour suppressor function and the fact that forced expression of ass in osteosarcoma cell lines suppresses growth adds weight to this notion [71] . another example of tumour specific ablation of proteins refers to glycine n-methyltransferase (gnmt). the enzyme catalyzes the transfer of a methyl group from s-adenosylmethionine (sam) to glycine thereby generating s-adenosylhomocysteine and n-methylglycine. this protein was completely downregulated in liver tumours. gnmt is known to play a role in the maintenance of genetic stability [44, 72] , and a novel tumour suppressor function was recently reported that is independent of its catalytic activity but does require its nuclear localization [73] . several of the proteins listed in table 1 were already reported for their tumour specific regulation while proteins so far unknown for their regulations in hcc, are marked with an asterisk (table 1) . these function in diverse biological processes including metabolism, translation and signalling. specifically, changes in carbohydrate metabolism are commonly observed in tumours where energy production relies on glycolysis rather than mitochondrial oxidative phosphorylation. in the present study induced expression of several glycolytic enzymes was observed, most notable [1] pyruvate kinase 3 that catalyzes the transfer of a phosphate group from phosphoenolpyruvate to adp and was shown to be a target of mi-rna122 in hcc [2, 74] aldolase, an enzyme that converts fructose 1,6-bisphosphate into dihydroxyacetone phosphate (dhap) and glyceraldehyde 3-phosphate and was reported to be a sensitive marker for benign and malignant liver disease [75] and [3] alpha glucosidase 2, a hydrolase that cleaves glycosidic bonds with the release of alpha glucose from carbohydrates. further important findings include the tumour specific expression of alanyl-, glycyl-and seryl-trna synthetases which catalyze the transfer of specific amino acids to trna, as well as regulation of eukaryotic translation elongation factor 2 and poly(rc) protein 2 that binds to oligo dc. note, knowledge on the role of aminoacyl-trna synthetases in cancer is just emerging [76] and through the use of a lentiviral mediated shrna vector, a link between aminoacyl-trna synthetases [aars]-interacting multifunctional protein 2 (aimp2) and repressed egfr signalling was established that resulted in repressed glucose uptake [77] . we also observed induced expression of heterogeneous ribonucleoprotein (hnrnp) that takes on diverse functions in the processing of mrna. its expression was reported to be increased in serum of hcc patients . in contrast, proteins involved in the synthesis and degradation of cholesterol, lipids, steroids and fatty acid were in part oppositely regulated and included induced expression of the aldo-keto reductase family 1. regulation of this protein has been reported for lung and pancreatic cancers [78] , and gene silencing of aldo-keto reductase family 1 b10 resulted in growth inhibition of colorectal cancer cells that might be of therapeutic utility [79] . the repressed expression of figure 9 pathways mapping of fussed network proteins. cytoscape 3.0.2 with plugins (see methods section) are used to generate functionally grouped network of pathways. grouping of significant pathway terms (p ≤ 0.05) were based on kappa score threshold of 0.4, initial group size of 2 and sharing group percentage of 50. the pathway network consisted of 35 significantly and 7 non-significantly regulated proteins involved in distinct pathways which are colour-coded. note, the two individual terms are grey-coloured. up and down-regulated proteins are coded as orange and green small discs, respectively. up-and down-regulated as well as non-significantly regulated proteins and connecting proteins of the network are given as orange, green, yellow and blue coloured discs, respectively. the network depicts protein-protein interactions in liver tumours of egfr transgenic mice and their relation to various pathways under the influence of egfr signalling. egfr is highlighted as blue triangle in this network. certain proteins may also be considered as an adaptive response and includes the enzyme enoyl coenzyme a hydratase 1. its activity was shown to contribute to lymphatic spread of liver tumours as was evidenced in gene silencing studies [80] . likewise, we observed repressed expression of dihydrodiol dehydrogenase in tumours. this enzyme plays an important role in the metabolism of steroids that leads to inactivation of circulating androgens, progestins and glucocorticoids and was repeatedly reported to be overexpressed in non-small cell lung cancer. amongst patients with high dhd expression the incidence of early tumour recurrence and distant metastasis is significantly higher and patients are highly resistant to chemo and radiotherapy [81] . intriguingly, complete ablation of mitochondrial butyryl coenzyme a synthetase 1, a gtp-dependent lipoateactivating enzyme was observed in tumours of egf transgenic mice. little is known about the possible link between butyrate metabolism and liver cancer. however, butyrate is well known to inhibit proliferation of human colon carcinoma cells in an epigenetic manner that involves histone acetylation [82] . note, it was recently reported that due to the warburg effect butyrate-mediated histone acetylation and cell proliferation is dictated [83] . several lines of evidence therefore suggest butyrate to act as a cytosolic sensor for histone acteylation and when transformed to intermediates by butyryl coenzyme a synthetase is unable to escape the mitochondria. moreover, we observed a highly significant repression of 2-hydroxyphytanoyl-coa-lyase. this peroxisomal thiamine pyrophosphate-dependent enzyme is rate limiting in the breakdown of 2-hydroxy fatty acids. the biological role of 2-hydroxy fatty acids has only recently become apparent [84] and cumulative evidence suggests intermediates of energy metabolism to specifically activate g-protein coupled receptors which are now classified as hydroxy carboxylic acid receptors (hca1-3). the hca2 receptor is involved in a complex negative feed-back loop whereby ketone bodies derived from fatty acid oxidation are sensed by hca2 via the activity of 3-hydroxybutyrate that leads to inhibition of lipolysis and to restriction of further fatty acid supply. in this way triglyceride use is diverted and energy demands for tumour growth are met more efficiently. specifically, during rapid tumour growth and the herewith associated ischemia the yield of high energy bonds (atp) from glucose oxidation is about twice that of fatty acid oxidation. our observation that proteins involved in the ß-oxidation of fatty acids were either repressed or unchanged agrees well with this principle (see also discussion below). the reduced expression of lysophosphopholipase signifies an adaptive response; it catalyses the production of lysophosphatidic acid, i.e. a second messenger known to contribute to tumour cell motility, survival and proliferation [85] . additionally, the repressed expression of mitochondrial acyl-coa thioesterase 1 in liver tumours which hydrolyzes acyl-coas to free fatty acids and coenzyme a, will influence the supply of ligands for nuclear receptors and the regulation of fatty acid oxidation in mitochondria and peroxisomes. equally, the regulation of farnesyl diphosphate synthetase, i.e. a key enzyme in the isoprenoid biosynthetic pathway is highly interesting and this enzyme is explored as a drug target of bisphosphonates to treat tumour growth [86] . it's up-regulation in colon cancers was reported [39] . in the present study repressed expression of the ribosom-compononent rps12 and enzymes of amino acid metabolism like branched chain ketoacid dehydrogenase e1 as well as dimethyl glycine dehydrogease was observed. conversely, expression of the proteasome 26s atpase subunit 5 (p45/sug) and its non-atpase regulatory subunit 11 (psmd11) was confined to tumour tissues (see table 1 ); the latter subunit is known to display high activity in embryonic stem cells. this multicomplex molecular machinery degrades intracellular proteins marked up by ubiquitin chains. psmd11 was reported to be up-regulated in breast cancer cells [87] . enhanced expression of cytoskeletal proteins such as tubulin β 5 and capza1 was also confirmed by ihc staining (see figure 3 ). differences in the localization of these proteins were obvious with tubulin ß 5 expression being primarily associated with cells proximal to the liver capsule, whereas expression of capping protein zline α1 (capza1) was strongly associated with tumour foci and this protein is known to play a pivotal role in cytoskeletal networks to support cell mobility, invasion and metastasis. additionally, gdi2, a protein functioning in the cycling of rab gtpases and arginase ii, i.e. a non-liver isoform of the urea cycle were up-regulated in tumours of egf transgenic mice (see figure 3 ). regulation of arginase ii was observed in various malignancies including lung cancer [88] . besides, the actin-binding protein lasp1 was uniquely expressed in tumours and is also up-regulated in breast cancer [89] to possibly support migration of cancer cells [90] . furthermore, pdia4, a disulfide bond isomerase and master regulator of the constructed networks (see below) was up-regulated as was kininogen that is part of the blood coagulation system and functions as a precursor of kinin. conversely, the serinproteinase inhibitor serpinb1a was repressed in expression to possible limited immunological responses in tumour growth and to influence inflammatory cytokine production by infiltrating monocytes [91] . the significant regulation of the calcium binding protein sorcin and nucleobindin 1 are further highly interesting results. sorcin is associated with multidrug-resistance in human leukemia cells [92] and nucleobindin 1 is evaluated as a biomarker of colon cancer [93] . in egf induced liver tumours transthyretin was also up-regulated. this protein is involved in the transport of thyroid hormones and was reported to be aberrantly regulated in thyroid cancer [94] . among the newly identified proteins is v-crk sarcoma virus ct10. this oncoprotein interacts with several tyrosinephosphorylated proteins and is part of the intracellular signalling cascades notably the phosphoinositide 3-kinase (pi3k)/akt pathway [95] . likewise, regulation of the 170 kda glucose-regulated protein grp170 is of great importance. this lumenal endoplasmic reticulum plays a role in immunoglobulin folding as was confirmed by coimmunoprecipitation in four different b cell hybridoma cell lines [11] . in our previous study several immunoglobulins were found to be either repressed or absent in serum of egf tumour bearing mice and this was particularly obvious for the ig k and l classes [7] . it remains to be determined whether repression of immunoglobulins can be attributed to aberrant grp170 activity. a summary of the biological functions in addition to their previous reported tumour association is given in additional file 16: table s14 while the regulation of genes coding for newly identified proteins and of genes coding for commonly regulated proteins in liver tumours and serum of egf2b-transgenic mice is given in additional file 17: table s15 and additional file 18: table s16 . initially the network construction was based on proteins exclusively expressed in tumours and by selecting master regulatory proteins linked to egfr signalling. thereafter, a fused hybrid network was developed in which tumour specific proteins were part of it. subsequently, the search was extended to all significantly regulated proteins (table 1) . this revealed 7 master regulatory proteins and its associated networks and encompassed 114 proteins of which 34 were disease regulated. eventually a fused network was developed; however not all disease regulated proteins are part of it. the performed pathway mapping over fused networks (see string analysis) defined protein interactions and grouped 76 disease regulated proteins into 6 distinct pathways of which platelet activation, signalling and aggregation is a major one (see figure 8) . specifically, the glycoprotein fibrinogen is a multimeric protein and consists of α, ß and y subunits. it is synthesized by hepatocytes and an essential blood coagulation factor with all polypeptide chains being highly regulated in tumours of egf transgenic mice. note, an association between coagulation factors and malignancies was established whereby fibrinogen functions as an extracellular matrix protein to interact with integrin receptors in the control of cell proliferation and cell migration [96] . accordingly, induced gene expression of the integrin receptors itgb1, itga3 and itgav was observed in egf induced liver tumours. in cancer progression a regulatory loop between fibrinogen, platelets and tumour cells has been determined that is activated by platelet cytosolic ca2+. this second messenger induces integrin receptor complex formation through an association of platelet glycoprotein chains iib and iiia (cd41/cd61) thereby creating an active binding site for fibrinogen. an association of tumour regulated proteins with the regulatory loop was confirmed in string analysis ( figure 8 ) and fibrinogen was reported to be an important determinant for metastasis of circulating tumour cells [97] . it is therefore of no surprise that elevated blood fibrinogen is a poor prognostic factor. haemostatic complications are commonly observed in cancer patients and future therapeutic strategies may focus on the hemostatic system by targeting tumour stroma. in this regard the tumour specific induction of plasminogen is of great importance. this zymogen [98] is converted to plasmin by urokinase (upa), a serine protease which itself was unchanged; however, gene expression of its receptor was significantly up-regulated in transgenic non-tumour livers. one report suggests the urokinase receptor to prime cells for proliferation in response to egf by promoting tyr845 phosphorylation and stat5b activation; nonetheless, this depended on intracellular c-src levels [99] . further studies established a link between induced expression of plasminogen activator, upa receptor and plasminogen activator inhibitor type-1 (pai-1) and invasiveness and metastasis of hcc [100, 101] . indeed, a fine balance exists between the plasminogen activating system and its inhibition by pai-1 and pai-2. based on transcriptomic data a highly significant induction of pai-i (up to 12-fold) in large tumours of egf transgenic mice was observed [6] ; consequently, the regulation of components of the plasminogen activating system may be considered as part of a strategy to degrade extracellular matrix thereby facilitating invasion and metastasis [102, 103] . to meet energy demands efficiently different sources are utilized and the induction of the proteins aldoa, aldoc, eno1, pkm and fbp1 is testimony to an altered glycolytic and pentose phosphate pathway. however, with the exception of acyl-coa thioesterase 2 that was below the limit of detection and functions in the hydrolysis of myristoyl-palmitoyl-, stearoyl-and arachidoyl-coa esters the regulation of enzymes linked to fatty acid metabolism in mitochondria and peroxisomes was hardly observed. in pursue of tumour growth and to sustain organelle and membrane biogenesis lipids are de novo synthesized and mobilized from stores and while the complex interaction of hepatic lipid and glucose metabolism in liver disease is the subject of intense research [104] the present study evidences significant regulation of several apolipoproteins, i.e. apoe, apoa1, apoa4 and isoforms of albumin. apart from lipid transport apolipoproteins play a wider role in cancers and are known to interact with diverse receptors to elicit cellular events as demonstrated for apoe to cause sustained proliferation and survival of cancer cells [105] . a further group of highly regulated proteins are aldoketo reductases. their quantitative evaluation in different hepatocellular carcinoma (hcc) cell lines was recently reported [106] . this superfamily of proteins comprises nad (p)(h)-dependent enzymes which catalyze oxidoreduction of a variety of prostaglandins, steroids and toxic aldehydes. their involvement in tumorigenesis is supported by several studies and they are explored as drug targets to overcome chemoresistance. in the present study the aldo-keto reductases akr1c14, akr1c18 and akr1c6 were uniquely expressed in tumours, however glutathione peroxidase 1 was repressed to 30% of healthy control livers to possibly support hif-1 signalling. indeed, the redox state and therefore glutathione participates in the hypoxic induction of hif-1 [107] , and two proteins of the glycolytic pathway, i.e. aldoa1 and eno1, which respond to hif-1 signalling, were regulated. moreover, glutathione peroxidase 1 was shifted in the gel as shown in figure 1 panel g iii as a result of post translational modifications that most likely involved c-abl and arg kinase activity at tyr 96 of gpx1 [108] . likewise, the genes coding for aldo1 and eno3 were significantly up-regulated in egf induced liver tumours. a complex interaction exists between egfr and rage signalling. this receptor for advanced glycation end-products is a member of the immunoglobulin family of cell surface molecules and was reported to significantly influence hepatic tumour growth in murine models of colorectal carcinoma [109] . there is strong evidence for rage to promote cancer growth upon ligand dependent activation and several proteins of the s100 family bind to the extracellular domain of rage [110, 111] . it is of considerable importance that gene expression of s100a4 and s100a11 was up to 34-fold induced in tumours of egf transgenic mice, however expression of s100a1 was repressed. likewise the tumour specific expression of the rage binding proteins lectin, galactoside-binding, soluble, 3 and capza1 in tumours of egf transgenic mice is highly suggestive for a sustained crosstalk between rage and egfr [112] . although the precise mechanism by which s100 proteins stimulate egfr signalling remains to be elucidated binding of s100a4 to egf and to other egfr ligands was reported to possibly facilitate interaction with the receptor [113] . similarly, the binding of s100a8/a9 to rage was shown to promote migration and invasion of human breast cancer cells through actin polymerization and epithelial-mesenchymal transition [114] . conversely, advanced glycation endproduct (age) receptor 1 suppressed oxidant stress-dependent signalling via the egfr and shc/grb2/ras pathway [115] . as depicted in figure 5 the amino acid metabolism was another distinct pathway to which several of the regulated proteins could be mapped to. note, the tumour specific regulations of arginine 1 and 2 as well as the regulation of subunits of the proteasome 26s atpase (psmc5 and psmd11) were already discussed (see above). in the following additional proteins regulated in this pathway are briefly summarized. specifically, 3-hydroxyanthranilate-3,4-dioxygenase (haao) catalyzes oxidation of 3-hydroxyanthranilate to quinolinate and this intermediate functions as a precursor in nad and pyridine biosynthetic pathways. expression of haao was significantly repressed in tumours of egf transgenic mice and hypermethylation of the coding gene was observed in ovarian cancer [116] . due to the fact that haao is significantly repressed at the gene and protein level in at least two different tumour entities (ovarian and liver cancer) the protein may function as a tumour suppressor that appears to be repressed by an epigenetic mechanism. a significant finding is the tumour specific expression of 3-phosphoglycerate-dehydrogenase which catalyses the production of 3-phosphoglycerate. this intermediate of glycolysis is an essential precursor of the serine biosynthetic pathway. importantly, a recent metabolomic study evidenced 3-phosphoglycerate to be diverted into serine and glycine metabolism and repressed expression of 3-phosphoglyceratedehydrogenase resulted in impaired tumour cell proliferation [117] . in support of tumour growth the diversion of intermediate of glycolysis affects protein, membrane lipid and nucleotide synthesis. moreover, the observed induction of creatine kinase in tumours of egf transgenic mice creates a circuitry for cellular energy homeostasis in conditions of high metabolic demands [118] . the enzyme catalyses the reversible transfer of phosphate from phosphocreatine to adp to yield atp and creatine. its induction has been observed in many cancers including liver cancer cell lines [119, 120] and a further study suggested a possible interplay between p53 mutations, hcc, ck expression with growth-inhibitory effects of cyclocreatine in hcc [121] . while the rationale of tumour cells in embarking on abnormal metabolism had already been discussed (see above) the finding that agmatine ureohydrolase was strongly repressed in egf induced liver tumours to about 10% of non-transgenic healthy livers is of great importance. this enzyme hydrolyzes agmatine (= decarboxylated arginine) to form putrescine and urea and repression of the enzyme will significantly increase agmatine tissue concentration to influence diverse cellular control mechanisms. importantly, in the study of battaglia and coworkers [122] 1 mm agmatine induced large amounts of superoxide production in rat liver mitochondria; however, it did not affect mitochondrial respiration or redox levels of thiols and glutathione. furthermore, atp synthesis remained normal and prevented ca(2+)-induced mitochondrial permeability transition in the presence of phosphate to suggest an intriguing regulatory loop whereby h2o2 induces hypoxia signalling that is linked to abberant metabolism, nonetheless by selecting interconnected physiological pathways tumour cells are equipped to avoid programmed cell death [122, 123] . thus, arginine deprivation is evaluated for its utility in cancer therapy [124] . a further enzyme repressed to 20% of healthy nontransgenic liver is carbamoyl phosphate synthetase 1 (cps1), i.e. a liver specific ligase to function in ammonia detoxification. it is perplexing that tumour cells disable such an important pathway of the urea cycle. however, a recent study demonstrated dna hypermethylation as a key mechanism of silencing cps1 gene expression in human hcc. note, forced expression of cps1 induced cell proliferation and the observed repression in human hcc may simply be the result of genomic instability as was observed in tumour cells [125] . the present study identified novel disease regulated proteins induced by overexpression of egf to provide new insight into the complex signalling events in hcc. six major pathways perturbed by egfr hyperactivity were identified and several of the regulated proteins are interesting drug target candidates and this includes tumour specific expression of kinases as well as proteins involved in aberrant metabolism. an identification of commonly regulated proteins in tumour and sera will be of great utility in the development of biomarkers to monitor disease progression and responses to therapy. the following additional data are available with the online version of this paper. aminoacyl-trna synthetases interacting multifunctional protein 2; alpha-hcca: alpha-cyano-4-hydroxycinnamic acid; aps: ammonium persulfate; ass: argininosuccinate synthetase 1; atp: adenosine triphosphate; ck: creatine kinase; ck2α: casein kinase ii subunit alpha; co: control specific protein dr: down-regulated protein; dr-t: down-regulated tumour; dr-tr-nt: down-regulated transgenic non-tumour; egf: epidermal growth factor egfr: epidermal growth factor receptor; eps15: epidermal growth factor receptor substrate 15; eps15r: epidermal growth factor receptor substrate 15r; er: endoplasmic reticulum; erk: extracellular signalling regulated kinase; fdr: false discovery rate; fgfr1: fibroblast growth factor receptor 1 go: gene ontology; grp170: 170 kda glucose-regulated protein; gtp: guanosine triphosphate h 3 po 4 : orthophosphoric acid; hca: hydroxy carboxylic acid; hcc: hepatocellular carcinoma; hdac2: histone deacetylase 2; hed: bis(2-hydroxyethyl) disulfide; hrp: horseradish peroxidase ihc: immunohistochemistry; il-1ra: interleukin-1 receptor antagonist ko: knockout mouse mek: mitogen-activated protein kinase kinase; nad: nicotinamide adenine dinucleotide; nad(p)(h): nicotinamide adenine dinucleotide phosphate nadp: nicotinamide adenine dinucleotide phosphate; no: nitric oxide; ogp: n-octyl β-d-glucopyranoside pai-1: plasminogen activator inhibitor type-1 pdgfr: platelet-derived growth factor receptors; plaur: plasminogen activator, urokinase receptor; pmf: peptide mass fingerprinting; ptbp1: polypyrimidine tract binding protein 1; rage: receptor for advanced glycation endproduct s100a1: s100 calcium binding protein a1; sam: s-adenosylmethionine short hairpin rna; stat5b: signal transducer and activator of transcription 5b; temed: tetramethylethylenediamine; to: tumour specific protein; trna: transfer rna; tyr845: tyrosine residue 845; upa: urokinase-type plasminogen activator; ur: up-regulated protein up-regulated transgenic non-tumour; vegfr: vascular endothelial growth factor receptor; xbp1(s): x-box binding protein 1 isoform surgery and ablative therapy for hepatocellular carcinoma improved method for proteome mapping of the liver by 2-de maldi-tof ms identification of specific protein markers in microdissected hepatocellular carcinoma soluble interleukin-2 receptor levels in hepatocellular cancer: a more sensitive marker than alfa fetoprotein discovery and development of sorafenib: a multikinase inhibitor for treating cancer 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antigens as markers in hepatocellular carcinoma a gene expression biomarker provides early prediction and mechanistic assessment of hepatic tumor induction by nongenotoxic chemicals positional expression profiling indicates candidate genes in deletion hotspots of hepatocellular carcinoma hormonal regulation of vitamin d-binding protein production by a human hepatoma cell line geneways: a system for extracting, analyzing, visualizing, and integrating molecular pathway data progression of hcc in mice is associated with a downregulation in the expression of hepatocyte nuclear factors boosting antitumor responses of t lymphocytes infiltrating human prostate cancers amphiregulin is a promising prognostic marker for liver metastases of colorectal cancer pcbp-1 regulates alternative splicing of the cd44 gene and inhibits invasion in human hepatoma cell line hepg2 cells tasp1, and prpf3 are novel disease candidate genes targeted by hnf4alpha splice variants in hepatocellular carcinomas a strategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment proteomics for hepatocellular carcinoma marker discovery liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice reduced argininosuccinate synthetase is a predictive biomarker for the development of pulmonary metastasis in patients with osteosarcoma methyl transfer in glycine n-methyltransferase. a theoretical study a novel tumor suppressor function of glycine n-methyltransferase is independent of its catalytic activity but requires nuclear localization mir-122 targets pyruvate kinase m2 and affects metabolism of hepatocellular carcinoma serum aldolase isoenzymes in benign and malignant liver disease aminoacyl trna synthetases and their connections to disease lentiviral vector-mediated shrna against 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3-kinase/akt pathway by utilizing focal adhesion kinase and h-ras tumors and fibrinogen. the role of fibrinogen as an extracellular matrix protein fibrinogen is an important determinant of the metastatic potential of circulating tumor cells plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease urokinase receptor primes cells to proliferate in response to epidermal growth factor invasion and metastasis of hepatocellular carcinoma in relation to urokinase-type plasminogen activator, its receptor and inhibitor urokinase-type plasminogen activator receptor transcriptionally controlled adenoviruses eradicate pancreatic tumors and liver metastasis in mouse models plasminogen activator system and its clinical significance in patients with a malignant disease the urokinase plasminogen activator system: role in malignancy the interaction of hepatic lipid and glucose metabolism in liver diseases apolipoprotein e is required for cell proliferation and survival in ovarian cancer quantitative evaluation of aldo-keto reductase expression in hepatocellular carcinoma (hcc) cell lines the redox state of glutathione regulates the hypoxic induction of hif-1 controlled elimination of intracellular h(2)o(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification rage signaling significantly impacts tumorigenesis and hepatic tumor growth in murine models of colorectal carcinoma binding of s100 proteins to rage: an update rage and rage ligands in cancer identification of galectin-3 as a high-affinity binding protein for advanced glycation end products (age): a new member of the age-receptor complex epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting s100a4 protein rage-binding s100a8/a9 promotes the migration and invasion of human breast cancer cells through actin polymerization and epithelial-mesenchymal transition advanced glycation end product (age) receptor 1 suppresses cell oxidant stress and activation signaling via egf receptor identification of candidate epigenetic biomarkers for ovarian cancer detection phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the'phosphocreatine circuit' for cellular energy homeostasis creatine and creatinine metabolism mitochondrial creatine kinase as a tumor-associated marker elevated creatine kinase activity in primary hepatocellular carcinoma different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species? hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways arginine deprivation as a targeted therapy for cancer submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we gratefully acknowledge support from the virtual liver network (grant 031 6154) of the german federal ministry of education and research (bmbf) to jb. additional file 1: table s1 . antibodies and dilutions used to study disease regulation by immunohistochemistry.additional file 2: table s2 . statistics and selection criteria for functional grouping of pathways terms of disease regulated proteins. a hypergeometric test followed by bonferroni correction was used with p-value ≤ 0.05. for grouping of pathway terms, the kappa score threshold was set to 0.4. additional file 3: table s3 . different master regulators and associated network for proteins expressed in tumours only (to) or significantly regulated when compared to healthy liver of non-transgenic control animals (t + ur + dr).additional file 4: table s4 . comparison of disease regulated proteins in mouse and human hcc. data were taken from 'the human protein atlas' database.additional file 5: table s5 . comparison of transcriptomic and proteomic data. this comparison revealed 19 significantly regulated genes of which 15 are regulated in common whereas for 4 genes transcript expression was opposite to that of the coded proteins.additional file 6: table s6 . biological processes ontology for significantly regulated proteins. additional file 7: table s7 . cellular component ontology for significantly regulated proteins.additional file 8: table s8 . molecular function ontology for significantly regulated proteins.additional file 9: table s9 . biological pathways and their cluster. cytoscape 3.0.2 with plugins (cluego and cluepedia) were used to generate functionally grouped network of pathways based on reactome, kegg and wikipathways databases. the grouping of significant pathway terms (p ≤ 0.05) is based on a kappa score of 0.4, initial group size of 2 and sharing group percentage of 50.additional file 10: table s10 . biological pathways information based each significantly regulated protein (to + ur + dr). this table depicts pathway terms of nearly 80% of regulated proteins (to + ur + dr) and are taken from reactome and kegg databases.additional file 11: figure s1 . master regulatory networks for proteins uniquely expressed in tumours with link to egfr signalling: (a) the plaur network consists of 44 proteins of which 20 are tumour-specifically regulated, (b) the fgfr1 consists of 41 proteins of which 18 are tumour-specifically regulated, (c) the ptbp1 network consists of 44 proteins of which 18 are tumour-specifically regulated, (d) the agtrap network consists of 43 proteins of which 19 are tumour-specifically regulated and (e) the s100a1 network consists of 38 protein of which 16 are tumour-specifically regulated. note all networks display connectivity to egfr signalling (yellow coloured inner node) and in the case of the s100a1 master regulatory protein egfr s ignalling is via the plaur/egfr network.the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression.additional file 12: table s11 . integrated hybrid network with master regulator information for proteins expressed in tumours only (to) and significantly regulated proteins when compared to heathy liver of non-transgenic control animals (t + ur + dr).additional file 13: figure s2 . master regulatory networks for regulated proteins with link to egfr signalling: (a) the pdia4 network consists of 68 proteins including 32 significantly regulated proteins, (b) the apeh network consists of 68 proteins including 32 significantly regulated proteins, (c) the pebp1 network consists of 63 proteins including 31 significantly regulated proteins, (d) the apoe network consists of 66 proteins including 31 significantly regulated proteins, (e) the arg1 network consists of 67 proteins including 31 significantly regulated proteins, (f) the fbp1 network consists of 69 proteins including 31 significantly regulated proteins, (g) the haao network consists of 66 proteins including 32 significantly regulated proteins. note, all networks display connectivity to egf protein (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression.additional file 14: table s12 . protein interaction information of the fused network. given are interacting proteins with association score from different prediction methods, i.e. neighborhood, gene fusion, co-occurrence, co-expression, databases and textmining.additional file 15: table s13 . biological pathways and their cluster for fused network proteins. reactome, kegg and wikipathways database information was used as input data file for cytoscape 3.0.2. the table shows grouping of significant pathway terms (p ≤ 0.01) and is based on a kappa score of 0.4, initial group size of 2 and sharing group percentage of 50.additional file 16: table s14 . biological function of newly identified proteins and their previously reported tumour association.additional file 17: table s15 . regulation of genes coding for newly identified proteins in egf2b-transgenic liver tumours.additional file 18: table s16 . regulation of genes coding for common proteins in tumour tissue and serum of egf transgenic mice. the authors declare that they have no competing interests.authors' contributions jb conceived the study and contributed the reagents, gg performed the experiments, ps performed the bioinformatics analysis. jb, gg and ps analysed the data, jb wrote the manuscript and all authors read and approved the final manuscript. key: cord-022300-9w0lehal authors: hoskins, johnny d. title: the liver and pancreas date: 2009-05-15 journal: veterinary pediatrics doi: 10.1016/b978-0-7216-7665-4.50015-2 sha: doc_id: 22300 cord_uid: 9w0lehal nan liver enzyme activities. the serum activities of alanine aminotransferase (alt) and aspartate aminotransferase (ast) in newborn and growing puppies and kittens are usually within the normal range or slightly higher than those of healthy adult dogs and cats (keller, 1981) . the serum alkaline phosphatase (alp) and gamma-glutamyltransferase (ggt) activities of 1-to l o-day-old puppies are 20-to 25fold greater than those of healthy adults. selected laboratory values used as indicators of hepatobiliary dysfunction in newborn and growing puppies and kittens are listed in table 11 -1. the source of the profound alp and ggt activity is probably of placental, colostral, and! or intestinal origin. these profound serum alp and ggt activities after 10 to 14 days postpartum will decrease to moderately increased activities. although the bone alp isoenzyme derived from active osteoblast activity may increase the serum alp activity in growing animals, the magnitude of increase usually is only two-to threefold the normal level (center et ai, 1995) . colostrum is rich in both alp and ggt activity, and it is possible that these enzymes may be absorbed from the colostrum or intestinal tract during the first days of life. alternatively, the ingestion of colostrum may stimulate intestinal growth and enzyme production. therefore, colostrum-deprived puppies can be identified by their serum alp and ggt activity level, particularly during the first week of 200 life, as an indicator of successful ingestion of colostrum. thus, increased serum alp and ggt activities cannot be used in the diagnosis of acute liver dysfunction during the first 10 to 14 days of life. such increases in serum alp and ggt activities cannot be used to detect colostrum-deprived kittens. serum bile acids. serum bile acids are used to identify hepatic and hepatoportal circulatory dysfunction. the use of the serum bile acid measurement as a test of hepatic dysfunction is of value for the young dog and cat using 12-to 24-hour fasted and 2-hour postprandial serum samples (center et al, 1985a,b) . the normal fasting and 2-hour postprandial values established for adults may be used for puppies and kittens as young as 4 weeks. this test is also reliable for detecting portal circulatory abnormalities (center et al, 1986a; meyer, 1986) . extramedullary hematopoiesis. extramedullary hematopoiesis is commonly observed in the livers of the puppy and kitten through 4 months of age. in older puppies and kittens, disorders associated with blood loss and a subsequent need for erythron mass replenishment may also be associated with hepatic extramedullary hematopoiesis. congenital gallbladder disorders. congenital division of the gallbladder is the most common anomaly and has been referred to as an accessory, cleft, or diverticular gallbladder (bartlett, 1951; boyden, 1926) . these anomalies may develop as an initial subdivision of the primary cystic diverticulum of the embryo or as a bud from the neck of the embryonic gallbladder. the gallbladder may become trilobed or bilobed ( fig. 11-1 ). other anomalies include accessory gallbladders; the development of two separated gallbladders with cystic ducts uniting in a common duct; ductular bladders developing as supernumerary vesicles derived from either hepatic, cystic, or common bile ducts; and trabecular bladders derived from vesicular outgrowths of liver trabeculae. these malformations are infrequently associated with signs of hepatobiliary dysfunction in puppies and kittens. congenital hepatic cysts. congenital hepatic cysts are infrequently found in the young dog and cat (black, 1983; crowell et ai, 1979; mckenna and carpenter, 1980) . cystic lesions may be parenchymal or ductal in origin and may be either solitary or multiple (polycystic). cystic lesions vary in size from a few millimeters to several centimeters. congenital hepatic cysts are typically asymptomatic. rarely, fluid may accumulate in the abdominal cavity as a consequence of cyst rupture or portal hypertension resulting from impingement of major vessels. cyst contents are usually a clear or modified transudate, although cysts may contain bile or blood. acquired hepatic cysts are usually solitary, whereas congenital or developmental hepatic cysts are commonly multiple. polycystic hepatic cysts may be associated with cystic lesions in other organs, most notably the kidneys. polycystic kidneys and liver have been identified in cairn terrier dogs and persian cats during the first few months of life (crowell et ai, 1979; mckenna and carpenter, 1980) . abdominal radiographs may reveal an irregular hepatic margin or focal densities if a few large hepatic or biliary cysts are present. ultrasonography readily reveals the cystic nature of these lesions and the extent of parenchymal or biliary tract involvement ( fig. 11-2 ). treatment is usually not needed for a congenital hepatic cyst unless a large cyst is causing abdominal discomfort or fluid accumulation. if cystic lesions are symptomatic, surgical resection of solitary lesions, partial cyst wall, or a liver lobe may be required. congenital biliary tract malformations. congenital anomalies of the biliary tract are rare in the young dog and cat. one case of suspected biliary atresia in the cat is reported (blood, 1947) . shunts. the hepatic portal system develops from the umbilical and omphalomesenteric systems. the mesenteric portions of the omphalomesenteric veins become the tributaries of the portal vein. small anastomoses develop between the portal and systemic circulation routes that become the normal portosystemic venous communications (heath and house, 1970; khan and vitums, 1971; sleight and thomford, 1969; viturns, 1959 ). in the fetus, blood from the umbilical vein flows directly to the caudal vena cava through the ductus venosus, thus bypassing the liver. by passively responding to changes in the systemic or hepatic circulation, the ductus venosus stabilizes the venous return to the fetal heart as the umbilical venous return fluctuates (edelstone, 1980) . functional and morphologic closure of the ductus venosus does not occur at the same time after birth (lohse and suter, 1977) . functional closure develops gradually during the second and third days after birth in the puppy. morphologic closure occurs as the ductus atrophies, resulting in the formation of a thin fibrous band, the ligamentum venosum, within the liver. the ductus closure depends on changes in pressure and resistance across the hepatic vasculature that follows the postnatal obliteration of the umbilical circulation. morphologic closure of the ductus occurs by 1 to 3 months after birth. congenital portosystemic venous shunts (pss) are abnormal vascular connections between the portal and systemic venous circulations boothe et ai, 1996; bostwick and twedt, 1995; carr and thornburg, 1984; gandolfi, 1984; scaveui et al, 1986) . several different types of congenital pss occur in young dogs and cats, including but not limited to (1) persistent patent fetal ductus venosus, (2) direct portal vein to caudal vena cava, (3) direct portal vein to azygos vein, (4) combination of portal vein with caudal vena cava into the azygos vein, (5) left gastric vein to vena caval shunt, (6) portal vein hypoplasia or atresia with secondary anomalous vessel, and (7) anomalous malformation of the caudal vena cava (center et ai, 1995) . the most cornman types of congenital pss are illustrated in figure 11 -3. in the cat, the most cornman congenital pss involves the left gastric vein . a congenital pss in puppies and kittens is usually a single anomalous vessel in extrahepatic or intrahepatic locations, whereas an acquired pss most commonly occurs as multiple extrahepatic smaller vessels that become patent during sustained portal hypertension. the consequences of the anomalous portal circulation are that the portal blood contains toxins absorbed from the intestines that is delivered directly to the systemic circulation without benefit of hepatic detoxification, contributing to signs of hepatic encephalopathy, and that hepatotrophic factors in the visceral circulation draining the gastrointestinal tract and pancreas do not circulate directly to the liver, causing inadequate liver development and reduced functional liver tissue. signs of hepatic dysfunction associated with congenital pss are usually exhibited at a young age in puppies and kittens. puppies may exhibit the signs as early as 6 to 8 weeks of age. the signs in puppies are variable but may include vomiting, diarrhea, anorexia, small body stature, weight loss, intermittent fever, polyphagia, polydipsia, hematuria, hypersalivation, intolerance to anesthetic agents or tranquilizers that require hepatic metabolism or excretion, atypical behavior, and, rarely, ascites or icterus. intermittent neurologic abnormalities associated with ingestion of protein-laden food or resolving hemorrhage are common and may include episodic aggression, amaurosis, ataxia, incessant pacing, circling, head pressing, and seizures. some puppies present with ammonium biurate uroliths located in the urinary tract (marretta et al, 1981) . most cases of congenital pss in kittens (by 6 months of age in 75% of affected kittens) occur in himalayan, persian, and mixed breed cats, although any breed may have a congenital pss (levy, 1997) . kittens may exhibit signs as early as ages 6 to 8 weeks. the signs of congenital pss are usually hypersalivation, seizures, ataxia, tremors, and depression. intermittent or permanent blindness and mydriasis are also ob-the liver end pencrces i 203 served. other less often noted signs may include vomiting, diarrhea, anorexia, tachypnea, dyspnea, and nasal discharge. polyuria and polydipsia are observed infrequently. dysuria may be observed in kittens with ammonium biurate calculi. about two thirds of affected kittens will have small body stature and be thin and unkempt. a definitive diagnosis of congenital pss in puppies and kittens is often not possible by routine laboratory evaluations. complete blood counts (cbcs), serum chemistry profiles, and urinalysis may help rule out other causes of presenting signs such as acute renal failure, electrolyte derangements, hypoglycemia, and urinary tract disorders. the cbc findings may include microcytic, normochromic erythrocytes and/or a mild nonregenerative anemia (griffiths et al, 1981) . poikilocytosis has been observed in peripheral blood films of some kittens with congenital pss. urinalysis may reveal ammonium biurate crystals when viewed under magnification (x 400) to determine their typical color and shape. serum chemistry profiles may reveal mild increases in the serum activities of alt, as'f, and alp. because of the young age of the animals at initial diagnosis, the serum alp activity is usually two-to threefold higher than normal. the serum activity levels of the alt and ast are less frequently increased. in some cases, active liver disease coexists with a congenital pss, and the animals thus affected have mildly to moderately increased liver enzyme activity and notable hepatic inflammation and/or fibrosis on histopathologic examination. in most animals with congenital pss, the total bilirubin value is normal. albumin values may be mildly decreased. coagulation profiles including prothrombin time, activated partial thromboplastin time, and fibrinogen are usually normal. serum glucose values may be normal, mildly reduced, or markedly hypoglycemic. in some cases, the hypoglycemia-produced neuroglycopenia may complicate the recognition of the congenital pss. animals with congenital pss may become hypoglycemic due to insufficient glycogen stores, abnormal responsiveness to glucagon, hyperglucagonemia, or abnormal insulin metabolism (lickley et al, 1975; magne and macy, 1984) . these abnormalities, coupled with the metabolic immaturity of the young animal's liver, may cause profound hypoglycemia during the first weeks of life. toy-breed puppies appear to be at increased risk for profound hypoglycemia. the blood urea nitrogen concentration may be low or in the low normal range in any young animal with hepatic dysfunction. the most reliable and consistent blood test for the detection of liver dysfunction in puppies and kittens with congenital pss is the 12-to 24-hour fasted and 2-hour postprandial serum bile acid concentrations (center et ai, 1986b; meyer, 1986) . diagnostic imaging of a puppy or kitten with abnormal serum bile acid values helps determine if a suspected congenital pss is present (table 11 -2) (lamb, 1996; lamb et ai, 1996) . animals with congenital pss frequently have reduced hepatic size (i.e., rounded contour of the caudal edge of the liver and cranial displacement of the stomach radiographically) (lamb, 1997) . in addition, these animals may have opaque ammonium biurate calculi. ultrasonographic findings in puppies with congenital pss include small liver, reduced visibility of intrahepatic portal vasculature, and an anomalous blood vessel draining into the caudal vena cava or sometimes into the azygos vein (lamb, 1996) . two-dimensional, gray-scale ultrasonography is used to image through a ventral abdominal wall; however, in most large puppies the optimal approach to the portal vein is through a lateral abdominal wall using the right intercostal spaces (lamb, 1997) . the portal vein is normally visible by ultrasound imaging as ultrasound waves enter the liver at the porta hepatis, ventral to the caudal vena cava. lobar branches of the portal vein have echogenic walls. congenital intrahepatic portocaval shunts are identified on the basis of their ultrasonographic appearance as left-divisional, central-divisional, or right-divisional intrahepatic shunts (lamb, 1997) . left-divisional intrahepatic shunts have a relatively consistent bent tubular shape and drain into the left hepatic vein. central-divisional intrahepatic shunts take the form of a foramen between dilated portions of the intrahepatic portal vein and caudal vena cava. rightdivisional intrahepatic shunts appear as large, tortuous vessels that extend far to the right of midline. the morphology of the left-divisional shunts is compatible with patent ductus venosus. the irish wolfhound and deerhound are predisposed to left-divisional intrahepatic shunts; the old english sheepdog and australian cattle dog are predisposed to central-divisional intrahepatic shunts; labrador and golden retrievers are affected by both left-divisional and central-divisional intrahepatic shunts (lamb, 1997) . animals with extrahepatic congenital pss typically have an anomalous vessel that drains into the caudal vena cava between the right renal vein and the hepatic veins; because of the dorsal location, this anomalous vessel may be visible only through the right dorsal intercostal spaces (lamb, 1997) . congenital portoazygos shunts may also be visualized using the right dorsal intercostal approach; looking for the shunting vessel at the point where it drains into the caudal vena cava is more accurate than trying to examine the various tributaries of the portal vein (lamb, 1997) . extrahepatic shunts may be difficult to identify ultrasonographically if access to the relevant structures is hindered by the skill of the person performing the ultrasana graphic study, the animal's large body size, a lack of acoustic windows as a result of reduced hepatic size, or the presence of excessive intestinal gas. duplex doppler ultrasonography may be used to measure portal blood flow velocity in puppies with suspected congenital pss (lamb and mahoney, 1994) . normal portal blood flow is relatively uniform and nonpulsatile, average portal blood flow velocity being approximately 15 cm/s in healthy, unsedated puppies. the caudal vena cava normally contains variable blood flow because of the influence of changing right atrial and pleural pressures. in most cases, congenital pss represents a low resistance pathway for blood to bypass the liver and enter the caudal vena cava. in affected puppies with congenital pss, the portal vein is exposed to right atrial and pleural pressure changes, and the pattern of portal blood flow may become similar to that noted in the caudal vena cava. puppies with portal hypertension have reduced mean portal blood flow velocity, which correlates with the presence of multiple extrahepatic anomalous vessels. puppies with portal hypertension as a result of hepatic arteriovenous fistula have pulsatile hepatofugal flow in the portal vein. most extrahepatic and intrahepatic congenital psss are detectable using two-dimensional, gray-scale ultrasonography; however, use of color-flow doppler ultrasonography aids in the detection of small extrahepatic shunting vessels. a congenital extrahepatic pss usually drains into the caudal vena cava close to the cranial pole of the right kidney, and on color-flow images a localized area of turbulent flow in the caudal vena cava indicates the shunt's location. when two-dimensional gray-scale, duplex doppler, and color-flow doppler modalities are used in combination, the accuracy for ultrasonographic diagnosis of congenital pss in puppies and kittens can be at least 94% (lamb, 1997) . various techniques for opacification of the portal vein and its hepatic branches have been used, including operative mesenteric portography, cranial mesenteric angiography, and percutaneous splenoportography (suter, 1975) . operative mesenteric portography is the most frequently performed opacification procedure for suspected congenital pss, where abdominal radiographs are made immediately after injection of contrast medium into a indwelling catheter placed surgically in a mesenteric vein . obtaining lateral and ventrodorsal portograms usually provides an excellent view of the intrahepatic or extrahepatic shunt. this technique can also be used in combination with surgery, in which case the mesenteric vein catheter is also used to measure portal blood pressure during the congenital shunt ligation or attenuation procedure. repeating the portogram after shunt ligation or attenuation enables the surgeon to assess the patency of the intrahepatic portal vessel and to check if there is any other congenital shunt(s) present. portal scintigraphy with tc 99m-pertechnetate that is absorbed into the portal circulation after administration per rectum is currently being used in puppies and kittens to detect congenital pss (forstervan hijfte et ai, 1996; koblik et al, 1990 ). by acquiring a dynamic series of gamma camera images of the thorax and cranial abdomen immediately after administration of tc 99m _ pertechnetate and comparing the rate of accumulation of tc 99m-pertechnetate activity in the liver and heart, congenital pss may be detected with a high degree of accuracy. tc 99m-pertechnetate activity in the portal vein normally accumulates first in the liver, but in animals with congenital pss the distribution of tc 99m_per_ technetate activity is altered as it bypasses the liver, reaching the heart before the liver. the severity of the congenital pss can be quantified as a shunt index that provides an estimate of the proportion of portal blood that bypasses the liver. normal puppies have a shunt index of less than 15%; most puppies with congenital pss have a shunt index greater than 60%. an alternative technique for portal scintigraphy involves ultrasound-guided injection of a radiochemical directly into a splenic vein (meyer et al, 1994) . this method of injection combined with the use of tc 99m-iabeled macroaggregates that are normally trapped in the cap-illaries or sinusoids of the target organ enables accurate quantification of the degree of congenital shunting. typical values of a shunt index using this technique are less than 5% for normal puppies and greater than 90% for puppies with congenital pss. whether the administration per rectum or splenic vein injection technique is used, portal scintigraphy can provide a comparison between the shunt index before and after surgical treatment (van vechten et al, 1994) . the shunt index is usually markedly decreased after surgery to attenuate or ligate an anomalous vessel, although it may not be in the normal range. a continued high shunt index is a poor prognostic sign. surgical ligation or shunt vessel attenuation is the definitive treatment for congenital pss and is the preferred method of long-term management (birchard, 1984; breznock et ai, 1983; vogt et al, 1996; wrigley et ai, 1983) . the extrahepatic congenital pss is more amenable to surgical ligation or shunt vessel attenuation than are intrahepatic congenital psss. medical management should be given to the affected animal before and after surgical correction until improvement in hepatic function is shown. mfected animals undergoing surgery should have their body temperature stabilized and should receive intravenous fluids supplemented with 2.5% or 5.0% dextrose solution and with potassium chloride. in addition, owners should be cautioned that any animal with a congenital pss probably would have a shortened life expectancy despite satisfactory surgical correction and their conscientious care. manometric determination of baseline portal blood pressure should be completed before shunt vessel ligation (hardie, 1997) . after visual observation of the anomalous vessel, a ligature is temporarily placed while manometric determination of the portal blood pressure is made. equilibration of the manometer pressure usually takes several minutes. if the relative change in portal pressure exceeds 10 cm h 20 or the postligation pressure exceeds 20 em h 20, the ligature should be loosened. assessment of visceral perfusion by color change (cyanosis and/ or injection) or of arterial vasospasm causing a throbbing of the mesenteric circulation is not a reliable method of determining the safe tautness of the shunt vessel ligature. in many cases, only a partial surgical ligation, or shunt vessel attenuation, can be completed. further ligation may be possible in several weeks or months. incomplete ligation of a congenital pss can result in marked clinical improvement of the animal and owner satisfaction. partial shunt vessel ligation may result in eventual complete shunt closure within 6 months in some animals. complications after shunt vessel ligation or attenuation are frequent and may lead to the animal's death (hardie, 1997) . it is important that the owner understand the potential complications before surgery is attempted because many times the financial and emotional costs of complications are great. the immediate complication rate for performing a laparotomy on puppies and kittens with congenital pss is between 14% and 25%. because many of the complications are life threatening, it is reasonable to tell the owner that this surgery carries a 15% risk of death due to unexpected complications. intrahepatic shunt ligation requires longer surgery times than does extrahepatic shunt ligation, but the risk of death is no higher than with extrahepatic shunt ligation when performed by a experienced surgeon. in the immediate postoperative period, the animal is closely monitored for signs of portal hypertension, as indicated by acute abdominal swelling, abdominal pain, shock, vomiting, or bloody diarrhea (holt, 1994) . if portal hypertension is suspected, shock therapy is initiated and the animal is returned to surgery for immediate ligature removal. feeding may precipitate portal hypertension, and animals should be given small amounts of food and monitored closely after each feeding. if thrombosis of the shunt vessel or the portal vein occurs, signs of portal hypertension may occur up to several days after surgery, and treatment is usually futile. bleeding after surgery can also result in abdominal distention and signs of shock. at reoperation these animals are usually found to have diffuse hemorrhage. conservative treatment with packed red blood cells, fresh frozen plasma, or whole blood may be a more appropriate treatment than reoperation, if bleeding is suspected (hardie, 1997) . serious complications encountered in animals undergoing congenital pss surgery include intraoperative cardiac arrest, life-threatening hemorrhage, portal hypertension, seizures that usually start 2 to 3 days after surgery and may progress to status epilepticus, hyperthermia, gastric dilation-volvulus, acute pulmonary edema, and biliary pseudocyst formation (hardie, 1997) . the more manageable complications include abdominal distention, hypotension, hypothermia, hypoglycemia, mild gastrointestinal disturbances, and postoperative pain. predictor signs of immediate postoperative complications include low packed cell volume before surgery, absence of arborizing intrahepatic vasculature the liver lind plincrells i 207 during the mesenteric portogram, partial shunt vessel ligation, and hypothermia in the postoperative period. kittens are especially prone to developing seizures after shunt vessel ligation and should be administered phenobarbital at therapeutic serum concentrations for the entire perioperative period (hardie, 1997) . seizures usually occur 12 hours to 4 days after surgery. kittens that have no evidence of seizures in the peri operative period are weaned off the phenobarbital 1 month after surgery. long-term complications are encountered in animals whose congenital pss is not completely ligated and in animals that are older than 2 years of age at the time of surgery (hottinger et ai, 1995) . approximately 50% of the single shunts cannot be completely ligated at the first surgery because complete occlusion results in portal pressures greater than 20 ern h 2 0 or a rise in portal pressure greater than 10 em h 2 0 . within this group of partial vessel ligation, recurrence of pss signs may occur in as many as 40% of animals if a further vessel ligation is not performed. multiple extrahepatic shunts may form even with partial vessel ligation and are often associated with recurrence of pss signs. for animals with multiple extrahepatic shunts due to portal hypertension, the 2-year survival rate is 50% regardless of whether they are treated medically or surgically (hardie, 1997) . postoperative improvement is apparent from observation of the animal's activity at home but should always be followed by assessment of serum chemistry profile and serum bile acid concentrations for hepatic dysfunction (hardie, 1997) . medical management should be maintained until postoperative improvement has been unequivocally demonstrated. if serum bile acid values remain increased and the shunt index is greater than 15% at 60 to 90 days after surgery, reoperation is indicated. reoperation, however, has its own complications, mostly associated with the risk of inadvertently cutting a structure surrounded by scar tissue. reoperation of congenital intrahepatic shunts can be extremely difficult. if a mattress suture was placed across the anomalous vessel, the long ends of the suture material can be identified and the mattress suture tightened further. in some instances, it is necessary, however, to place additional sutures. to avoid the risks associated with reoperation of congenital intrahepatic shunts, a new technique has been developed in which an extrahepatic shunt is created between the portal vein and the vena cava using a jugular vein graft . this shunt is created at the time of the first surgery, and the intrahepatic shunt is completely closed. the venous graft prevents portal hypertension from developing at the time of the first surgery. the venous graft may slowly occlude, resulting in a normal shunt ndex .60~o 9~days after surgery. if the shunt mde~is std.l hi~h, reoperation is simply a matter of .either ligating or placing an ameroid constrictor band on the vein graft. in some cases, serum bile acid values remain abnormal despite a remarkable improvement in the animal's signs (hardie, 1997) . if serum chemistry profile and serum bile acid concentrations. indicate ongoing hepatic dysfunction, then medical management should be continued. medi~al manageme~t is directed at minimizing the signs of hepatic encephalopathy and includes manipulation of dietary proteins and intestinal flora and avoidance of medications or s?bstances capable of inducing encephalopathic signs. a restricted protein diet (2.0 to 2.5 mg/ kg) composed of proteins rich in branchedchain amino acids with comparatively smaller amounts of aromatic amino acids is recom-m~nded. foods containing milk protein (dried milk or cottage cheese) are best. the bulk of the caloric intake should consist of simple carbohydrates such as boiled white rice. meals should be frequent and in small amounts to maximize digestion and absorption so that minimal residue is passed into the colon where intestinal anaerobic bacteria degrade~itrogenous compounds to ammonia. commercial diets formulated for liver or renal dysfunction and a diet formulated for intestinal disease are used with success in most puppies and kittens with congenital or acquired pss. manipulation of intestinal flora with antimicrobial a~e.nts.and lactulose also produces mar~ed :limcallmprovement. for animals presennng m encephalopathic crisis intravenous isotonic electrolyte solutions suppl~mented with 2.5 %. or 5.0%~extrose solution and potassium chloride, cleansing enemas with warmed 0.9% saline solution, or enemas with added neomycin (15 to 20 ml of 1% solution three to four times daily), lactulose (5 to 10 ml diluted 1:3 with water three to four times daily), or betadine solution (10% solution, rinse after 10 minutes with wa~water) are recommended. for long-t~rm medical m~nagement of encephalopathic signs, lactulose is given orally at a dosage of 0.~5 to 1.0 ml per 4.5 kg body weight, the dose adjusted to the frequency and consistency of the stools passed each day. two to three soft or pudding-consistency stools indicate an optimal dose. too great a dose may result in flatulence, severe diarrhea, dehydration, and acidemia. to f\1rther manipulate the intestinal flora, neomyem (22 mglkg orally two to three times daily) etronidazole (7.5 mg/kg orally two to threå¨ rnes daily), aijolpicillin (5 mglkg orally two to three urnes. dally), or amoxicillin (2.5 mg/kg orally two ttmes a day) may be used intermittently for several weeks. congenital hepatic arteriovenous fistulas. congenital hepatic arteriovenous fistulas etween the~epatic artery and portal vein occur in both puppies and kittens (easley and carpenter, 1975; legendre et al, 1976â· moore and rogers et al, 1977) . these congenital malformations are the result of failure of e com~on embryologic anlage to differentiate l1~to artenes and veins. congenital hepatic artenovenous fistulas are associated with portal hyertension and shunting of blood through multtple portosystemic venous collaterals (table 11 -3): in~reased pressure in the portal vein, hepa~c. vel?, and hepatic sinusoids is caused by arterialization of the portal circulation. arteriovenous fistulas located in other areas of the body increase cardiac output and produce signs of heart failure (gomes and bernatz 1970â· moore and whiting, 1986) . the interpositioãµ f the hepatic sinusoids between the heart and fistula. cushions the hemodynamic effects, influencing heart function in animals with congenital hepatic arteriovenous fistulas. animals with congenital hepatic arteriovenous fistulas have multiple portosystemic shunts and ascites. portal venography by splenic pulp injection or mesenteric vein catheterization demonstrates multiple anomalous shunts but does not show e fistulas. diagnosis is made by nonselective jugular venography , selective celiac angiography, technetium scintigraphy, ultrasonography, or observation of an abnormal liver lobe during laparotomy. affected liver lobes are large and may have numerous pulsating surface vessels. unaffected liver lobes are small. a continuous murmur accentuated during systole may be auscultated near the lesion. palpation of the area may reveal a thrill. ultrasonographically, congenital hepatic arteriovenous fistulas are identified on the basis of finding multiple large, tortuous, and pulsatile hepatic vessels and en-lar~ement of the celiac and common hepatic artenes, congenital hepatoportal microvascular~p lasia. hepatoportal microvascular dyspla-sl~is char~ct~nzed by the presence of multiple tnlcroscopic intrahepatic shunts (phillips et al, 1993; schennhorn et ai, 1996) . the microvascular dysplasia occurs in the same dog breeds that have congenital pss, possibly being an inherited disorder in cairn terriers. most dogs with microvascular dysplasia are asymptomatic, probably because only a small amount of blood is being shunted away from the liver. when signs are present, they are similar to those seen in dogs with congenital extrahepatic and intrahepatic pss, with the exception that most dogs with microvascular dysplasia usually present at an older age. the most prominent laboratory abnormality is increased serum bile acid concentrations. there is no ultrasonographic, sur-gical, or portographic evidence of a congenital pss, and the rectal portal scintigraphy is normal. medical treatment is the same as for any suspected congenital pss. asymptomatic dogs with increased serum bile acids as their only detectable abnormality do not require treatment. congenital storage disorders. congenital storage disorders affecting the function or availability of lysosomal enzymes or effector proteins essential for catabolism of glycoproteins, glycolipids, glycosaminoglycans (mucopolysaccharides), gangliosides, and glycogen have been identified in puppies and kittens. these disorders are characterized by tissue accumulation of undegraded storage products. signs are usually progressive in association with tissue accumulation of storage material. hepatomegaly may develop from the undegraded storage product accumulating in hepatocytes and kupffer cells. mannosidosis resulting from a deficiency in acid mannosidase activity that causes the intralysosomal accumulation of a mannoside oligosaccharide occurs in kittens (jezyk et al, 1986; vendevelde et ai, 1982) . clinical findings include hepatomegaly, neurologic dysfunction (including tremors, ataxia, hypermetria, and/or weakness), stunted growth, facial dysmorphia, and early death. histopathologic examination reveals extensive cytoplasmic vacuolation in hepatocytes and neurons and the presence of unusual axonal spheroids. the mucopolysaccharide storage disorders are caused by a defect in lysosomal enzymes responsible for the degradation of dermatan sulfate, heparan sulfate, or keratan sulfate-normal constituents of the connective tissue matrix. these disorders are clinically progressive and are associated with tissue accumulation of glycosaminoglycans. clinical features vary with the specific enzyme deficiency. hepatosplenomegaly may develop from the accumulation of incompletely degraded mucopolysaccharides in parenchymal and reticuloendothelial cells. clinical findings may include facial dysmorphia (rounded broad forehead, small ears, and dished face), corneal opacity, bone and joint lesions (including odontoid hypoplasia, intervertebral disk degeneration, spinal canal and vertebral exostoses, osteoporosis, coxofemoral luxation, lytic areas in long bones and vertebrae, joint effusions, and degenerative joint disease), cardiac murmurs, stunted growth, metachromatic granules in leukocytes, neurologic abnormalities (mental slowness, cervical or thoracolumbar myelopathy), and early death. mucopolysaccharidosis i (a-l-iuronidase deficiency) has been described in a kindred of plott hounds and in domestic short-haired cats (haskins et ai, 1983; shull et ai, 1984) . mucopolysaccharidosis vi (arylsulfatase b deficiency) has been described in siamese and domestic short-haired cats (breton et al, 1983; haskins et ai, 1980 haskins et ai, , 1981 . mucopolysaccharidosis vii has been described in a dog (haskins et al, 1984) . a presumptive diagnosis of mucopolysaccharidosis can be made by a positive urine toluidine blue spot test. definitive diagnosis is made by measurement of the activity of specific enzymes in fresh serum, cultured dermal fibroblasts, or leukocytes. treatment with bone marrow transplantation has been reported to result in clinical improvement (dial et al, 1985; . gangliosidosis occurs from incomplete catabolism of certain gangliosides and glycolipids and retention of these substrates within lysosomes. gangliosidosis has been reported in the puppy and kitten (alroy et al, 1985; baker and lindsey, 1974; barnes et al, 1981; cork et al, 1977 cork et al, , 1978 n euwelt et al, 1985; read et ai, 1976; wenger et al, 1980) . affected animals develop neurologic signs as early as 2 or 3 months of age. progressive, fine generalized muscle tremors, ataxia, and paresis are the usual clinical findings. gangliosides accumulate in the central nervous system and in visceral organs, including the liver. membrane-bound cytoplasmic bodies are observed in cells from affected individuals. glycogen storage disorder associated with hepatomegaly has been diagnosed in a kindred of lapland dogs and in a german shepherd dog (rafizuzzaman et ai, 1976; walvoort et al, 1982 walvoort et al, , 1984 . affected animals showed signs as early as 2 months of age that were slowly progressive over many months. signs included weakness, weight loss, and gradual abdominal distention associated with profound hepatomegaly. glycogen is freely dispersed in the hepatocellular cytoplasm. a deficiency of amylo-l,6-g1ucosidase was demonstrated in a german shepherd dog (ceh et ai, 1976) , and a deficiency of a-glucosidase was demonstrated in a lapland dog (walvoort et al, 1982) . copper storage disorders of the bedlington terrier. a chronic active liver disease associated with an age-related accumulation of hepatic copper occurs in bedlington terrier dogs (hultgren et al, 1986 ). an autosomal recessive the liver .nd p.ncre.s i 2ii mode of inheritance is involved; only individuals homozygous for the recessive gene develop the excess copper accumulation in hepatic lysosomes. the adverse effects of retained copper are not noted during the first few years of life in affected bedlington terrier dogs by the protective lysosomal sequestration of copper. once lysosomal storage is overwhelmed, a progressive hepatopathy and clinical evidence of chronic active liver disease ensue. in affected dogs, copper accumulation begins before 1 year of age and continues for at least 5 or 6 years. hepatic copper concentrations exceeding 2000 j.lg/g dry tissue are consistently associated with morphologic and functional evidence of the progressive hepatopathy that over time progresses to chronic active hepatitis and cirrhosis (fig. 11 -6a) (hultgren et al, 1986; twedt et al, 1979) . affected dogs can be identified at 6 months of age on the basis of hepatic biopsy results (lohnson et ai, 1984) . liver tissue can be qualitatively and quantitatively evaluated for copper accumulation. routine staining with hematoxylin and eosin reveals dark cytoplasmic granules in hepatocytes of affected dogs early in the disease. tissue-bound copper can be stained with rubeanic acid, rhodanine, or timm's stain for qualitative and semiquantitative estimation of the degree of copper retention (fig. 11-6b) (johnson et al, 1984; . tissues should be stored embedded in paraffin blocks rather than formalin solution if examination is delayed for several months, because copper staining is reduced after prolonged storage in formalin solution . quantitative assessment of hepatic copper is accomplished by atomic absorption spectroscopy of tissue previously preserved in formalin or paraffin block or frozen. normal dog liver has less than 400 j.lg copper per gram of dry tissue twedt et al, 1979) .affected bedlington dogs may develop hepatic copper content up to 2000 j.lg/g during the first year of life before developing histopathologic evidence of hepatocellular injury. dogs showing evidence of abnormal copper storage in hepatic biopsy material by 1 year of age should not be used for breeding. affected bedlington dogs may have evidence of increased hepatic copper as early as 8 to 12 weeks of age. diagnosis of copper-associated hepatopathy in bedlington terriers can be made by examination of hepatic tissue for excessive copper storage or by performing genetic tests on dna samples collected from suspected dogs. an autosomal recessive mode of inheritance is invalved in copper-associated hepatopathy in bedlington terriers. the frequency of the recessive gene in bedlington terriers is estimated to be as high as 50% in the united states, with a similar frequency in england. this means that more than 25% of bedlington terriers are "affected," and another 50% are "carriers." the dna samples can be collected with a soft cheek brush that is provided by a commercial genetic laboratory (vetgen, 3728 plaza drive, suite 1, ann arbor, mi 48108; 1-734-669-8440, toll free: 1-800-4-vetgen, fax: 1-734-669-8441; or see their web site: www.vetgen.com). by gently brushing the inside of the dog's cheek, cells containing dna are removed. the collected dna samples then are analyzed to determine the genetic status of the suspect dog. useful for dogs of any age, the dna sample collection and analysis activities can be completed before puppies are purchased at 6 to 10 weeks. the results of the dna testing also can be formally registered with the orthopedic foundation for animals. (for further information about the orthopedic foundation for animal's registry for copper toxicosis in bedlington terriers, contact orthopedic foundation for animals, 2300 e. nifong boulevard, columbia, mo 65201-3856 or telephone 1-573-442-0418.) n-penicillamine, a copper chelator, is recommended for the treatment of the copper-associated hepatopathy in bedlington terriers as soon as the disorder is confirmed. the recommended dose of n-penicillamine is 125 to 250 mg/day (adult dogs), given 30 minutes before feeding (hardy, 1983) . the most common adverse effects from o-penicillamine administration are vomiting and anorexia. vomiting may be manageable by dividing the daily dose into two or three doses. if o-penicillamine is not tolerated, another copper chelator, 2,3,2-trientine, administered at 10 to 15 mg/kg orally one to two times daily or zinc acetate administered at 50 to 200 mg orally once a day, decreases intestinal absorption of copper and may be used. in addition to the decopper drugs, vitamin c and prednisolone have been recommended. vitamin c is known to facilitate the excretion of copper in urine, and large doses may reduce the intestinal absorption of copper. dosages of 500 to 1000 mg/day have been suggested (hardy, 1983) . prednisolone at 0.5 to 1.0 mg/kg per day is recommended only for those dogs showing evidence of active hepatic necrosis. limitation of the dietary intake of copper is usually not possible. most dog foods contain 5 to 10 mg/ kg of copper, which may result in a higher copper intake per kilogram than is appropriate. hepatopathies associated with increased concentrations of hepatic copper have been recognized in young dogs and cats with chronic active hepatitis, cirrhosis, and chronic bile duct obstruction (rolfe and twedt, 1995) . copper may aggravate the underlying pathologic process in these disorders by direct injury to cellular organelles or by promotion of fibrogenesis (hultgren et al, 1986) . the decreased ability to excrete biliary copper probably underlies abnormal hepatic copper retention when a primary cholestatic disease exists. primary hepatobiliary disease associated with an increased accumulation of hepatic copper, albeit smaller amounts of tissue copper than in bedlington terriers, has been described in doberman pinscher, skye terrier, west highland white terrier, and american and english cocker spaniel dogs (crawford et al, 1985; thornburg and rottinghaus, 1985; thornburg et al, 1986) . the chronic active hepatitis associated with an increased liver copper content in doberman pinschers occurs primarily in middle-aged females. although the youngest dog reported with this disorder was 1.5 years old, it is unknown whether younger dogs are symptomatic. it is suspected that affected dogs could be identified at a younger age on the basis of routine screening serum chemistry profiles revealing increased liver enzyme activity. a familial copper-associated liver disease occurs in west highland white terrier dogs (thornburg et ai, 1986) . increased hepatic copper concentrations are detected in asymptomatic dogs as young as 7 months of age. in three affected dogs younger than 9 months of age, the hepatic copper concentration ranged between 1500 and 1750 f.lg/g dry weight. hepatic copper concentrations in affected dogs have ranged as high as 3500 ppm, considerably lower than the maximal values recorded for bedlington terriers. in an attempt to decrease the perpetuation of this disorder, it has been recommended that relatives of west highland white terrier dogs dying of liver disease be evaluated by hepatic biopsy before 1 year of age. those animals with increased hepatic copper content should not be used for breeding purposes. liver disease has also been observed with unexpected frequency in american and english cocker spaniel dogs (thornburg and rottinghaus, 1985) . dogs as young as 9 months have been diagnosed as having chronic active hepatitis. the liver disease appears to be progressive, and dogs dying of cirrhosis have had hepatic copper concentrations three to five times normal. a genetic defect in the dalmatian dog's liver results in an inability to convert uric acid into allantoin, the soluble excretory product of purine metabolism in non-dalmatian dogs (briggs, 1985; briggs and harley, 1986; giesecke and tiemeyer, 1984) . this genetic defect is transmitted by homozygosity for a recessive trait. serum uric acid concentrations in dalmatian dogs are consistently increased, and urinary excretion of uric acid is markedly greater than in non-dalmatian dogs. typical serum uric acid concentrations in dalmatian dogs range between 2 and 4 mg/dl versus less than 1 mg/dl the liver .nd p.ncre.s i 213 in other breeds of dogs (kruger and osborne, 1986; schaible, 1986) . urine excretion of uric acid in dalmatians ranges between 400 and 600 mg in 24 hours versus 10 to 60 mg in 24 hours in non-dalmatians (kruger and osborne, 1986) . urine uric acid-to-creatinine values have ranged between oj and 0.6 for normal puppies and 1.3 and 4.6 for pedigree dalmatian puppies at 3 to 7 weeks of age and between 0.2 and 0.4 for normal dogs and 0.6 and 1.5 for purebred adult dalmatians (schaible, 1986) . the increased urinary excretion of uric acid puts the dalmatian at increased risk for the formation of urate uroliths, although not all affected dogs develop uroliths. hepatic lipidosis. most puppies and kittens that present with hepatic lipidosis have primary disease in other organ systems or an infectious disease, and therefore it is possible that the hepatic lipidosis was the consequence of acquired nutritional inadequacies. a variety of metabolic disorders can disturb the mobilization of triglycerides from the liver. whenever intrahepatic lipid synthesis or the hepatocellular uptake of fat exceeds the dispersal of triglycerides from the liver, hepatic lipidosis develops (fig. 11-7) (miettinen, 1981; pulito et ai, 1976) . severe hepatic lipidosis occurs most commonly in toy-breed puppies, which become hypoglycemic and die after prolonged anorexia or fasting (van toor et ai, 1991) . clinically, kittens appear to be more susceptible to hepatic triglyceride accumulation than puppies. any serious medical problem in the kitten can be associated with excessive hepatic lipid accumulation characterized by cytoplasmic vacuole formation that adversely influences hepatic function. nutritional management that ensures adequate intake of calories, essential amino acids, and essential fatty acids is the best recommended symptomatic therapy. in addition, nutritional management for the mother during pregnancy can be important in possibly preventing hepatic lipidosis in the newborn. neonatal icterus. neonatal icterus often occurs in puppies and kittens as a result of immunohemolytic anemia (cain and suzuki, 1985; giger et al, 1991; young et al, 1951) . icterus often occurs in kittens within 3 days of birth with hemolysis from neonatal isoerythrolysis (see chapter 3). noncirrhotic portal hypertension in young dogs. most young dogs with noncir-rhotic portal hypertension are younger than 19 months old, pedigree dogs, and female (see table 11 -3) (bunch, 1997; demarco et ai, 1998; rand et al, 1988; rutgers et al, 1993; van den ingh and rothuizen, 1994; van den ingh et al, 1995) . typical signs are apathy, ascites, vague gastrointestinal upset (anorexia, vomiting, diarrhea), neurologic derangements, and polydipsia! polyuria. the affected dogs typically have smallsized livers, acquired pss, and splenomegaly. common trends in serum chemistry profiles are increased liver enzyme activities and evidence of hepatic dysfunction (e.g., hypoalbuminemia, increased serum bile acid content, and hyperammonemia). microcytosis is a consistent finding. liver biopsy is required for an accurate diagnosis in the affected dog. histopathologic findings include preserved to altered liver architecture, portal hypoperfusion, and variable degrees of fibrosis; there are usually no cytopathic indications of destructive processes such as necrosis or inflammation. responses to symptomatic and specific hepatic treatment of affected dogs vary with the degree of portal hypertension present and the length of time hypertension has existed. symptomatic measures to decrease ascites and signs of hepatic encephalopathy are indicated. colchicine (0.025 mglkg orally once daily) and/or prednisone (0.5 to 1.0 mglkg orally daily initially, then every other day) have been the medications reported to be useful in a small number of cases (rutgers et ai, 1990) . it seems that affected dogs have the potential to have a good quality of life for an indefinite period of time. feline inflammatory liver disease. inflammatory liver diseases of young cats is probably best referred to as feline cholangitis/cholangiohepatitis syndrome (cchs) (center, 1997) . this syndrome can then be described as being either a suppurative cchs or a nonsuppurative cchs. affected cats with suppurative cchs usually are 3 months and older and usually are males. a sudden-onset history of vomiting and diarrhea is common. older cats are icteric, febrile, lethargic, and dehydrated on initial presentation. less than 50% of cats have hepatomegaly. the most common organisms associated with suppurative cchs are escherichia coli, staphylococcus, a-hemolytic streptococcus, bacillus, actinomyces, bacteroides, enterococcus, enterobacter, and clostridium species. most cats with suppurative cchs show a moderate increase in serum alt, as'f, alp, and ggt activities. some cats have left-shifted leukograms with an accompanying leukocytosis. on ultrasonography, severe ascending cholangitis associated with thickening of the extrahepatic biliary system and inflammation within the lumen of the intrahepatic bile ducts may be observed. ultrasonography also may show coexisting extrahepatic bile duct obstruction (enlarged gallbladder, distended and tortuous common bile duct, and obvious intrahepatic bile ducts), cholecystitis (thickened, laminar appearance of the gallbladder wall, adjacent fluid accumulation), and pancreatitis (prominent, easily visualized enlarged pancreas with adjacent hyperechoic fat). cytologic evaluation of liver aspirates or imprints may reveal suppurative inflammation. most cats with nonsuppurative cchs are 1 year of age or older and have been ill for several months (center, 1997) . clinical signs are subtle and may include only episodic vomiting, diarrhea, and anorexia. most cats have hepatomegaly, are icteric, and may have ascites. concurrent disorders frequently include inflammatory bowel disease, low-grade lymphocytic pancreatitis, and cholecystitis. cats with lymphoplasmacytic inflammation tend to have greater magnitudes of increased serum alt, ast, alp, and ggt activities than cats with just lymphocytic inflammation. cats with lymphocytic inflammation may develop a lymphocytosis (total lymphocyte counts greater than 14,000/f..d) without other evidence of malignant lymphoproliferative disease. similar to cats with suppurative cchs, abdominal radiographs rarely show important diagnostic information. in most cats with nonsuppurative cchs, a multifocal hyperechoic pattern is recognized ultrasonographically, which represents peribiliary inflammation and fibrosis. in some cats, ultrasonography may fail to show any abnormalities. cytologic preparations from liver aspirates may lack evidence of inflammation or may disclose only a few inflammatory cells. a wedge biopsy of the liver for histopathology is preferable for a definitive diagnosis because it more reliably demonstrates whole acinar units and portal triads (center, 1997) . treatment of suppurative cchs incorporates appropriate antimicrobial therapy based on identification of infectious organisms. if bacteria are cytologically observed, a gram's stain facilitates selection of antimicrobial agents. cats with extrahepatic bile duct obstruction should have their biliary occlusion decompressed, if possible. if biliary tract decompression cannot be accomplished, the biliary pathway may be rerouted by a cholecystoenterostomy. biliary diversion is a vital early therapeutic intervention in the prevention or control of sepsis in obstructive suppurative cholangitis. aerobic and anaerobic bacterial cultures should be collected from bile, tissue adjacent to any focal lesion, gallbladder wall, and liver tissue. any icteric cat suspected of having suppurative or nonsuppurative cchs should be evaluated for coexistent extrahepatic bile duct obstruction, pancreatitis, and inflammatory bowel disease as well as coexistent hepatic lipidosis. if lipid vacuolation is detected, nutritional support with a commercially prepared feline diet should be included in the treatment plan. immunosuppressive therapy for cats with nonsuppurative cchs includes a combination of prednisolone (initial dose of 2 to 4 mglkg orally once a day or divided twice daily), with titration to the lowest effective dose over the the liver ind plncfels i 215 next several months, and metronidazole (7.5 mglkg orally two to three times daily) (center, 1997) . supplementation with l-carnitine 250 mg/cat per day, water-soluble vitamins (two times the normal maintenance dose), and vitamin k 1 (0.5 to 1.5 mglkg) subcutaneously or intramuscularly for three doses at 12-hour intervals and then once a week for 1 or 2 additional weeks may be provided. oral vitamin e can also be added as a supplement to ensure its adequacy as a free radical scavenger; a dose of 100 to 200 iu per day is used. ursodeoxycholic acid (10 to 15 mglkg orally per day) is given to all cats with cchs once extrahepatic bile duct obstruction is corrected. monthly serum liver enzyme activities and total bilirubin concentrations may be used to monitor treatment response as well as how well the cat is doing at home. hepatic abscessation. hematogenous, omphalogenic, biliary, and peritoneal extension are sources of infecting organisms that cause hepatic abscesses to appear in puppies and kittens (hargis and thomassen, 1980; valentine and porter, 1983) . postpartum umbilical infection appears to be the most common cause of hepatic abscessation. once clinical signs develop, animals deteriorate and die within 2 to 4 weeks. occasionally, seemingly healthy puppies die unexpectedly, the cause being discovered on histopathologic examination. most affected puppies are between 3 and 70 days of age and are from large litters (hargis and thomassen, 1980) . organisms frequently isolated from hepatic abscesses in puppies and kittens include escherichia coli and staphylococcus, streptococcus, and salmonella species. puppies and kittens with hepatic abscesses are usually stunted, emaciated, and dehydrated and may have enlarged abdomens due to hepatomegaly and peritonitis. unaffected liver lobes usually show multifocal necrosis on histopathologic examination. suspected hepatic abscesses in puppies and kittens should be managed with antimicrobial drugs and other supportive care (see chapter 5). hepatic parasitism. hepatic trematode infection may be diagnosed in kittens as young as 4 months of age. the most common liver fluke in cats in north america is platynosomum amcinnum. other species of flukes that may infect cats include amphimerus pseudofelineus, opisthorchis tenuicollis, metorchis albidus, and metorchis conjuctus. cats acquire platynosomum con-cinnum infection by the ingestion of the second intermediate hosts: a land snail (subulina octona) and a lizard or marine toad. once ingested, the infective stage of the parasite migrates up the common bile duct into the gallbladder and bile ducts, where in 8 to 12 weeks it matures into the adult fluke. embryonated eggs are passed in the feces and are the basis for diagnosis. clinical signs are noted by 7 to 16 weeks after infection and may include inappetence, lethargy, weight loss, hepatomegaly, emaciation, mucoid diarrhea, depression, vomiting, and abdominal tenderness. many naturally infected cats show no clinical signs. in heavy infections, clinical signs may develop before the fecal shedding of ova, which occurs as early as 8 weeks after infection. concentration of eggs in feces by sedimentation is the most reliable diagnostic test. transient increases in the serum ast and alt activities develop during fluke migration through the liver. the serum alp activity may remain normal or may increase. cats with heavy fluke infection may become jaundiced. persistent fluke infections and bile duct obstruction may result in biliary cirrhosis. treatment with praziquantel (20 to 40 mglkg daily for at least 3 days) is clinically effective. hepatobiliary lesions produced by ascarid larval migration are commonly observed during necropsy of young dogs and cats. these lesions are usually not associated with clinical signs or laboratory abnormalities. severe hepatic and peritoneal migration, gallbladder rupture, and bile peritonitis may, however, occur in a few puppies. in young dogs and cats, after ingesting eggs, the larval forms of 'toxocara canis and 'toxocara cati penetrate the wall of the alimentary canal and pass by way of lymphatics or the portal circulation to the liver. ascarids may also migrate from the gastrointestinal tract directly through the peritoneal cavity to the liver. diseases. canine herpesvirus infection is an acute, rapidly fatal disease that is associated with hepatic necrosis. puppies acquire canine herpesvirus in utero, during passage through the birth canal, by exposure to infected littermates, or from orona sal secretions of the dam. abortions and stillbirths may occur if infection is acquired in utero (poste and king, 1971) . generalized, fatal infections develop in puppies during the first 3 weeks of life. puppies infected when older than 3 weeks are comparatively resistant and develop mild or inapparent infection. an incubation period of 4 to 6 days follows initial exposure. a diffuse necrotizing vasculitis and spread of virus into parenchymal organs, including the adrenals, kidneys, lungs, spleen, and liver, results in multifocal organ necrosis. meningoencephalitis that causes seizure activity is common in canine herpesvirus infections. in survivors, permanent neurologic deficits may persist, most common of which are cerebellar vestibular defects. ocular involvement causing panuveitis, cataracts, keratitis, retinitis, and subsequent blindness may occur. clinical signs of canine herpesvirus infection in puppies may include depression, diminished suckling response, persistent crying, yellowgreen diarrhea, abdominal pain, and incoordination. petechial hemorrhages may be notable on mucous membranes. cutaneous lesions may include an erythematous rash with red papules progressing to vesicles. papular or vesicular lesions may develop in the vulvovaginal orifice, prepuce, and/or oral cavity. neurologic signs may occur during the terminal stages of the disease. death frequently occurs within 24 to 48 hours after onset of clinical signs in infected puppies. definitive diagnosis of canine herpesvirus infection in puppies is made on the basis of history, clinical signs, histopathologic changes, and virus isolation. hematologic and biochemical abnormalities are nonspecific and variable. thrombocytopenia may be present in ill puppies. widespread hepatic necrosis causes increased serum activities of alt and ast. icterus does not occur. gross pathologic findings include disseminated multi focal petechial hemorrhages ( fig. 11-8 ) and areas of necrosis that are distinctly circumscribed in the liver, kidney, and lungs (greene and kakuk, 1984) . hepatomegaly, splenomegaly, and lymphadenopathy are common. histopathologic lesions are characterized by perivascular necrosis associated with a mild neutrophil and lymphocyte infiltration, hemorrhages, and occasional intranuclear inclusions (fig. 11-9 ). treatment for canine herpesvirus infection is usually unrewarding owing to its rapidly fatal progression. rectal temperature elevation to about 37.7 0 c (100 0 f) and adequate nutritional support may improve puppy survival during an outbreak. focal hepatitis and hepatic cord disorganization may develop in puppies and kittens infected with canine or feline parvovirus. two-to fivefold increases in serum activities of alt and ast may develop. in some cases, hepatic involvement is progressive, resulting in icterus. seemingly, a poor prognosis is warranted when hepatic involvement becomes clinically apparent. coronavirus infection causing feline infectious peritonitis (fip) most often affects cats between 6 months and 2 years of age. coronavirus infection has been diagnosed as a cause of stillborn kittens and fading kittens and as an effusive disease in kittens younger than 4 weeks of age. clinical signs of fip usually develop in several siblings in a litter, and death losses may span a 6-to 12-month interval. cats with liver involvement may demonstrate cranial abdominal pain and hepatomegaly. serum alt and ast activities are usually increased from 2-to 10-fold in cats with liver involvement. icterus may develop in those cats with severe, diffuse hepatic lesions. a coagulopathy and thrombocytopenia develop in cats with diffuse vascular the liver and pancreas i 217 injury, in those with severe inflammation and subsequent activation of clotting factors, or in severe hepatic involvement (weiss et ai, 1980) . immunosuppression may help prolong the survival of some cats. unfortunately, kittens showing signs of hepatic involvement are usually poor candidates for immunosuppressive therapy. infection of kittens with feline leukemia virus or feline immunodeficiency virus may occur by horizontal or vertical transmission. by virtue of their oncogenic potential and ability to immunologically compromise the host, these viruses may be associated with neoplastic conditions and infectious diseases involving the liver. lymphosarcoma and myeloproliferative disease can develop in infected cats within weeks or months of exposure. affected kittens demonstrate hepatomegaly when they have liver involvement. icterus develops with diffuse hepatic involvement, periportal infiltration, or major bile duct occlusion. serum chemistry profile abnormalities are variable, depending on the extent of hepatic involvement. bacterial-induced hepatic diseases. enteric organisms such as salmonella species and escherichia coli can be a source of hepatic parenchymal and biliary tract infections in young dogs and cats (greene, 1984) . salmonella species may exist in young dogs and cats as a part of the normal enteric flora. transmission of salmonella species from carrier animals to susceptible hosts may result in gastroenteritis, bacteremia, parenchymal organ or lymph node colonization or abscessation, endotoxemia, stillbirths, or a fading puppy or kitten syndrome. signs of gastrointestinal infection may develop after 3 to 5 days of exposure or after some unusual environmental or physical stress. initial signs may include fever (104 0 to 106 0 f), malaise, anorexia, vomiting, abdominal pain, and diarrhea. diarrhea can be voluminous and usually contains mucus and fresh blood. further signs may develop, including weight loss, severe dehydration, weakness, hypotension, pale mucous membranes, and, in some cases, evidence of neurologic involvement. icterus may develop as a result of endotoxemic effects on the liver, hepatic infarction, or bacterial colonization of hepatic tissue. serum chemistry profile evidence of liver involvement includes increases in the serum activities of al'f, as'f, and alp. hyperbilirubinemia is an inconsistent finding. multifocal necrosis is the most common histopathologic lesion of salmonellosis. a necrotizing pneumonia may also occur in puppies with hepatic involvement. definitive diagnosis of salmonellosis relies on culture of the organism from involved tissues or body fluids that are normally free of this organism. positive culture of fecal specimens does not confirm the causal relationship of the organism to the animals' clinical disease. successful treatment requires attention to supportive nursing care, plasma transfusion for severe hypoproteinemia, and selection of an appropriate antimicrobial agent. the prognosis for puppies and kittens with salmonellosis is generally poor. efforts to improve kennel or cattery sanitation, to improve nutrition, and to reduce stress on puppies and kittens may curtail further infection. bacillus piliformis, the causative agent of tyzzer's disease, is a gram-negative, spore-forming, obligate intracellular bacterium that can cause enteric and hepatic infections in young dogs and cats, most commonly seen at the time of weaning. animals subject to infection develop necrotizing enteritis and multifocal hepatitis. infection in most young dogs and cats is thought to occur by their ingestion of bacterial spores passed in rodent feces. signs of natural disease in young dogs and cats include a sudden onset of lethargy, depression, anorexia, diarrhea, and abdominal tenderness. icterus may occur in some affected kittens. within 24 to 48 hours after the onset of illness, affected animals become hypothermic and severely depressed. death rapidly follows. identification of the bacterium is aided by the use of special stains such as giemsa's stain or gomori's methenamine silver stain (greene, 1984) . isolation of the causative agent cannot be accomplished on routinely used bacterial culture media. toxoplasma gondii infections vary depending on the chronicity of infection, immune status of the host, mode of infection, and target organs affected. young dogs and cats are particularly at risk when immunocompromised and debilitated. in utero infection can occur and lead to stillbirths or neonatal disease and death. mfected puppies and kittens may appear normal at birth but become depressed, inappetent, and dyspneic and develop a mucopurulent oculonasal discharge and progressive neurologic disease; they eventually die. dissemination to multiple organs usually occurs. hepatic lesions are typified as a multifocal necrotizing hepatitis. hepatic inflammation may be associated with cranial abdominal pain and peritoneal effusion and is usually associated with vomiting, diarrhea, and inappetence. animals may become icteric o.wing to the diffuse nature of the hepatic necro-sis. laboratory abnormalities associated with toxoplasmosis are variable, depending on the target organs affected and the chronicity of infection. early hematologic features may include a panleukopenia with a degenerative left shift. a leukocytosis may follow during the recovery period (greene and prestwood, 1984) . serum chemistry profile abnormalities indicating hepatic involvement include marked increases in serum al'f, ast, and alp activities and hyperbilirubinemia. definitive diagnosis of toxoplasmosis is made on the basis of tissue examination for toxoplasma gondii organisms or demonstration of a rising serologic antigen and/or antibody titer. recommended treatments for toxoplasmosis include the use of pyrimethamine, trimethoprim-sulfonamide, and clindamycin (greene and prestwood, 1984) . the pancreas is a unique organ possessing both exocrine (digestive) and endocrine (hormonal) functions. inflammatory pancreatic disease affecting only the exocrine portion is extremely uncommon in young dogs and cats (strombeck and guilford, 1990) . consequently, inflammatory pancreatic disease, that is, acute pancreatitis or relapsing pancreatitis that more commonly affects older dogs and cats, has been rarely identified in dogs and cats younger than 6 months of age. the likely causes of inflammatory pancreatic disease in the young dog and cat are abdominal trauma and infectious agents. abdominal trauma may induce pancreatitis in dogs that are traumatized by motor vehicles and in cats that have fallen or jumped from high places (high-rise syndrome) (drazner, 1986 ). in addition, abdominal surgery may result in acute pancreatitis due to traumatic injury to the pancreas (spearing the pancreas with a surgical instrument) or excessive manipulation of the pancreas. infectious agents can occasionally contribute to inflammatory pancreatic disease. pancreatic necrosis can be found on postmortem examination of an occasional dog afflicted with canine parvovirus infection (drazner, 1986) . it is not known whether the canine parvovirus is directly cytotoxic to the pancreatic tissue or pancreatitis occurs secondary to the invasion of enzymes and bacteria of the intestinal tract into the pancreas. in cats, pancreatitis may be associated with the effusive form of fip (barlough and weiss, 1983) . other infectious agents directly associated with inflammatory pancreatic disease in the young dog and cat would be extremely unusual and most likely a one-time occurrence. although seldom required, laboratory confirmation of inflammatory pancreatic disease includes a complete blood count, serum chemistry profile, serum amylase and lipase determinations, serum trypsin-like immunoreactivity (tli) assay, and survey radiographs and/or ultrasonography of the abdomen. normal values for serum amylase and lipase activities in dogs and cats younger than 6 months of age are generally indicative of normal adult values. hyperamylasemia and hyperlipasemia combined with typical clinical features of inflammatory pancreatic disease, as seen in adult animals, establish the diagnosis of inflammatory pancrefigure 11 -10. photograph of the pancreas from a young dog with congenital pancreatic hypoplasia. note the generalized reduction in amount of pancreatic tissue present and the absence of any inflammatory pancreatic disease. the liver end pencreas i 219 atic disease until proved otherwise. the serum tli assay may be increased-tli values of more than 35 ij.gll in young dogs and more than 50 ij.gll in young cats are consistent with pancreatitis. its treatment is entirely supportive and is managed in a manner similar to that for the afflicted older dog or cat. by far the most common cause of noninflammatory pancreatic disease in the young dog is congenital pancreatic hypoplasia (harris, 1985; jubb, 1983; sherding, 1979) . this disorder of young dogs is characterized by generalized reduction in pancreatic exocrine (acinar) cells, but the islets of langerhans remain intact (fig. il-10) . the disorder is more common in large breeds of dogs, that is, german shepherd (alsatian), doberman pinscher, irish setter, labrador retriever, and saint bernard, but has also been seen in the beagle (hill et ai, 1971; prentice et al, 1980) . there may be a sex predilection favoring females (anderson and low, 1965) , and young dogs that are symptomatic generally present before 1 year of age. congenital pancreatic hypoplasia has not been recognized in the young cat. most dogs affected with congenital pancreatic hypoplasia present with signs of weight loss or failure to gain adequate weight and poor physical appearance (i.e., dull, dry haircoat and excessive shedding) despite exhibiting a good to voracious appetite (sherding, 1979) . varying degrees of frequent (6 to 10 stools per day), foul-smelling, bulky, greasy, loose stools are de-scribed by the dog owner. often, coprophagia is noted. affected dogs commonly eat their stools because of their high fat content and because of a dietary energy deficit. the diarrheic stools contain undigested sugars and fats that are being altered by intestinal bacteria to become osmotically active particles (drazner, 1983) . the marked increase in osmotically active particles and the subsequent efflux of water into the lumen of the intestinal tract result in the colon's inability to resorb the increased volume, and diarrhea ensues. the volume of unabsorbed intraluminal water produces marked intestinal distention and altered motility, which may be severe enough to cause intestinal and colonic bacterial overgrowth (drazner, 1983) . unabsorbed fatty acids may also impair the absorptive capacity of the small intestine by damaging the brush border, blunting the villi, and inhibiting colonic water absorption. the diagnostic evaluation of dogs suspected to have congenital pancreatic hypoplasia differentiates this disorder from intestinal mucosal malabsorption. diagnosis of congenital pancreatic hypoplasia is usually not difficult because the presenting signs are rather characteristic and the laboratory test results are helpful in its diagnosis. the serum tli assay values are classically decreased-tli values are consistently less than 2.5 f.lgll in affected dogs. "when the diagnosis is still in question or serum tli assay results are not available, an exploratory laparotomy can be used for confirmation. treatment of dogs with congenital pancreatic hypoplasia depends mainly on dietary management and supplementation with pancreatic digestive enzymes (lewis et al, 1987) . efforts to treat these dogs are usually rewarded with a favorable response. the expense of treatment and an unconscientious owner, rather than the ineffectiveness of the treatment regimen itself, are most often the reasons that successful treatment is not accomplished. the most effective dietary management for dogs with congenital pancreatic hypoplasia is a highly digestible, lowfiber, moderate-fat diet supplemented with pancreatic enzymes (lewis et ai, 1987) . commercial diets formulated for gastrointestinal disease may be fed. the dog's daily food intake is divided into two or three feedings or is fed free choice. the dietary replacement of pancreatic digestive enzymes is given orally with each meal. various pancreatic enzyme products are commercially available for this purpose (sherding, 1979) . reliable commercial products are available in both powder and tablet form. the usual effective dosage of the powder preparation is 1 to 2 tsp per meal for each 20 kg body weight. the pancreatic enzyme product is mixed with the commercially prepared canned or well-moistened dry dog food and fed without necessarily any preincubation time. "when diarrhea is in remission and the animal is gaining weight, the pancreatic enzyme product should be titrated to the minimum effective maintenance dose per feeding. "when pancreatic digestive enzymes are given orally, a high percentage of them are inactivated by gastric acid. even though only a fraction of the pancreatic enzymes administered reach the small intestine in an active state, they are still effective because only a slight increase in duodenal digestive enzyme activity is needed to achieve marked improvement in nutrient assimilation (drazner, 1986) . in some dogs, antimicrobial agents may be a helpful adjunctive therapy for the bacterial overgrowth of the small intestine that often accompanies malassimilation in congenital pancreatic hypoplasia (drazner, 1986) . medium-chain triglycerides may be added to the dog's diet if additional dietary energy is needed to increase weight gain or maintain condition in the dog that fails to respond otherwise. the medium-chain triglycerides can be used to provide up to 25% of the dog's caloric need and, when fully utilized, provide 8 kcal/ml (lewis et ai, 1987) . the dog's body weight, general condition, and stool character should be monitored weekly during the treatment of congenital pancreatic hypoplasia. stool volume should decrease precipitously, and gains in body weight should begin soon after initiation of dietary management and the supplementation of pancreatic digestive enzymes (sherding, 1979) . the dietary replacement of pancreatic digestive enzymes is generally required for the rest of the dog's life. neurovisceral and skeletal gml-gangliosidosis in dogs with betagalactosidase deficiency hepatic beta galactosidase and feline gm 1 gangliosidosis a divided intrahepatic gallbladder in a cat congenital feline portosystemic shunts ea: the accessory gall-bladder: an embryological and comparative study of aberrant biliary vesicles occurring in man and the domestic mammals a case of mucopolysaccharidosis vi in a cat briggs om: serum urate concentrations in the dalmatian coach hound presumptive neonatal isoerythrolysis in cats congenital portacaval shunt in two kittens 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significance of hepatic copper values in dogs with cirrhosis hereditary copper toxicosis in west highland white terriers clinical, morphologic, and chemical studies on copper toxicosis of bedlington terriers van den ingh tsgam, rothuizen j: lobular dissecting hepatitis in juvenile and young adult dogs experimental induction of fasting hypoglycaemia and fatty liver syndrome in three yorkshire terrier pups use of transcolonic portal scintigraphy to monitor blood flow and progressive postoperative attenuation of partially ligated single extrahepatic portosystemic shunts in dogs hereditary neurovisceral mannosidosis associated with alpha-mannosidase deficiency in a family of persian cats portosystemic communications in the dog gradual occlusion of extrahepatic portosystemic shunts in dogs and cats using the ameroid constrictor canine glycogen storage disease type ii: a biochemical study of an acid alpha-glucosidase deficient lapland dog canine glycogen storage disease type ii: a clinical 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progression of liver disease can cause several physiologic derangements that may precipitate hepatic failure and require admission to an intensive care unit. the underlying pathology may be acute, acute-on chronic, or chronic in nature. liver failure may manifest with a variety of clinical signs and symptoms that need prompt attention. the compromised synthetic and metabolic activity of the failing liver affects all organ systems, from neurologic to integumentary. supportive care and specific therapies should be instituted in order to improve outcome and minimize time of recovery. in this chapter we will discuss the definition, clinical manifestations, workup, and management of acute and chronic liver failure and the general principles of treatment of these patients. management of liver failure secondary to certain common etiologies will also be presented. finally, liver transplantation and alternative therapies will also be discussed. accepted theories that revolve around impaired detoxification of substances normally cleared by the liver. • ammonia • the metabolism of nitrogen-containing compounds in the gastrointestinal system results in the production of ammonia. in its normal state, the liver converts this neurotoxic product into glutamine and urea. impaired liver function results in elevated blood ammonia. astrocytes contain the enzyme glutamine synthetase in their endoplasmic reticulum as a means of handling excessive ammonia. accumulation of glutamine within the astrocytes results in cell swelling which leads to a series of events that result in a neuroinhibitory state [5] . • false neurotransmitters • the failing liver results in the production of false neurotransmitters. these molecules may interfere with normal brain functioning and have a net inhibitory effect [6] . • amino acid imbalance • patients with hepatic failure have decreased plasma levels of the branched-chain amino acids (bcaa) valine, leucine, and isoleucine while experiencing increased levels of aromatic amino acids (aaa) phenylalanine, tryptophan, and tyrosine. this is thought to be related to increased muscle catabolism and therefore increased bcaa metabolism as well as decreased breakdown of aaa by the compromised liver. the end result is an imbalance that leads to an increased influx of aaa in the brain which has an inhibitory effect in the nervous system [7] . • gaba receptor • thought to be mediated by inflammatory cells, neurosteroids are produced by myelinated glial cells. this results in positive modulation of gaba receptors that in turn enhance the inhibitory tone [8] . besides the astrocyte swelling that is seen with the accumulation of glutamine explained above, overall neurologic dysfunction results in loss of autoregulation of intracranial pressure as well as reduced cerebral blood flow. the result of these changes may result in further neurologic derangement and compromise [9] . besides hepatic encephalopathy, patients with alf can also present with cerebral edema. there is an overlap with the clinical features that are seen with encephalopathy and include nausea, vomiting, headache, and agitation. in advanced cases which can progress to brain herniation, hypertension, bradycardia, changes in pupillary exam or reflexes, as well as respiratory depression can be seen [10] . patients with alf may present with nonspecific respiratory symptoms including dyspnea on exertion, orthopnea, anxiety, and air hunger. the affecting processes involved are very broad and can range from a simple pleural effusion to acute respiratory distress syndrome (ards) [11] . the spectrum of respiratory pathology that is seen can be grouped in to two major categories: infectious and noninfectious (table 18.3) . pulmonary edema can be of cardiogenic or noncardiogenic etiology. the prevalence of pulmonary edema appears to be higher in those patients with cerebral edema, suggesting the accumulation of osmotic substances within the lung parenchyma and outside the vasculature [12] . molecular imbalance and injury to endothelial cells, accompanied by a decrease in oncotic pressure, may play a role in the development of this disease. hepatopulmonary syndrome can be seen in both alf and chronic liver failure. it is thought to arise from microscopic shunting from arteriovenous dilations that occur in the pulmonary vasculature [13] . the precise mechanism is unknown; however, it is thought that the elevated levels of nitric oxide seen in patients with liver failure may mediate the abnormal vasodilation that occurs in the pulmonary parenchyma. the result is an overperfusion with maintenance of ventilation; a vq mismatch occurs that ultimately leads to hypoxemia [14] . as part of the pathophysiology associated with alf, there is low systemic vascular resistance and a hyperdynamic circulation with elevated cardiac output. the pathophysiology is multifactorial but vasoactive substances are thought to mediate the process [15] . while the underlying pathophysiology may differ, hemodynamic variables appear very similar to those seen in sepsis and septic shock. in the failing liver, there is an increase in splanchnic blood pooling that is associated with the increased resistance of flow through the liver. this results in increased shear stress in the splanchnic circulation that causes upregulation of endothelial nitric oxide synthase (enos) and ultimately nitric oxide (no) production [15, 16] . there is further systemic vasodilation causing a low effective circulating volume and relative hypotension despite an overall elevated intravascular volume. the systemic baroreceptors are unloaded and there is a compensatory increase in cardiac output as well as activation of the renin-angiotensinaldosterone system (raas) that may ultimately affect the renal system ( fig. 18.1 ) [15] . patients with alf usually present with varying degrees of coagulopathy. as the liver fails, there is decrease in the synthesis of factors involved in both coagulation and anticoagulation, specifically fibrinogen, prothrombin, protein c, protein s, and factors v, vii, viii, ix, x, and xi. the end result is an increased in prothrombin and activated partial thromboplastin times as well as elevation of inr [17] . overt bleeding is not typically seen, as there is a decrease in both coagulation and anticoagulation factors. however, mucosal bleeding from the oropharynx or the gastrointestinal mucosa can be frequently seen. this is compounded by the underlying platelet dysfunction that can occur in patients with liver failure. right upper quadrant pain, gastrointestinal bleeding, ascites, nausea, and vomiting can be seen in patients with alf. these symptoms are nonspecific and can be multifactorial. patients with acute viral or autoimmune hepatitis may experience liver parenchyma inflammation as part of the normal response to infection. this leads to an increase in the overall volume of the liver. the liver capsule may be unable to accommodate acute volume changes, and stretching of it results in activation of pain receptors and right upper quadrant pain. discomfort in this area can also be related to direct trauma causing bleeding. abdominal distention may be associated with ascites. the neurohumoral alterations are seen with alf leading to excessive sodium retention and ultimately plasma volume expansion. this, combined with a decrease in the overall circulating proteins due to compromised liver function, leads to overflow of fluid into the peritoneal cavity [18] . tense ascites can result in compromise of respiratory, renal, and cardiovascular function due to direct compression of the diaphragm and vasculature. as part of its normal physiologic function, the liver is responsible for gluconeogenesis as well as glycogen storage. as liver function worsens, these two key metabolic functions are compromised. in up to 40 % of patients, hypoglycemia is seen and treatment is warranted [19] . compromised flow through the liver secondary to fibrosis or intrinsic disease acute kidney injury can be present in 30-70 % of patients with alf [20, 21] . the etiology can be variable: prerenal azotemia, drug toxicity, and acute tubular necrosis have all been implicated. hepatorenal syndrome, especially type 1, has also been associated with the progression of this disease. acute kidney injury can be divided into oliguric vs. anuric failure, with the latter making fluid management difficult in the critical care setting [15] . accompanying this derangement we can also see electrolyte disturbances: hyperkalemia, hyperphosphatemia, hypophosphatemia, hypercalcemia, and hypomagnesaemia that can lead to secondary arrhythmias and mental status changes [22] . lactic acidosis can be seen in patients with alf. the accumulation of tissue lactate is multifactorial. the effective blood pressure is usually lower in those patients with liver failure. this causes a generalized tissue hypoxia that leads to the production of lactate. the compromised liver is unable to uptake and process the lactate, leading to its accumulation [23] [24] [25] . in addition, acute kidney injury can further contribute to the underlying acidosis due to failure of fixed acid clearance [22] . kupffer cells can be found around the hepatic sinusoids. because of their location, they are constantly exposed to gut bacteria and endotoxins. they play a key role in clearing these pathogens and in maintaining normal homeostasis. in patients with liver failure, their function is impaired, and there is an increased susceptibility to develop gram-positive and gram-negative bacterial infections as well as possible fungal and viral infections [26] . hepatic encephalopathy has been linked to an increased incidence of infection [27] . although the mechanism behind this has not been clearly elucidated, it is thought that cns depression alters the immune system modulation. in alf, there is also a change in the production as well as clearance of different cytokines in patients with liver failure and compromised neutrophil function. these problems will lead to decreased bacterial opsonization and clearance. these alterations ultimately contribute to the immunologic impairment [26] [27] [28] . up to three quarters of patients with alf will develop a bacterial infection. the organisms that are most commonly seen include gram-negative-bacteria, streptococcus, staphylococcus, and candida. they may develop a systemic inflammatory response syndrome (sirs) that will be undistinguishable from noninfectious conditions including necrotic hepatocytes from the failing liver [29] [30] [31] . jaundice and pruritus are common complaints of patients with alf. although not specific to liver failure, the presence of both symptoms should raise suspicion of compromised excretion of bilirubin by hepatocyte failure. a normal by-product of the metabolism of heme, bilirubin is usually excreted in bile and urine. the liver is responsible for conjugating glucuronic acid with bilirubin in order to make a soluble compound. as a result, conjugated bilirubin passes into the colon and is eventually eliminated. in the failing liver, there is a severe compromise of the ability to metabolize and excrete bilirubin secondary to the undergoing cell necrosis. there is buildup of unconjugated bilirubin in the blood resulting in eventual deposition of these molecules in mucous membranes, skin, and conjunctiva, what is known as jaundice [32] . because of the yellow color of the pigment, the physical appearance of the patient changes, directly correlating with bilirubin levels. besides bilirubin, there is also accumulation and deposition of bile acids in the skin. this has been associated with pruritus. other mechanisms that may explain this symptom include the endogenous opioids theory which proposes that the liver failure patient has elevated opioid levels secondary to decrease clearance and metabolism. these molecules activate the mu opioid receptor which may produce pruritus [33] [34] [35] . as explained throughout this chapter, the management strategies for patients with alf are different from those of patients that have chronic liver failure with an acute decompensation. it is imperative to determine what form of failure the patient is experiencing. for those with alf, early recognition and transfer to a transplant center will improve outcomes and mortality. on initial presentation, a patient's mental status will be affected to different degrees; however it may deteriorate further. getting a thorough history during the first encounter is therefore important as it can elucidate the possible cause of the acute failure. the intensivist should review all medications that the patient ingested in the last 7 days. specific questions about ingestion of acetaminophen should be asked. dietary intake should also be explored, playing close attention to any exposure to mushrooms. exact time of ingestion is key in order to determine treatment and further steps in management. social history should also be reviewed in detail. recent travel to viral hepatitis endemic areas as well as contact with other patients that have required hospital visits should be evaluated. focus on alcohol and drug use, sexual behaviors, and vaccination status can help determine the causative mechanism for the liver failure. past medical history plays a key role in determining if the patient has chronic liver disease or if they are experiencing an acute failure. a history of hepatitis, ascites, jaundice, asterixis, and gynecomastia and family history of a metabolic disorder favor chronic liver disease with an acute exacerbation. history of malignancy and lack of screening for colorectal cancer should also make the intensivist suspicious for metastatic malignancy. physical exam may disclose important findings that can elicit cause. an effort to identify the clinical manifestations described previously should be done. laboratory values that should be routinely obtained are listed in table 18 .4. when testing for hepatitis b, it is important to evaluate for immunity (hepatitis b surface antibody), infectivity (hepatitis b e antigen), and the presence of an acute infection (hepatitis b core antibody igm). although hepatitis c can cause alf, it is usually associated with chronic liver failure [36] . bun and co2 can usually be lower than reference values in patients with alf. this is secondary to poor muscle mass as well as a respiratory alkalosis experienced by these patients. presentation with concomitant renal failure will alter most serum electrolytes. elevation of liver enzymes can be indicative of acute hepatitis and alf. however, values that are within reference range may be markers of poor prognosis as it may be reflective of decreased effective liver mass [26, 34] . workup should be started on presentation, even if patient is going to be transferred to a liver center. early identification of the etiology and early treatment can significantly improve outcome. it can also identify those patients that will need liver transplantation in order to treat their disorder. if during the history and physical assessment a cause can be clearly identified, treatment should be started empirically. waiting for laboratory values can be detrimental and result in further deterioration of the patient. consultation with hepatology/gastroenterology, transplant surgery, and the intensivist should be done upon determination of liver failure of any cause. the development of alf has very different etiologies as well as presentations. as such, the management may differ from patient to patient. identification of the causative agent and treatment of it is important. however, supportive care in the intensive care unit is critical for ensuring a positive outcome. patients that have evidence of encephalopathy will require intensive care unit (icu) admission and management while those with no neurologic derangement can be followed on a regular ward with close monitoring. patients should have frequent checks of their coagulation parameters, arterial blood gases, complete blood counts, metabolic panels, serum aminotransferases, alkaline phosphatase, and bilirubin levels. derangements warrant further investigation. hemodynamic monitoring, precise fluid management, and monitoring for infections are all essential. the grade of hepatic encephalopathy guides the management and treatment of the neurologic system in alf. this is because intracranial hypertension (ich) and cerebral edema characterize the severity of patient presentation. those with mild forms (grades i and ii) very rarely develop these devastating complications while 25-35 % of patients with grade iii and 65-75 % of those with grade iv present with ich [11] . for those patients with grades i and ii, frequent neurologic assessments should be performed to follow possible neurological progression. maintaining the patient in a quiet environment helps minimize agitation. sedation should be minimized; however, if needed minimal doses of short-acting benzodiazepines should be used [37] . for patients who present with or develop grade iii and iv neurological symptoms, securing an airway should be the first treatment strategy followed by mechanical ventilation. for sedation, propofol should be used since there is evidence that it decreases cerebral blood flow and allows for frequent ongoing neurological assessment [38] . intracranial pressure (icp) monitoring devices are used in some icus in patients with alf and grade iii or grade iv encephalopathy [39] . the main reason for its use is the early identification of ich and subsequent treatment. also, not all patients present with cushing's trial of systemic hypertension, bradycardia, and irregular respirations. several trials have shown that icp monitoring can be performed safely and successfully be used to manage ich [40] [41] [42] . however, no trial has demonstrated a survival benefit. bleeding has been associated with the placement of monitors; however, recent literature reports that there is a decrease prevalence of this particular complication. the incidence of bleeding after placement of icp monitor device has been less than 1 % [43] . ct scan of the brain should be considered in those patients with an acute mental status change and those with coagulopathy in order to rule out intracranial bleed. this imaging modality does not diagnose cerebral edema or ich in all patients, and therefore, it is not needed in every case of encephalopathy. patients at risk of encephalopathy should also have the head of their bed elevated at 30° [44] , minimize et suctioning, and minimize pain as these factors can lead to ich [37] . for those patients with elevated ammonia levels (greater than 75 ug/dl) and alf, administration of lactulose can lower the incidence of cerebral edema and decrease mortality [45] . prior to prescribing this drug, the route of drug administration must be considered as the patient's ability to tolerate po intake may be compromised. other compounds studied include l-ornithine l-aspartate but have failed to demonstrate any survival improvement [46] . phenytoin has been proposed as a possible prophylactic measure to prevent cerebral edema. an initial study that involved evaluation of brain at autopsy showed that patients who were treated with prophylactic phenytoin had a decrease in cerebral edema [47] . follow-up trials were unable to replicate these results and more importantly, there was no survival improvement when this agent was used prophylactically [48] . the administration of intravenous mannitol has been shown to transiently decrease cerebral edema and may be helpful in cases in which ich is <60 mmhg [49] . a dose of 0.5-1 g/kg may be beneficial and it may be repeated if serum osmolality is below 320 mosm/l. the use of hypertonic saline has also been suggested. there is a lower incidence of ich in patients with alf that are treated with hypertonic if it is used to achieve a serum sodium level between 145 and 155 meq/l [50] . use of hypertonic saline can be limited by renal failure. a newer treatment technique that has been proposed to prevent ich is hypothermia. it is thought to mediate this benefit by preventing hyperemia [51] . concerns regarding the use of hypothermia in the treatment of alf include worsening coagulopathy and compromise of hepatocyte recovery [52] . hyperventilation and use of corticosteroids have been proposed as a management option to reduce icp. the former may achieve this goal via vasoconstriction. however, trials suggest that although there is a delay in the onset of cerebral herniation, there is no reduction in the incidence of cerebral edema and no survival benefit [53] . hyperventilation should only be used after all other resources have failed. while hypoxemia in patients with alf arises from many causes, it is treated with supplemental oxygen. if the patient has grade iii or iv hepatic encephalopathy, a definite airway should be established. during intubation, cis-atracurium is the agent of choice since it does not increase icp [54] . pleural effusions can be observed and may or may not be contributing to hypoxemia or other respiratory problems. the use of diuretics should be carefully considered as these patients are usually in a very delicate hemodynamic state. overuse of diuretics can precipitate renal failure [34] . hepatopulmonary syndrome (hps) has been traditionally resistant to medical therapies [15] . oxygen supplementation for hypoxemia is recommended. transjugular intrahepatic portosystemic shunt (tips) has been reported to improve hps; however, it is not currently recommended as its outcomes are variable [55, 56] . liver transplantation is the only therapy that has been shown to improve oxygenation and decrease oxygen requirement [57] . the diagnosis of hps should prompt immediate referral to a transplant center. decreases in blood pressure lead to compromised renal and brain perfusion. it is imperative to be attentive to blood pressure and heart rate values in order to ensure adequate hemodynamics and, most importantly, adequate perfusion. patients with alf should be resuscitated initially with crystalloid before considering vasoactive agents. the generally accepted goal mean arterial pressure is 65 mmhg [58] . if after adequate volume resuscitation the patient is still hypotensive and not meeting blood pressure goals, vasopressors should be considered. norepinephrine should be initiated and titrated to effect [59] . for resistant hypotension consideration to vasopressin should be given, although it should be used with caution as it has been associated with cerebral vasodilation and increased ich [60, 61] . terlipressin has also been suggested as adjuvant treatment but it is currently not available in the united states [60] . other causes of hypotension resistant to vasopressor therapy should also be entertained including adrenal failure and severe acidosis. during liver transplantation, ich and hemodynamics improve immediately after hepatectomy, probably secondary to removal of vasoactive cytokines. hepatectomy can improve these derangements for up to 48 h [62] . hepatectomy is currently recommended only as a last resort and when a liver graft in the process of being delivered to the transplant institution [37] . despite the derangements of coagulation laboratories in patients with alf, their coagulation status remains in equilibrium and overall hemostasis. in the absence of bleeding, no correction of laboratory parameters should be performed [63] . transfusion should be discouraged because treatment with ffp may precipitate pulmonary problems including hypoxia, and transfusion also prevents the use of inr as a marker of hepatocyte recovery [37] . if an invasive procedure is planned or if there is evidence of significant bleeding, correction of coagulopathy should be done. ffp can be used for this purpose; however, careful volume management should also be achieved. the use of plasmapheresis and recombinant activated factor vii (rfviia) can help in the correction of coagulopathy. rfviia has been proposed as it effectively corrects derangements without volume overload [64] . however, administration does carry the risk of myocardial infarction and portal vein thrombosis [65] . alf has also been associated with vitamin k deficiency and it should be administered routinely in these patients [66] . thrombocytopenia has also been reported in patients with alf. platelets should not be administered in the absence of bleeding. if the patient has platelet counts that are greater than 10,000/mm 3 , no prophylactic transfusion should be given [67] . if an invasive procedure is planned, platelets between 50,000/mm 3 and 70,000/mm 3 have been proposed, and in those bleeding, the intensivist should consider transfusion if platelets drop below 50,000/mm 3 [67, 68] . bleeding from intestinal mucosa is rare but has been reported in patients with alf. histamine-2 receptor blockers have been used in critically ill patients as prophylaxis of gastrointestinal (gi) bleeding with great success [69] . also, proton pump inhibitors (ppi) have contributed to the reduced incidence of upper gi bleeding in patients with liver dysfunction [70] . it is therefore recommended that alf patients are started on prophylaxis while in the icu. nutrition can be compromised in patients with alf; therefore, enteral feedings should be started early unless there are contraindications. there is no evidence that using branched-chain amino acid formulas has benefits over other enteral tube feeds [71] . protein supplementation should not be restricted but rather limited to 60 g per day in most patients. if gastrointestinal feeding is contraindicated, parenteral nutrition may be considered. there is also evidence that the risk of gi bleeding is reduced in patients that are on enteral feeding [72] . hypoglycemia should be actively treated in patients with alf. the intensivist should consider adding dextrose to crystalloids in the form of d5. if hypoglycemia is severe, central replacement with d20 concentration should be used. frequent glucose checks should be performed in order to assess the response to glucose administration. improvement and eventually weaning can be achieved in those patients that experience hepatocyte recovery. right upper quadrant pain can be treated with narcotics. judicious doses should be used as metabolism of medications can be compromised with the failing liver [37] . the management of ascites will be discussed with chronic liver failure. close urine output monitoring is paramount in patients with alf. hemodynamic changes and alterations in the cardiovascular system make the kidneys susceptible to injury. insertion of a urinary catheter should be performed upon determination of hepatic failure. besides serum electrolytes, measurement of urinary sodium and creatinine is necessary. high or normal urine sodium may indicate the presence of acute tubular necrosis, while a low urine sodium may indicate prerenal azotemia or hepatorenal syndrome. several electrolyte derangements may occur and correction should be attempted. accumulation of lactate may result from tissue hypoxia and combined with renal failure may cause life-threatening acidosis. renal replacement therapy may be necessary in these patients. when indicated, continuous dialysis should be used as studies have shown that it provides cardiovascular as well as intracranial pressure stability when compared to intermittent dialysis [73] . the development of an infection in a patient with alf has been associated with worsening encephalopathy and cerebral edema. also, the presence of bacterial or fungal infections may compromise any attempts at performing a liver transplantation. because of the impact that it has, prophylactic antimicrobials have been proposed as a prevention strategy for these patients [74] . prophylactic antibiotics have been used and shown to decrease the incidence of infections in patients with alf. in a prospective control trial by rolando n et al., patients with fulminant liver failure were randomized to receive either selective parenteral and enteral antimicrobials vs. no treatment until clinically indicated. 104 patients were included in this study. thirty-four percent of those patients randomized to receive prophylactic antibiotics developed an infection compared to 61 % of those that were treated when clinically indicated (p < 0.005). however, this did not translate into a survival benefit [75] . it is currently recommended that if no prophylactic antibiotics are used, periodic sputum, urine, and blood cultures are performed to determine if there are bacterial infections [37] . the use of antifungals has also been studied [76] . it is routine practice of the authors to use prophylactic enteric fluconazole in patients that are expected to be in the icu for more than 3 days, given that there is a decrease in fungal infections in high-risk critically ill surgical patients [77] . it is paramount to perform an infectious workup to any patient with liver failure that develops a change in mental status as it may be a change precipitated by infection. the most common cause of alf in the united states is acetaminophen (paracetamol) toxicity [78] . over-the-counter availability and the fact that it can be found in combination with other medications make it the cause of voluntary or involuntary overdoses that compromise liver function and may result in fulminant liver failure. acetaminophen is usually taken orally and absorbed via the gastrointestinal system. its half-life is usually 2-4 h with one exception being extended release preparations in which it is increased to more than 4 h. total doses should not exceed 4 g per day. ingesting doses less than 7.5 g per day is unlikely to result in acute toxicity; however, it can vary depending on underlying liver function [79] . the metabolism of acetaminophen is performed in the liver. most of the compound, approximately 90 %, is conjugated with sulfate or glucuronide and excreted in the urine. five percent of the remaining medication is excreted unchanged in the urine. the remaining acetaminophen is subject to metabolism by the cytochrome p450 pathway. it is converted into n-acetyl-p-benzoquinoneimine (napqi), a highly reactive and toxic compound that is immediately conjugated with hepatic glutathione and excreted in the urine. when glutathione levels drop below 20 % physiologic levels, napqi forms covalent bonds via cysteine groups with hepatic molecules and proteins, leading to irreversible hepatocyte damage. a decrease in glutathione levels, enhanced cytochrome p450 activity secondary to medication use, acetaminophen overdose, or decreased liver function from chronic disease make patients more susceptible to developing toxicity. the clinical presentation of acetaminophen toxicity can be divided into four different stages (table 18 .5). stage i includes a series of nonspecific gi symptoms that start shortly after ingestion. no liver abnormality can be seen. during stage ii, there is usually transaminitis with a high ast/alt ratio. stage iii is characterized by the clinical evidence of liver failure and, in some patients, renal failure. mortality is higher at this stage. those patients that survive this stage progress to stage iv in which there is normalization of most of their lab derangements. because patients may not show symptoms up to 24 h after ingestion, it is very important to obtain a detailed history. standard workup should be initiated as discussed previously. contacting poison control will help coordinate efforts to treat and eventually transfer patient to a liver center [37] . in order to determine the severity of the poisoning, a serum acetaminophen concentration (4 h post ingestion or later) should be plotted against time on the modified rumack-matthew nomogram (fig. 18. 2) [80, 81] . patients with acetaminophen levels below the treatment line can be discharged home after psychiatric and social evaluation. all other patients should be admitted to the intensive care unit [82] . for those patients that ingested a single dose of acetaminophen of more than 7.5 g less than 4 h prior to presentation, administration of activated charcoal should be considered. review of several small studies demonstrated that activated charcoal was the best available option to reduce absorption [83] [84] [85] . also, there is a decreased risk of developing liver injury if charcoal is given prior to other forms of treatment [85] . if patient has an unstable airway, charcoal should not be administered until the airway is controlled. the antidote of choice for acetaminophen toxicity is n-acetylcysteine (nac). the exact mechanism of action is unclear; however, it appears to restore glutathione levels [86, 87] . indications for administration include a serum acetaminophen level above the treatment line, ingestion of more than 7.5 g, serum acetaminophen level >10 mcg/ml if time of ingestion is unknown, evidence of liver injury, and a history of acetaminophen ingestion regardless of time of ingestion [86] [87] [88] . oral and iv administration of nac have been studied and both appear effective [86] . the main factor determining the mode of treatment should be the mental status of the patient. if the patient is confused or has evidence of encephalopathy, oral administration should be avoided. if the oral protocol is used, a loading dose of 140 mg/kg should be given followed by 17 doses of 70 mg/kg given every 4 h. if iv nac is used, a loading dose of 150 mg/kg is given over 1 h. a second dose of 50 mg/kg is then given over 4 h and finally a third dose of 100 mg/kg is given over 16 h. an alternative to nac is hemodialysis. this method effectively removes acetaminophen [89] . however, because of the effectiveness of nac, it should be reserved for cases in which the antidote is not available. acetaminophen toxicity is best managed in a multidisciplinary setting with assistance from hepatology and surgery teams. ingestion of poisonous mushrooms can lead to lethal emergencies including alf. amanita phalloides, amanita bisporigera, amanita verna, and other mushroom species may cause alf. these mushrooms do not express repulsive smells or tastes, and they can be found throughout midsummer in moist oak forests. alpha-amanitin is the amatoxin responsible for liver failure. after gastrointestinal absorption, enterohepatic circulation is responsible for transportation into the liver, where via active transport it concentrates in hepatocytes. the toxin will bind to rna polymerase and inhibit protein synthesis, ultimately leading to apoptosis [90] . the clinical presentation of patients that ingest amatoxin includes an initial asymptomatic period of a few hours. this is followed by gastrointestinal symptoms that include abdominal pain, nausea, vomiting, and diarrhea that can be bloody. liver enzymes will be elevated and will continue to increase. one to two days after ingestion, the second phase of the presentation begins with an apparent recovery with continuing elevation of ast and alt. in severe poisonings, coagulopathy and possible dic and renal failure may ensue. the last phase includes alf and typically starts 3 days after ingestion. hypoglycemia and multi-organ failure can be seen. workup of a patient with suspected amanita ingestion should proceed as indicated earlier in this chapter. detection of amatoxin can be performed in urine samples using enzyme-linked immunoassay (elisa); this test is not readily available in all institutions and awaiting results should not preclude supportive treatment [91] . supportive treatment should be started immediately after presentation. in addition, an effort to minimize toxin absorption should be attempted. activated charcoal can bind amatoxin, and if given in repeated doses, it can reduce mortality significantly by increasing elimination via gastrointestinal tract [37] . medications that can inhibit uptake of this toxin have also been described. these include penicillin g and silymarin. the former is given as a continuous infusion and has been show to decrease mortality [92, 93] . the latter is a more potent inhibitor and is available in iv and po formats. silymarin has been shown to minimize damage to hepatocytes [92, 94, 95] . nac has also been used in the treatment of amatoxin intoxication. mortality appears to improve with implementation of protocols very similar to those of acetaminophen toxicity [92, 96] . wilson's disease poses a different presentation from frank alf. it normally occurs in the background of chronic liver disease that has been unrecognized. treatment varies when presentation of this disease is acute, and this will be the focus of this section. a genetically recessive disease, it is estimated that 2-3 % of alf cases are related to wilson's disease [97] . the majority of copper that is ingested is transported into the liver where it is incorporated into enzymes and copper-binding proteins (ceruloplasmin). excess copper is combined with apometallothionein and excreted into bile. in wilson's disease, the incorporation into ceruloplasmin is compromised and copper is accumulated in the liver. as the disease progresses, other organs are affected. besides parkinsonian movements and tremors, kayser-fleischer rings, psychiatric alterations, and renal problems, wilson's disease will present with liver disease: cirrhosis, chronic failure without cirrhosis, and acute liver failure. laboratory workup should include serum ceruloplasmin, which is usually low, as well as serum copper level (above 200 mcg/dl) [97] . in patients with evidence of alf, low transaminases, low alkaline phosphatase, hypokalemia, glycosuria, hypophosphatemia, and renal tubular acidosis, the diagnosis of wilson's disease should be considered. in patients with acute failure, the aim should be to remove copper. hemodialysis and peritoneal dialysis can successfully achieve this goal [98] . albumin dialysis and the molecular absorbent recirculating system (mars) device have also been used with promising results [99, 100] . penicillamine, zinc, and other medications used for treatment of wilson's disease do not play a role in alf. the development of alf from viral hepatitis may occur after acute infection; ostapowicz et al. estimated that the etiology of 12 % of those patients that were diagnosed with alf was viral hepatitis [101] . most of the clinical deteriorations that are seen in patients with this etiology of disease are related to chronic liver infection. alf is more common with hepatitis b but it can also present in patients with hepatitis a, c, and e [34] . presentation of viral hepatitis is described in four phases. phase 1 is characterized by lack of symptoms but changes in laboratory studies that may be suggestive of viral hepatitis. phase 2 marks the development of symptoms that include nausea, vomiting, abdominal pain, arthralgias, and possible fevers. the next phase includes clinical characteristics of alf including right upper quadrant pain, becoming icteric, and possible coagulopathy. the last phase, 4, leads to the normalization of laboratory values and resolution of symptoms. diagnosis of viral hepatitis relies on serum laboratories. hepatitis b has several important antigens and antibodies. hepatitis b surface antigen (hbsag) is usually found in patients with acute infection. a second antigen, associated with infectivity, is hepatitis b e antigen. the first antibody that can be detected in patients acutely infected and that indicates acute presentation of disease is igm anti-hbcag. resolution of acute infection and recovery results in igg antibodies against this antigen. finally, anti-hbsag appears in the serum several months after infection, indicating resolution. they will also be found in patients with hepatitis b vaccine. igg anti-hepatitis c virus has been used to diagnose exposure to this viral infection. it can usually be found in the serum several months after an acute infection and contrary to anti-hbsag, it does not confer immunity to hepatitis c. use of elisa and riba testing for diagnosis has fallen out of favor. hcv rna pcr assays were developed in order to detect the presence of the virus. it has been successful in not only establishing the diagnosis but also the presence of an acute infection. treatment of acute hepatitis a is limited to supportive care as there are no medications that improve outcome. hepatitis b treatment usually follows the same principles as most antiviral therapy is directed toward treatment of chronic disease. however, recent studies have suggested that acute hepatitis b may benefit from administration of lamivudine [102] . finally, acute hepatitis c has been treated with ifn therapy with resolution of hcv rna after several months of treatment [103] . low perfusion pressure to the liver may result in clinical manifestations of alf known as ischemic or hypoxic hepatitis. it is an uncommon cause of liver failure, with a prevalence of 1 per 1,000 hospital admissions [104] . this can be a direct consequence of global hypoperfusion, hemodynamic instability, direct vascular occlusion during surgical procedures, hepatic artery disease (occlusion, dissection, thrombosis) in patients with portal vein thrombosis, or hepatic sickle cell crisis [105] . hepatocytes in zone 3 become ischemic and eventually necrotic leading to liver insufficiency. prognosis of ischemic hepatitis is poor. raurich et al. described an in-hospital mortality of 61.5 % in all patients that were diagnosed with this disease process. in those patients with concomitant septic shock and those that experienced cardiac arrest, mortality rates were higher, at 83.3 % and 77.7 %, respectively. risk factors for mortality included an elevated inr, need for renal replacement therapy, and diagnosis of septic shock. non-survivors were more likely to be on vasopressors and to require mechanical ventilation [106] . patients with hepatitis secondary to shock present with several symptoms related to their hemodynamic instability including altered mental status, respiratory distress, severe hypotension, and renal failure. patients with a history of cardiac compromise may present with nausea, vomiting, right upper quadrant pain, and malaise. up to 14 % of patients with septic shock will also have ischemic hepatitis, presenting with fevers and severe hypotension [106] . laboratory examination reveals elevated aminotransferase levels, usually above 1,000 iu/l. the ratio of serum alanine aminotransferase to ldh less than 1.5 suggests ischemic hepatitis [107] . if hypoperfusion is chronic in nature, synthetic function may be preserved and coagulation studies may be normal; however, in acute cases, there is severe derangements that continue to progress with time. if ischemic hepatitis is suspected, a right upper quadrant ultrasound with doppler should be immediately performed as it may reveal the etiology of the insufficiency. there is no specific treatment for ischemic hepatitis. management is centered around restoring cardiac output and reestablishing hepatic perfusion. appropriate resuscitation is necessary. excessive fluid administration may lead to vascular congestion which can in turn compromise perfusion of hepatocytes and aggravate the presentation. judicious use of diuretics should be exercised as diuresis may exacerbate hypoperfusion and therefore liver failure. intensivists should rule out ischemic hepatitis in any patient that presents with septic shock and has elevated aminotransferases [106] . prompt recognition of hypoperfusion state may lead to early intervention and possible better outcomes. continuous hepatic injury that persists for more than 6 months is considered chronic liver disease (cld). the liver parenchyma suffers continuous inflammation and potential destruction. the hepatic insult does not only result in damage but also in attempts of repair. ultimately this leads to a broad spectrum of clinical manifestations including fibrosis, cirrhosis, and hepatocellular carcinoma. these changes are accompanied by alterations in serum liver function tests and can include physical exam finding suggestive of physiologic alterations. in the united states, the most common causes of cirrhosis leading to liver transplantation are alcoholic liver disease, chronic viral hepatitis, and nonalcoholic liver disease (table 18 .6) [108] . this last etiology has increased significantly in incidence. most patients are generally asymptomatic until decompensation occurs, making the calculation of prevalence difficult. approximately 49,500 deaths in 2010 where associated with cld [109] . patients with cld may present with compensated or uncompensated hepatic failure. the former may be asymptomatic prior to evaluation, but patients usually report nonspecific symptoms such as weight change, fatigue, and lack of appetite. those patients with an acute decompensation may show signs of active bleeding, confusion, and skin changes. because of the broad spectrum of the disease, presentation will vary between different patients. due to similar underlying pathophysiology, symptoms and findings may be similar to those described previously during the acute liver failure presentation. patients with cld may present with varying degrees of hepatic encephalopathy. classification and underlying pathophysiology are similar to those described previously in the alf section. an acute exacerbation with an underlying chronic liver dysfunction can cause rapid progression from confusion to coma. shortness of breath, dyspnea, and other nonspecific respiratory symptoms may also be reported. as with acute dysfunction, the etiology may be of infectious, metabolic, or of cardiac etiology. hepatopulmonary syndrome can also play a role in underlying hypoxemia [15] . the mechanisms that lead to the respiratory derangements in cld are similar to those described in acute liver compromise. figure 18 .1 explains the molecular mechanism behind the underlying decreased effective perfusion pressure seen in patients with liver failure. as a result, patients will have a lower than baseline blood pressure, with some of them transitioning from hypertensive to normotensive. the cardiac output in patients with liver disease is usually high; however it is important to understand that myocardial cells are actually depressed from exposure to the changes in cytokines and other molecules. there is a slightly elevated heart rate that compensates for the depression and overall results in increase cardiac output, in a normal-sized man, often in the range of 10-12 l/min [15] . patients with cld may present with anemia, leukopenia, thrombocytopenia, and coagulopathy [110] . the pathophysiology behind anemia is multifactorial, and it may include episodes of gastrointestinal bleeding associated with portal hypertension and coagulopathy. there may also be nutritional deficiencies such as folate deficiency that can lead to compromised production of red cells and vitamin k deficiency that can lead to decreased production of coagulation factors [17] . aplastic anemia, hypersplenism, and hemolysis may contribute to the anemia experienced by patients with chronic failure [111] . thrombocytopenia is associated with portal hypertension: an enlarged spleen can sequester the majority of the circulating platelet mass and lead to a decrease platelet count. it has also been described that patients with liver disease have decreased levels of thrombopoietin that will also lead to thrombocytopenia [112] . patients experiencing cld can present with abdominal distention and pain, anorexia, nausea, and vomiting. physical exam may also show ascites, hypogonadism, hypersplenism, and evidence of gastrointestinal (gi) bleeding such as hematemesis, hematochezia, and melena. gi bleeding can be the result of mucosal injury and thrombocytopenia or a more severe and life-threatening event such as variceal hemorrhage. an umbilical hernia may be seen when ascites becomes prominent. for those patients with cld, there are significant changes in the hemodynamics of the portal vein. the hepatic microcirculation, sinusoids, undergoes constriction secondary to architectural changes that compromise the lumen of these systems. furthermore, there is active contraction of myofibroblasts and active smooth muscle secondary to cytokine changes (increased levels of intrahepatic et-1) that cause even more restriction in the radius of these sinusoids [113, 114] . these changes lead to an increase in portal pressure. a second factor that impacts the pressure of the portal vein is the increased in blood flow in the portal vein. as shown in fig. 18.1 , there is a splanchnic arteriolar vasodilation that leads to increase venous outflow and, therefore, increased flow that results in further increases of portal pressure and eventually portal hypertension (pht) [15] . the elevated blood pressure and flow are partially relieved by decompressing the inflow into the portal vein into systemic collaterals. the esophageal submucosal veins are a preferred method of decompression and may result in esophageal varices. as flow increases so does the vessel radius [115] . this ultimately leads to an increase in wall tension that may end up in rupture and variceal bleeding [114, 116] . ascites is also closely related to pht. in fact, patients without evidence of pht do not develop ascites even in the presence of cirrhosis. the threshold for formation of ascites appears to be 12 mmhg at the level of the portal vein [117] . as a response to this increase in pressure, there is splanchnic vasodilation leading to a decrease in effective arterial blood volume that is mediated by several molecules including nitric oxide (no). there is subsequently an activation of the renin-angiotensin-aldosterone system that increases renal sodium retention and plasma expansion that ultimately leads to accumulation of fluid in the peritoneal cavity [118] . the low levels of circulating protein secondary to liver compromise may also favor the formation of ascites. on physical exam, we can find evidence of pht by placing a stethoscope over the epigastrium. if there are collateral connections between the portal system and the umbilical vein, a murmur can be auscultated. this finding is known as cruveilhier-baumgarten murmur. dizziness, diaphoresis, and overall malaise may be reflective of underlying hypoglycemia. patients with cld undergoing an acute exacerbation may see decreased levels of circulating glucose with corresponding changes in neurologic exam. male and female patients with cld can report abnormalities related to infertility, impotence, and in the case of women chronic anovulation. physical exam may show evidence of testicular atrophy in men, while ultrasound and other imaging may show atrophic ovaries and uterus. there are several possible mechanisms that explain these findings. the increased levels of follicle-stimulating hormone (fsh) and luteinizing hormone (lh) observed in some patients suggest the primary dysfunction of the testicles or ovaries. an alternative mechanism suggests suppression of the hypothalamicpituitary function. the dysfunction may be secondary to decreased clearance of estrogen, testosterone, prolactin, and other substances [119, 120] . male patients with cld may complain of loss of male pattern pubic hair, chest and axillary hair loss, and gynecomastia. this finding is thought to be related to an overall increase in estradiol: the adrenal glands produce and increase quantities of androstenedione that undergoes aromatization into estrone and eventually to estradiol [120] . similar to patients with alf, patients with cld can present with renal pathology. these may manifest as decreased urine output, arrhythmias, generalized body edema, and overall malaise. most of the changes are associated with the underlying liver dysfunction. in hospitalized patients with cld, it is estimated that approximately 10 % of them will develop hepatorenal syndrome (hrs). the pathophysiology of hrs follows the development of pht. as explained in fig. 18.1 , there is dilation of the splanchnic circulation, leading to a decrease in perfusion pressure. the response is cardiac compensation as well as activation of the renin-angiotensin-aldosterone system. there is also vasoconstriction mediated by the sympathetic nervous system. these changes ultimately lead to low renal perfusion and a significant decrease of the glomerular filtration rate [16] . electrolyte abnormalities can accompany the changes that are seen on the renal system. hyperkalemia, hyperphosphatemia, and hyponatremia can be detected in serum electrolytes. symptoms may be variable and depend not only on severity of derangement but acuity. dizziness, weakness, and palpitations may be reflections of these abnormalities. cld leads to acquired immune deficiency and makes these patients prone to developing infections. the mechanism by which the immune response is compromised includes the deficiency of serum complement [121] as well as the compromised activity and function of phagocytes such as macrophages, pmns, and kupffer cells [122, 123] . certainly, the presence of fevers should make the intensivist suspicious for an infectious process and further investigation is warranted in order to determine additional symptoms that may guide further treatment. however, patients who present with decompensated liver failure may have an infection causing the decompensation. thus, suspicion for the presence of infection should be high, and the threshold for obtaining cultures is low in any patient with liver failure who is acutely ill. abdominal pain that worsens and fevers should raise the suspicion for spontaneous bacterial peritonitis (sbp) in those patients with evidence of ascites. up to 30 % of these patients may develop sbp [124] . patients with cirrhosis have an increased intestinal permeability as well as altered intestinal motility. this may lead to the bacterial overgrowth and infection of ascites [125] . the most common organism seen is escherichia coli; however, other organisms have also been described [126] . typically sbp is monomicrobial and a polymicrobial infection should prompt consideration of a perforated viscous. similar to alf, skin and urine color can change in patients with cld. the increase in bilirubin secondary to compromised liver function leads to the accumulation in the skin leading to jaundice as well as dark appearance of urine. these changes are usually undetectable if the serum bilirubin is less than 2 mg/dl. another change that can be appreciated in the skin of patients with cld includes palmar erythema. it is thought to be the consequence of altered sex hormone metabolism which may lead to capillary vasodilation [127] . careful examination of the skin can also reveal vascular lesions characterized by the presence of a central arteriole with surrounding smaller vessels. these are called spider angiomata and their appearance is related to an increase in estradiol levels. the number as well as size of these lesions is related to the severity of liver disease although they are not specific for it [128] . as an additional route to decompress the portal vein during pht, the umbilical vein may open leading to shunting into abdominal wall veins. these vessels engorge significantly making them very easy to identify during physical exam. this finding is known as caput medusa. initial workup and management of patients with cld should begin with a thorough history. onset of symptoms and identification of disease progression helps determine the pathophysiologic manifestations of the disease. previous medical diagnosis including viral hepatitis should be assessed. a thorough review of all medications that the patient takes can help identify potential additional mechanisms of liver injury. hospitalizations and transfusions should be reviewed. social history including exposure to high-risk behaviors such as intravenous drug use and alcohol abuse should be performed. family history of liver disease and personal history of malignancy (including oncologic treatment and surveillance studies) also play a key role in the development of disease and should be explored. a complete physical exam should be performed and an attempt to determine if any of the clinical manifestation discussed previously are present. the exam should include neurologic, rectal, and skin exam. assessment of vital signs in order to identify possible hypotension, hypoxemia, as well as end-organ perfusion should be performed. there is no serologic test that can diagnose cld accurately. laboratory abnormalities that are identified could be related to alf or another etiology with some degree of liver dysfunction. besides serologic tests, evaluation of the degree of liver fibrosis and additional characteristics of cld can be investigated with radiologic studies. the initial serologic studies that are performed as well as initial management are similar to those described in table 18 .4 in the alf section. in addition, studies from ascitic fluid should also be performed when it is desired to identify etiology of fluid and possibility of infection. after paracentesis with removal of 50 ml of ascites in a sterile fashion, the intensivist should send the fluid for cell count, cytology, albumin, total protein, triglycerides, amylase, adenosine deaminase, as well as culture [129] . this should be accompanied by a serum albumin in order to calculate the serum-ascites albumin gradient (saag). this is done by subtracting the albumin in the ascitic fluid from the serum value. based on such studies, the etiology of ascites can be determined (table 18.7) . imaging studies that are routinely used include ultrasonography (us), ct scan, and magnetic resonance imaging (mri). us can help identify morphologic changes such as nodularity. with doppler us, patterns of flow as well as possible occlusions can be identified. ct and mri are able to identify nodularity and changes in volume of liver mass (hypertrophy or atrophy) as well as assess the portal vasculature [130] . evaluation of collateral circulation, varices, and tumors can also be performed. since us does not use contrast, this can be very helpful in those patients with renal compromise [131, 132] . if after a thorough workup, the diagnosis of cld cannot safely be established, liver biopsy should be considered. identifying changes consistent with cld may be very beneficial as it may prevent delays in therapy and potential worsening of the patient [133] [134] [135] . surgery and interventional radiology teams should be involved in order to determine the safest and least invasive method that can render a diagnosis. suspicious findings for cld should prompt consultation with hepatology/gastroenterology and transplant surgery in order to determine if the patient will benefit from additional therapies and workup including possible transplantation. evidence of encephalopathy, compromised ventilation, hypotension, hypoperfusion, active bleeding, sepsis, and sbp should prompt admission to the icu. consideration of additional hemodynamic monitors such as an arterial line and central access may be considered in every patient. a foley catheter should be placed in all patients with hemodynamic instability or with poor renal function but avoided in those with anuria to prevent a urinary tract infection. it is also helpful to classify the severity of liver disease. the child-turcotte pugh (ctp) classification divides patients into three groups based on serum labs and clinical presentation. it can help in determining possible surgical treatments or additional therapies [136, 137] . this specific scoring system is presented in table 18 .8. another classification system that is used for the allocation of organs in the unites states is the model for end-stage liver disease (meld). it consists of a formula that will assign a score to a patient and that accurately predicts mortality within 3 months. the formula is based on three laboratory values (bilirubin, inr, and creatinine) and it is modified by etiology. the formula is shown below [138] : if the disease process is alcohol, 1 is assigned to etiology. if the liver failure is secondary to a cholestatic process, 0 is assigned instead. several factors can modify the calculated meld score for allocation purposes, and these include dialysis and the presence of hepatocellular carcinoma. the ctp and meld system have been compared in several studies in order to determine which provides a better answer to prognosis for patients. although some studies show superiorities of meld, others show no difference and good predictions with both systems [139] [140] [141] [142] . a systematic review, suggested that the meld was better for predicting 3-month mortality but otherwise the systems were similar [143] . because of its use with united network for organ sharing (unos) lists for allocation of organs, meld has become more popular. hepatic encephalopathy (he) is a diagnosis of exclusion, and therefore, an effort to identify other etiologies of altered mental status should be performed. it is also necessary to determine the precipitating event leading to the neurologic derangement which includes bleeding, renal failure, electrolyte abnormalities, changes in diet, and changes in medication [144] . treatment principles are similar to those described in the alf section. they should be based on supportive care, attempts to correct precipitating factors, minimizing gi nitrogen intake, and establishment of therapy. admission to an icu is important as patients with he need constant neurologic assessments for progression or resolution. for grade iii and grade iv he, establishment of definite airway should be the first step in management. laboratory studies are key in order to identify possible precipitating events. a decrease in nitrogen production as well as nitrogen delivery should be attempted with medication. the most common therapy used is lactulose, which reduces the absorption of ammonia. twenty-five milliliter should be given twice a day and should be titrated to achieve two soft bowel movements [145] . rifaximin has also been used as an add-on therapy to lactulose. it is an antibiotic with activity against grampositive and gram-negative aerobes and anaerobes. the usual dose is 400 mg three times a day. trials have shown benefit in the treatment of he when rifaximin is used in addition to lactulose [146] . another antibiotic that has been use is neomycin. this alternative treatment has been used for the treatment of overt hepatic encephalopathy [147] . however, because it has been associated with complications such as ototoxicity and nephrotoxicity, neomycin is used less commonly today [145] . an assessment of nitrogen intake by assessing a patient's diet is also very important. if a patient's he is unresponsive to the therapies described above, oral branched-chain amino acids (bcaa) should be considered in an attempt to reduce the hepatically metabolized nitrogen load. a recent metaanalysis showed that bcaa-enriched formulations may be beneficial in some patients with he and cld [71] . the daily protein intake should be 1.2-1.5 g/kg/day as severe restriction may be detrimental in the catabolic state of cld [145] . the first step in management of a patient with cld and ascites should be sodium restriction to no more than 2,000 mg per day [129] . this should also be accompanied by oral spironolactone and possibly furosemide in order to perform natriuresis while maintaining normokalemia. spironolactone inhibits sodium reabsorption in the distal tubule and collecting ducts but it can lead to gynecomastia and hyperkalemia. furosemide is a loop diuretic and inhibits the luminal na-k-2cl symporter causing natriuresis and also hypokalemia when used alone. combination therapy has been used more effectively in achieving sustained results. if the serum sodium is less than 125 mmol/l, fluid restriction to no more than 1.2 l per day should also be done [148] . for those patients that are not responsive to diuretic therapy, serial paracenteses can be performed in order to relieve symptoms [149] . in carefully selected patients, transjugular intrahepatic portosystemic shunt (tips) should be considered. trials have demonstrated that there is better control of ascites and overall survival with this procedure; however, there is worsening hepatic encephalopathy [150] . referral to a transplant center should be done for patients with refractory ascites. tense ascites with respiratory compromise and abdominal discomfort can also be the initial presentation of patients with cld. prior to sodium restriction, paracentesis should be performed. for large volume (>5 l) removal, albumin replacement should be done [151] . replacement of 6-8 g of albumin per l of fluid removed has been shown to improve survival [129] . replacement after paracentesis has remained a controversial topic. in one study performed by gines et al., patients with tense ascites were randomized to receive albumin or no replacement. those that did not receive albumin had more changes in serum electrolytes, plasma renin, and creatinine but had no survival advantage [152] . there has been no study up to date demonstrating decreased survival in patients without replacement when compared to albumin [153] . in a meta-analysis by bernardi et al., 1,225 patients from 17 trials were analyzed. albumin was shown to be superior to other plasma expanders, with an infusion between 5 and 10 g of albumin per liter removed [154] . angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aspirin, and nonsteroidal anti-inflammatory agents should be avoided in patients with cld and ascites: prostaglandin inhibition can severely affect renal hemodynamics as well as natriuresis. it is important to evaluate patients with ascites for ventral and umbilical hernias. for those patients with ascites, hernia repair should only be attempted after medical treatment of ascites. for those with refractory ascites, repair should be deferred until after liver transplantation. if the patient has an incarcerated or strangulated hernia, emergency repair is warranted, but special attention to the ascites postoperatively must be made. the diagnosis of spontaneous bacterial peritonitis (sbp) is established with studies sent from ascitic fluid revealing one of the following three findings: 1. leukocyte count of more than 500 per mm 3 2. polymorphonuclear count of more than 250 per mm 3 the causative organism is usually a gram-negative enteric bacteria; if more than one organism is identified, secondary peritonitis should be considered. escherichia coli and klebsiella are responsible for more than 50 % of the cases [155] . therapy is tailored based on the most likely causative agent. if the patient has not been on empiric antibiotics prior to presentation, an intravenous third-generation cephalosporin should be started, preferably cefotaxime 2 g every 8 h. if the patient has been exposed prior to this medication, coverage should be based on hospital antibiogram [129] . therapy should be started if there is a high suspicion for infection while cultures are pending. the recurrence rate of sbp can be as high as 70 % and therefore prophylaxis is advocated. long-term antibiotic therapy, norfloxacin 400 mg daily, is recommended [156] . trimethoprim/sulfamethoxazole can be used as a secondline agent for those patients with sensitivities [129] . the presence of esophageal varices in patients with cld warrants prophylactic therapy. the most effective medication has been propranolol that inhibits stimulation of the beta-2 venodilator receptors seen in varices. it should be started at low doses, 5 mg orally twice a day, and titrated to reduction of pulse rate by 25 %. if patients cannot take propranolol, isosorbide mononitrate can be used. if the patient is unable to tolerate medical therapy, esophagogastroduodenoscopy (egd) and variceal banding should be performed [157] . three principles govern the management of an acute variceal bleed: stabilization and resuscitation, identification and treatment of bleeding, and prevention of recurrence. if a patient presents with evidence of gi bleeding, immediate type and cross should be performed, and if needed, transfusion of untyped and uncrossed blood should begin. waiting for laboratory values to show anemia may worsen the overall clinical condition of the patient. upper gi bleeding in a patient with presumed cld prompts urgent endoscopy to identify possible bleeding esophageal or gastric varices. if during endoscopy, no varices are seen, repeat evaluation should be done in 3 years. if varices are identified but not bleeding, follow-up endoscopy should be done after 1 year. if active bleeding is encountered and it appears to involve esophageal varices, an attempt at controlling the bleeding varices should be done. banding followed by sclerotherapy are the two most common methods of achieving control. if after appropriate attempts bleeding does not stop, a sengstaken-blakemore tube should be inserted. tips and surgical shunts should be considered if all previous methods fail. tips has shown improved outcomes [129] ; however, it is associated with he [157] . surgical shunts carry a high morbidity and should be considered a last resort. cld patients with gi bleeding are at risk of developing bacterial infections. some advocate the use of ceftriaxone for 7 days while patients are gi bleeding [158, 159] . if the patient stabilizes and tolerates oral intake, changing to norfloxacin is reasonable. the diagnostic criteria for hepatorenal syndrome (hrs) are shown in table 18.9. hrs is a diagnosis of exclusion and it is important to rule out other etiologies including prerenal azotemia, intrinsic renal disease, and post renal failure. in order to diagnose hrs, all major criteria in table 18 .9 must be met. minor criteria are not required; however, they provide supportive evidence that the pathophysiology is consistent with hrs. identification of precipitating event is also instrumental in the management of hrs as additional therapy can be instituted. when performing large volume (>5 l) paracentesis, it is recommended to replace volume with albumin (see ascites section above) as this procedure may lead to hrs. evaluation for possible sbp as well as workup for gi bleeding should be considered as they are well-established risk factors for the development of this syndrome. there are two manifestations of hrs: type i and type ii. the former shows a rapid decline in renal function with either an initial creatinine of greater than 2.5 mg/dl or a 50 % reduction in the creatinine clearance. type ii usually leads to moderate renal failure that progresses slowly and is manifested as diuretic-resistant ascites [160] . liver transplantation is the preferred treatment for patients with hrs. any patient with evidence of this syndrome should be referred to a liver transplantation center in order to be listed for transplantation [161] . bridging with pharmacotherapy is necessary in most patients as there is rapid decompensation, especially in those with type i hrs. the basic principle behind the management of hrs is reversal of renal vasoconstriction and splanchnic vasodilation. dopamine, fenoldopam, and prostaglandins have been used in an attempt to cause direct renal vasodilation [15] . results of several trials have not favored any of these agents as none have improved outcome [160] [161] [162] . splanchnic vasoconstriction, in an attempt to reduce portal blood flow and decrease pressure, has been attempted with vasopressin, ornipressin, terlipressin, norepinephrine, and midodrine [15] . ornipressin, with some promising results, resulted in an increase rate of ischemic events [163] . terlipressin in combination with albumin has shown the most promising results, with improvements in renal function although its use has not been approved in the united states [164] . norepinephrine and vasopressin have been used with improvement of renal function and successful bridging to transplantation [60] . hemodialysis may be required in the treatment of these patients, especially those with type 1 disease. those patients that are hospitalized in an icu should receive continuous dialysis rather than intermittent as it minimizes changes of abrupt hemodynamic changes and further compromise of these frail patients [73] . patients with alf and cld may benefit from liver transplantation. this therapeutic option should be considered when medical therapy has failed and when there is progression of disease. referral to transplant center should occur once the patient has experienced ascites, variceal hemorrhage, hrs, and he. consultation with hepatology and transplant surgery teams ensures early consideration for transplantation. table 18 .10 presents poor prognostic factors from the king's college criteria that may suggest that the need for transplantation is increased. prior to transplantation, a thorough evaluation is performed on patients regardless of etiology. this includes assessment of cardiac function, possible occult malignancy, identification of infection, contraindications to chronic steroid therapy, and appropriate social support. the rapidly progressive nature of alf designates that these patients are currently listed as status 1 by the united network for organ sharing (unos) [165] . approximately table 18 .9 criteria for diagnosis of hepatorenal syndrome chronic or acute liver disease with advanced hepatic failure and portal hypertension low glomerular filtration rate serum creatinine >1.5 mg/dl or 24 h creatinine clearance <40 ml/min absence of shock, ongoing bacterial infection, and current or recent treatment with nephrotoxic drugs absence of gi fluid losses absence of renal fluid losses in response to diuretic therapy no sustained improvement in renal function after diuretic withdrawal and expansion of plasma volume with 1.5 l of plasma expander proteinuria <500 mg/day no obstructive uropathy, parenchymal renal disease, microhematuria minor criteria urine volume <500 ml/day urine sodium <10 meq/l urine osmolality greater than plasma osmolality urine rbcs <50/high-power field serum sodium concentration <130 meq/l non-acetaminophen inr greater than 6.5 or three of the following five criteria: patient age of less than 11 or greater than 40 serum bilirubin of greater than 300 μmol per liter time from onset of jaundice to the development of coma of greater than 7 days inr greater than 3.5 drug toxicity, regardless of etiology of alf acetaminophen arterial ph <7.3 inr greater than 6.5 creatinine greater than 300 μmol per liter encephalopathy (grade iii or iv) 40 % of patients with alf will undergo liver transplantation, 25 % of them will improve with supportive care, and 35 % will not survive their presentation; of those that have a liver transplant performed, the 3-year survival is approximately 75 % [165] . patients with failure secondary to viral hepatitis usually have better outcomes than those with drug reactions or metabolic causes. also, patients with alf have worst outcomes when compared with patients with cld. the 1-year survival for patients with cld that undergo liver transplantation is 90 % [166] . timing is not standard and is usually dependent on severity of meld. living donors have been used secondary to decrease in organ availability and it has been successful. this therapy has not been studied in patients with alf. liver replacement therapies (lrt), also known as liver dialysis, have been studied and used as a bridging therapy to transplant [167] [168] [169] [170] . several methods have been developed and they can be grouped into artificial and bioartificial devices. regardless of the mode of action, they attempt to clear toxins that are free and protein bound, as well as to regenerate or replace proteins that are affected by the liver failure process. among the artificial methods, the most studied is the molecular adsorbent recirculation system (mars). it effectively clears several toxic compounds and causes a dramatic improvement in serum laboratories and in some symptoms such as pruritus [171] . unfortunately, this has not translated into clinical benefits [172] . biologic methods include devices with porcine hepatocytes and with human hepatoblastoma cells [167, [171] [172] [173] . their theoretical advantage is the production of proteins and compounds produced by a normal liver as well 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controlled clinical trial the effect of mannitol on cerebral white matter water content the effect of hypertonic sodium chloride on intracranial pressure in patients with acute liver failure moderate hypothermia prevents cerebral hyperemia and increase in intracranial pressure in patients undergoing liver transplantation for acute liver failure moderate hypothermia in patients with acute liver failure and uncontrolled intracranial hypertension a double-blinded, randomized trial of hydrocortisone in acute hepatic failure. the acute hepatic failure study group atracurium infusions in patients with fulminant hepatic failure awaiting liver transplantation effect of transjugular intrahepatic portosystemic shunt on pulmonary gas exchange in patients with portal hypertension and hepatopulmonary syndrome transjugular intrahepatic portosystemic shunt: a successful treatment for hepatopulmonary syndrome improved survival after liver transplantation in patients with hepatopulmonary syndrome acute liver failure in adults: an evidence-based management protocol for clinicians management of acute liver failure comparison of terlipressin and noradrenalin on cerebral perfusion, intracranial pressure and cerebral extracellular concentrations of lactate and pyruvate in patients with acute liver failure in need of inotropic support worsening of cerebral hyperemia by the administration of terlipressin in acute liver failure with severe encephalopathy two-stage total hepatectomy and liver transplantation for acute deterioration of chronic liver disease: a new bridge to transplantation minimal effects of acute liver injury/acute liver failure on hemostasis as assessed by thromboelastography recombinant activated factor vii for coagulopathy in fulminant hepatic failure compared with conventional therapy fviia corrects the coagulopathy of fulminant hepatic failure but may be associated with thrombosis: a report of four cases pharmacokinetics and efficacy of oral versus intravenous mixed-micellar phylloquinone (vitamin k1) in severe acute liver disease the expert panel on preventive services: continuing the work of the uspstf coagulopathy of acute liver failure oral ranitidine as prophylaxis for gastric stress ulcers in intensive care unit patients: serum concentrations and cost comparisons proton pump inhibitors versus histamine 2 receptor antagonists for stress ulcer prophylaxis in critically ill patients: a systematic review and meta-analysis parenteral nutrition with branched-chain amino acids in hepatic encephalopathy. a meta-analysis the value of early enteral nutrition in the prophylaxis of stress ulceration in the severely burned patient improved cardiovascular stability during continuous modes of renal replacement therapy in critically ill patients with acute hepatic and renal failure prospective study of bacterial infection in acute liver failure: an analysis of fifty patients prospective controlled trial of selective parenteral and enteral antimicrobial regimen in fulminant liver failure fungal infection: a common, unrecognised complication of acute liver failure double-blind placebo-controlled trial of fluconazole to prevent candidal infections in critically ill surgical patients annual report of the american association of poison control centers toxic exposure surveillance system assessment and treatment of acetaminophen overdose acetaminophen poisoning and toxicity acetaminophen hepatotoxicity: the first 35 years a review of acetaminophen poisoning simulated acetaminophen overdose: pharmacokinetics and effectiveness of activated charcoal impact of activated charcoal after acute acetaminophen overdoses treated with n-acetylcysteine a prospective evaluation of the effect of activated charcoal before oral n-acetylcysteine in acetaminophen overdose comparison of oral and i.v. acetylcysteine in the treatment of acetaminophen poisoning treatment of paracetamol (acetaminophen) poisoning with n-acetylcysteine acetylcysteine for acetaminophen poisoning hemodialysis as adjunctive therapy for severe acetaminophen poisoning: a case report amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous amanita mushrooms phalloidin syndrome: role of elisa-based assay for the detection of alpha-and gamma-amanitins in urine. preliminary results treatment of amatoxin poisoning: 20-year retrospective analysis a rare case of amatoxin poisoning in the state of texas the use of silymarin in the treatment of liver diseases effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial amatoxin poisoning treatment decision-making: pharmaco-therapeutic clinical strategy assessment using multidimensional multivariate statistic analysis aasld practice guidelines: wilson disease fulminant wilson's disease with haemolysis and renal failure: copper studies and assessment of dialysis regimens molecular adsorbent recycling system (mars): clinical results of a new membrane-based blood purification system for bioartificial liver support albumin dialysis: effective removal of copper in a patient with fulminant wilson disease and successful bridging to liver transplantation: a new possibility for the elimination of protein-bound toxins results of a prospective study of acute liver failure at 17 tertiary care centers in the united states lamivudine treatment for acute severe hepatitis b: a pilot study treatment of acute hepatitis c with interferon alfa-2b hypoxic hepatitis hypoxic liver injury hypoxic hepatitis in critically ill patients: incidence, etiology and risk factors for mortality serum lactic dehydrogenase in the differential diagnosis of acute hepatocellular injury nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the united states us burden of disease collaborators. the state of us health, 1990-2010: burden of diseases, injuries, and risk factors incidence, prevalence, and clinical significance of abnormal hematologic indices in compensated cirrhosis splenomegaly, hypersplenism and coagulation abnormalities in liver disease thrombopoietin in acute liver failure endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats hemodynamic mechanism of esophageal varices pathophysiology of portal hypertension and esophageal varices portal hypertension and its complications is sinusoidal portal hypertension a necessary factor for the development of hepatic ascites? pathogenesis of ascites in cirrhosis amenorrhoea in women with non-alcoholic chronic liver disease patterns of hypothalamic-pituitary-gonadal dysfunction in men with liver disease due to differing etiologies low-protein-concentration ascitic fluid is predisposed to spontaneous bacterial peritonitis role of kupffer cells in host defense and liver disease role of kupffer cells in the pathogenesis of liver disease spontaneous bacterial peritonitis: recent data on incidence and treatment intestinal permeability in cirrhotic patients with and without spontaneous bacterial peritonitis: is the ring closed? spontaneous bacterial peritonitis: pathogenesis, diagnosis, treatment clinical anatomy of the umbilicus aasld practice guideline management of adult patients with ascites due to cirrhosis: update 2012 ct and mri findings of cirrhosis-related benign nodules with ischaemia or infarction after variceal bleeding ultrasound of the liver the real capabilities of contrast-enhanced ultrasound in the characterization of solid focal liver lesions noninvasive tests for liver disease, fibrosis, and cirrhosis: is liver biopsy obsolete? sampling variability on percutaneous liver biopsy sampling variability of liver biopsy in nonalcoholic fatty liver disease resection for hepatocellular carcinoma is a good option in child-turcotte-pugh class a patients with cirrhosis who are eligible for liver transplantation evaluation of hernia repair operation in child-turcotte-pugh class c cirrhosis and refractory ascites the model for end-stage liver disease (meld) meld vs child-pugh and creatinine-modified child-pugh score for predicting survival in patients with decompensated cirrhosis meld score is better than child-pugh score in predicting 3-month survival of patients undergoing transjugular intrahepatic portosystemic shunt comparison and improvement of meld and child-pugh score accuracies for the prediction of 6-month mortality in cirrhotic patients comparison of meld, child-pugh, and emory model for the prediction of survival in patients undergoing transjugular intrahepatic portosystemic shunting systematic review: the model for end-stage liver disease -should it replace child-pugh's classification for assessing prognosis in cirrhosis? hepatic encephalopathy: from pathophysiology to therapeutic management hepatic encephalopathy in chronic liver disease: 2014 practice guideline by aasld and easl rifaximin treatment in hepatic encephalopathy neomycin reduces the intestinal production of ammonia from glutamine peritoneovenous shunting as compared with medical treatment in patients with alcoholic cirrhosis and massive ascites comparison of paracentesis and diuretics in the treatment of cirrhotics with tense ascites. results of a randomized study randomized controlled study of tips versus paracentesis plus albumin in cirrhosis with severe ascites recommendations for the use of albumin and immunoglobulins randomized study of therapeutic paracentesis with and without intravenous albumin in cirrhosis randomized trial comparing albumin, dextran 70, and polygeline in cirrhotic patients with ascites treated by paracentesis albumin infusion in patients undergoing large-volume paracentesis: a metaanalysis of randomized trials spontaneous bacterial peritonitis norfloxacin prevents spontaneous bacterial peritonitis recurrence in cirrhosis: results of a double-blind, placebo-controlled trial propranolol plus placebo versus propranolol plus isosorbide-5-mononitrate in the prevention of a first variceal bleed: a double-blind rct improved survival after variceal bleeding in patients with cirrhosis over the past two decades antibiotic prophylaxis for the prevention of bacterial infections in cirrhotic patients with gastrointestinal bleeding: a metaanalysis definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis hepatorenal syndrome oral misoprostol or intravenous prostaglandin e2 do not improve renal function in patients with cirrhosis and ascites with hyponatremia or renal failure reversibility of hepatorenal syndrome by prolonged administration of ornipressin and plasma volume expansion terlipressin improves renal function in patients with cirrhosis and ascites without hepatorenal syndrome etiology and outcome for 295 patients with acute liver failure in the united states analysis of long-term outcomes of 3200 liver prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure the treatment of acute liver failure with fractionated plasma separation and adsorption system: experience in 85 applications in vitro comparison of the molecular adsorbent recirculation system (mars) and single-pass albumin dialysis (spad) extracorporeal liver support-albumin dialysis with the molecular adsorbent recirculating system (mars) treatment of resistant pruritus from cholestasis with albumin dialysis: combined analysis of patients from three centers extracorporeal albumin dialysis with the molecular adsorbent recirculating system in acute-on-chronic liver failure: the relief trial pilot-controlled trial of the extracorporeal liver assist device in acute liver failure foreword: prospects of liver cell transplantation and liver-directed gene therapy treatment of the crigler-najjar syndrome type i with hepatocyte transplantation key: cord-023033-tgt69ir6 authors: nan title: poster session (pp. 78a–178a) date: 2006-02-10 journal: hepatology doi: 10.1002/hep.1840380503 sha: doc_id: 23033 cord_uid: tgt69ir6 nan , ala-60 (n=4), tyr-77 (n=2), gly-42 ( n = l ) and lys-89 ( n = l ) . the mean time from onset of symptoms to diagnosis was 3.4 years (0-10) and patients were listed for olt an average of 10.8 months (0-60) after diagnosis. the mean time from listing to olt was 8.2 months (1-18). five patients died after olt, during a mean follow-up of 3.5 years (4 months -6 years). one-year patient survival was 100% and threeyear patient survival was 92%. one patient required retransplantation for hepatic artery thrombosis. neurologic symptoms were the initial clinical manifestation in the majority of patients (7/12), followed by cardiopulmonary (4112) and gastrointestinal symptoms (1112). most patients experienced multiple symptoms. subjective evolution of symptoms as assessed by chart review demonstrated that symptoms referable to amyloidosis worsened after olt in seven patients, and improved or stabilized in five. following olt, neuropathy symptoms improved in four patients, worsened in seven patients and were unchanged in one patient. pre and post-olt nerve conduction velocities were available for 5 patients and the post-olt studies showed progression of disease in 3, improvement in 1 and were unchanged in 1 patient. prior to olt, all twelve patients had an increased ivst on echocardiogram. post olt cardiac symptoms improved in six patients (five of whom also had cardiac transplantation), worsened in three patients, and were unchanged in three patients (one of whom also had cardiac transplantation backgr0und :accurate delineation of the scope and magnitude of peri-operative donor risk is necessary to better allow for informed consent, to maximize the potential for donor safety, for comparative outcome analysis and ultimately to serve as a key determinant of the utility of adult-to-adult living donor liver transplantation (aaldlt). however, at present, there is lack of uniformity regarding what constitutes a complication in this setting. moreover, a system to stratify adverse events with respect to their life altering (quantity or quality) impact is lacking. aims: 1) to define a graded, inclusive classification schema for both early (e) and late (l) adverse operation-related events in live liver donors a n d 2) to apply this system to a retrospective review of events in individuals undergoing partial hepatectomy for live liver donation at our center. two patients (6.5%) suffered cut-surface bile leaks and one (3.2%) a right colon injury (grade 3e complications). one patient (3.2%) developed a transient bilateral ulnar neuropathy (grade 2e). two patients (6.5%) were readmitted within 30 days of operation for nausea and dyspnea respectively (grade 1e). one patient underwent repair of an incisional hernia 6 months post donation (grade 2l). one patient suffered positioning-related brachial plexopathy (grade 3l). conclusions: the definition and adoption of a graded, scale-based system of operation-related adverse events in live liver donors will allow for an inclusive, consistent and universally applicable method to collect, analyze and report donor complications. all aaldlt programs must be encouraged to fully review and report their donor related morbidity, ideally through the creation of a national donor registry initiation of antiviral therapy in the early post-transplant period, prior to overt evidence of hcv recurrence (i.e. preemptive therapy) may enhance rates of hcv eradication. aims: to determine the efficacy and tolerability of preemptive ifn versus ifnlrbv in anti-hcv positive lt patients. methods: consecutive and eligible lt recipients from a single center were enrolled. the goal was to initiate treatment within 6 wks of lt. patients were randomized to ifn or ifn plus rbv (400mg daily x 2wks, then 800mg daily x 2 wks, then 1.0-1.2g daily based upon body weight 75 kg). induction therapy with daily ifn was used for the first 8 wks (1.5mu daily x 2wks, then 3mu daily x 6 wks) followed by 3 mu tiw (n=39) or peg-ifn 1.5 uglkglwk (n=10) for 40 wks. key inclusion criteria were stable clinical status, cr45,ooo and wbc>3.0. dose reductions for side effects, especially cytopenias were standardized. growth factors were given for neutropenia (anc<1000) and anemia (hgb<9.0gldl) beginning 7l2001. virological (vr) and biochemical responses (br) were evaluated at end-of-treatment (et) and 6 mos post-treatment (sustained virological (svr) and biochemical (sbr) responses (table below) were associated with response. histological disease was mild in the majority of patients at treatment end (78% stage 0 fibrosis and 72% grade 1 or less necroinflammatory activity). conclusions: preemptive antiviral therapy was applicable to -60% of transplanted patients. biochemical responses were frequent but svrs uncommon. treatment discontinuation and lowering of rbv doses due to side effects likely reduced svrs and may have limited our ability to detect differences between treatment groups. the only predictor of svr was a negative qhcvrna pre-treatment, which implies that patients with low vl early post-lt may be the best candidates for preemptive therapy. compared to historical reports, the severity of histological disease was very mild at 1-year post-lt in the majority of treated patients, suggesting preemptive antiviral therapy may provide important histological benefits. the cadaveric organ shortage has led to the development of alternative strategies for organ transplantation. living donor liver transplant (ldlt) is one strategy that has offered many individuals transplantation before they die or become too sick for transplant.chronic hepatitis c (hcv) is the leading indication for liver transplantation. preliminary results from small single center studies suggest that recurrent hcv is more common after ldlt compared to cadaveric transplant with concern that graft survival may be lower after ldlt. &: to compare patient and graft survival in hcv recipients who undergo ldlt to cadaveric liver transplant recipients. methods: we analyzed the united network for organ sharing (unos) liver transplant database from january, 1999 -december, 2002. inclusion criteria included liver transplant recipients 218 years old transplanted from 1999-2002 with the transplant diagnosis of chronic hepatitis c. exclusion criteria included subjects who were hepatitis b surface antigen positive, history of prior organ transplant, concurrent kidney or heart transplant. the logrank test was used to compare survival between the two groups. results: from 1999-2002 6.6% of adult liver transplants were ldlt. 279 ldlt recipients and 3,955 cadaveric recipients transplanted for end stage liver disease from hcv were identified. the results are shown in the table: ldlt ( a greater proportion of ldlt recipients were female and ldlt recipients were less ill at the time of transplant.1 and 3 year patient and graft survival were not significantly different between the 2 groups, (p=o.11). if only the pre-meld era is considered (01l99 thru 2/02) 1 year graft survival for ldlt (n=240) and clt (n=3322) are 78% and 83%, respectively, p=o.lo. conclusions: our analysis demonstrates that short-term patient and graft survival are equivalent in ldlt and cadaveric recipients with hcv suggesting recurrent hepatitis c does not adversely affect survival over this time period. ldlt recipients are less ill at transplantation which did not confer a short-term survival advantage. continued follow-up should determine the impact of less advanced liver disease at the time of transplant and recurrent hepatitis c after transplant on long-term graft and patient survival in ldlt recipients. disclosures: introduction: histologic injury due to recurrent hcv has been reported in up to 90% of hcv infected patients who undergo liver transplantation with a cadaveric graft. however, the natural history of hcv following living donor liver transplantation (ldlt) is not as well described. anecdotal evidence suggests that hcv recurrence may be more severe in ldlt recipients. we hypothesize that post-operative liver regeneration in the partial graft procured from living donors may facilitate hcv replication, or increase the vulnerability of hepatocytes to infection. methods: we performed a retrospective analysis comparing outcomes, and the incidence, timing, and severity of histologic recurrence of hcv following transplantation in patients who underwent living donor liver transplant compared to recipients of cadaveric organs. between 12/98 and 3/02,68 hcv infected adult patients (age > 18 years old) underwent liver transplantation for hcv associated cirrhosis. 45 patients received a cadaveric graft (cad) and 23 received a graft from a living donor (ldlt). mean time of patient follow up was 25 months, with a range of 6 to 39 months. elevated serum transaminases, positive hcv rna, and liver biopsy consistent with histologic evidence of hcv defined recurrence. cholestatic hepatitis c (chc) was confirmed if all of the following criteria were met: serum total bilirubin greater than lomg/dl with no evidence of extra-hepatic biliary obstruction on ultrasound and cholangiography, and histologic features on liver biopsy consisting of portal expansion, ductular proliferation, and bile stasis with or without hepatocyte ballooning. immunosuppression, definition and treatment of rejection were standardized for both cad and ldlt. results: when comparing cad to ldlt, both the incidence of hcv recurrence and time to recurrence were not different. the overall incidence of "severe" sequelae of hcv recurrence, either cholestatic hepatitis, grade 111-iv inflammation, and/or hcv induced graft failure requiring re-transplantation was also not different when comparing cad to ldlt. however, when comparing cad versus ldlt, no cad patient developed cholestatic hepatitis c, compared to 17% of ldlt who developed this complication (p = 0.001). conclusions: in this patient population, the timing and overall incidence of hcv recurrence were not different when comparing cad versus ldlt, but the incidence of cholestatic hepatitis was significantly greater in patients with hcv who underwent ldlt. further multicenter studies are warranted to determine the incidence and risk factors for cholestatic hepatitis c following liver transplantation. ucsf, sun francisco, ca; fabien zoulim, inserm, lyon, france; carol l brosgart, michael wulfsohn, michael d miller, shelly xiong, gilead sciences, inc., foster city, ca background lamivudine (lam) resistance occurs in approximately 40% of chronic hepatitis b (chb) patients after 2 years of lam monotherapy. in contrast, resistance to adefovir dipivoxil (adv) occurs infrequently with 1.6% of chb patients developing the adefovir resistance mutation rtn236t after 2 years of adv therapy. pre-existing lam resistance mutations or concurrent use of immunosuppressive therapy by lt patients may increase the risk of resistance to adv. objective: to determine the incidence of adv resistance in a clinical trial of liver transplantation (pre and post) patients with lam-resistant hbv treated with adv for 96 weeks (lam therapy was maintained in most patients). methods: the hbv reverse transcriptase domain was sequenced for lt patients with detectable hbv dna by pcr (>lo00 copieslml) after 96 weeks of adv therapy (n=114). in vih-o drug susceptibility was determined following transfection of hepg2 cells with patient-derived hbv clones from baseline and week 96 serum samples. results: the rtn236t mutation wa!j observed in 21114 patients (1.8%) at week 96. lam had been discontinued at weeks 16 and 28 after initiation of adv in these two patients respectively. the baseline lam-resistant ymdd mutation reverted to wildtype prior to week 96 in both patients. emergence of rtn236t was associated with rebound in serum hbv dna and alt elevation in both patients. in vitro phenotypic analysis showed approximately 4-fold reduced susceptibility to adefovir with rtn236t. however, these adefovir-resistant hbv clones were fully susceptible to lam and entecavir in vitro. lam therapy was re-initiated, in addition to the ongoing adv therapy, after emergence of rtn236t in these patients resulting in a > 2.9 loglo copies1ml reduction in serum hbv dna in both patients. one patient also had a significant reduction (>80%) in alt within 5 months of lam treatment. no other mutations potentially associated with adefovir resistance were detected. conclusions: emergence of the adefovir resistance mutation rtn236t was observed infrequently after 2 years in liver transplantation patients (1.8%, 2/114) infected with lam-resistant hbv, similar to observations in treatment nayve non-liver transplantation patients. the adv-resistant hbv was sensitive to lam and addition of lam resulted in clinical stabiliation in both patients. local liver immune responses are thought to play a major role in chronic autoimmune diseases directed at biliary epithelium.using the apical sodium dependent bile acid transporter (asbt) promoter to drive biliary epithelial cell -specific expression of a membrane form of ovalbumin (ova), we have previously developed ova-bil transgenic mice. because these mice are tolerant to ova, we use ova-specific t cells from ot-1 and ot-i1 transgenic mice, restricted by mhc class i and class 11, respectively, with well defined peptide epitopes specific for ova to induce biliary damage in a dose dependent manner. aim: 1) to determine the liver mononuclear cell populations (mnc) involved in necroinflammatory disease, and 2) to determine where adoptively transferred cells home and proliferate. methods: 10 million ot-i and 2 million ot-i1 nayve t cells were adoptively transferred to ova-bil mice by intraperitoneal injection. at days 0, 5, and 7, liver mnc were isolated by collagenase digestion, purified by discontinuous percoll gradient centrifugation, and analyzed by flow cytometry. tail bleeds were performed at days 0, 3, 5, and 7 to follow serum alt. in a subset of experiments, ot-1 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (cfse) and analyzed on day 3 and 5 by flow cytometry after adoptive transfer with unlabeled ot-i1 t cells into ova-bil mice. results: ova-bil mice develop normally without evidence of disease up to 2 years. after adoptive transfer of ova-specific t cells, there was a marked increase in serum alt. cd8+ ot-i t cells were required for liver damage and ova-specific cd4+ t cells markedly augmented this inflammation. adoptive transfer of ot-i1 cd4 t cells alone did not induce liver injury. there was extensive portal inflammation in every portal triad, centered around the bile ducts with infiltrating lymphocytes in the bile duct epithelia, apoptotic cells, loss of biliary epithelial cells as well as interface hepatitis. liver mnc were abundant in ova-bil mice and increased after adoptive transfer of ova-specific t cells. serum alt peaked at day 5 (mean 263 iulml), coincident with liver mnc peak. cfse labeling studies revealed robust homing of adoptively transferred ot-i cd8 cells to the liver, but not to the spleen, of ova-bil mice. the ova-specific cd8 cells, but not cd4, nk1.l, or cd19 cells, underwent cell division. conclu-sion: recognition of biliary epithelial antigen in ova-bil mice induces a necroinflammatory response in the liver as assessed by serum alt, liver mnc numbers and immunohistochemistry. the magnitude of this response correlates with influx of nahe ovaspecific cytotoxic t cells, which are activated and divide in the liver but not in the spleen. t cell recognition of antigen expressed on bile duct epithelium occurs rapidly, causes biliary specific inflammation with interface hepatitis, which may be a model of autoimmune bile duct injury or cholangiopathy. the etiology of autoimmune hepatitis (aih) is only poorly understood although the major autoantigens, such as cytochrome p450 2d6 (cyp2d6), could be identified and immunodominant epitopes have been mapped. one major reason for this lack of comprehension is the fact that there are currently only few valid animal models for aih and none of them involves the autoantigen targeted in humans. thus, we generated an animal model for human aih using a natural autoantigen (cyp2d6). self-tolerance in transgenic mice that express human cyp2d6 in the liver was challenged by infecting these cyp2d6-mice with an adenovirus-cyp2d6 vector (ad-2d6) in order to deliver large amounts of the critical antigen to the liver and to provide a inflammatory environment that would favour autoimmunity. infection with an empty adenovirus vector resulted in a transient form of focal necrosis 4 days after infection that subsequently disappeared after 2 weeks. in contrast, infection with ad-2d6 resulted in extended and persistent infiltration of cd4, cd8 lymphocytes as well as macrophages resulting in confluentlbridging necrosis at week 10 post-infection. in addition, the overall morphology of the liver was massively disturbed after ad-2d6 infection. at week 10 postinfection, the liver was approximately half the size of the control and its lobules were fused together and rounded up. first indications of fibrosis and hyperplasic nodules become apparent. the overall architecture of the liver was disrupted i disorganized and the liver parenchyma was partially collapsed. furthermore, theliver of ad-2d6 infected mice was surrounded by multiple layers of connective tissue as seen in some stages of liver cirrhosis. additional signs of cirrhosis started to become apparent in the form of nodules that are entrapped in fibrous tissue. in addition, accumulation of infiltrating cells are visible directly under the liver capsule. these observations indicate that the cyp2d6-mouse displays a persistent form of hepatitis after infection with ad-2d6 that may form the basis for a novel model system which would allow to study mechanisms involved in the initiation, propagation and precipitation of autoimmune processes involved in human autoimmune hepatitis. disclosures: urs christen -no relationships to disclose eric f johnson -no relationships to disclose michael p manns -no relationships to disclose antje rhode -no relationships to disclose matthias g von herrath -no relationships to disclose herkel, peter r galle, edgar schmitt, ansgar w lohse, johannes gutenberg university, mainz, germany background: in clinical hepatitis, hepatocytes express mhc class i1 molecules; we recently reported that mhc i1 expressing hepatocytes can function as antigen presenting cells that stimulate specific cd4 t cells (hepatology 2003; 37: 1079-85) . to understand the relevance of hepatocellular antigen presentation for hepatic immunity, we now examined whether hepatocytes may induce the differentiation of primary cd4 t cells. because inflammatory cd4 t cells in the liver are most likely primed by dendritic cells of the draining lymph nodes, we also examined whether hepatocytes may also re-differentiate dendritic cell-primed cd4 t cells. methods: primary cd4 t cells from ovalbumin-specific t cell receptor-transgenic mice were stimulated by antigen presenting hepatocytes from hepatocyte-specific ciita-transgenic mice or splenic dendritic cells.the response type was determined by measuring secreted interferon-gamma (ifn) and interleukin-4 (il-4). results: we found that antigen presenting hepatocytes by default induced differentiation of primary cd4 t cells to th2 cells (ifn: 200 ulml; 1l-4: 1800 ulml). in contrast, primary cd4 t cells stimulated by dendritic cells differentiated to thl effector cells (ifn: 2400 ulml; 1l-4 20 ulml). however, these dendritic cellprimed thl type cells, when re-stimulated by hepatocytes, had a decreased capacity to produce ifn (580 ulml vs. 2800 ulml after dentritic cell stimulation); and after repeated re-stimulation by hepatocytes, the dendritic-cell primed t cells even differentiated into th2 cells (ifn: 112 ulml; il-4: 1270 ulml). conclusions: these data show that antigen presenting hepatocytes induce th2 differentiation of undifferentiated cd4 t cells. most notably, even dendritic cell-primed cd4 t cells that are committed to thl differentiation could be reverted by hepatocytes to th2 type. thus, hepatocytes seem to have an extraordinary capacity to promote antiinflammatory hepatic immune responses. it is therefore conceivable that antigen presentation by hepatocytes associated with clinical hepatitis, in the absence of pro-inflammatory stimuli, seems to downregulate inflammatory infiltrating t cells. background nkt cells are a unique subset of regulatory lymphocytes with important immune modulatory effects. these cells recognize exogenous glycolipids anchored by a ceramide tail to the mhc-like cdld molecule, expressed by various antigen presenting cells. glycosylceramides, including the marine sponge-derived a-galactosylceramide can activate nkt cells, leading to exacerbation of hepatitis. concanavalin a induces immune mediated hepatitis in which nkt cells are key participants. glucocerebroside (gc) is a naturally occurring glycolipid. aims: to determine the immune modulatory effect of gc in a murine model of con a hepatitis. methods: five groups of balblc mice were studied group a and b mice were treated with gc (1.0 pg ip) two hours prior to and two hours following administration of 500 pg con a, respectively; group c mice were treated with con a alone; group d mice were treated with gc alone; group e mice did not receive any treatment. the degree of liver damage was evaluated by serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels, and by liver histology. the immunmodulatory effect of gc was determined by facs analysis of intrahepatic and intrasplenic lymphocytes for nkt markers, and by elisa measurements of serum ifny, il2, il4, il10, and il12. the effect of gc on nkt lymphocyte proliferation was assessed in vitro. results: treatment with gc markedly reduced serum ast and alt levels in group a compared to group c (143 vs. 600 iu in group a and group c, respectively, for ast; 57 vs. 801 iu in group a and group c, respectively, for alt, p<0.05). administration of gc alone did not change ast or alt levels compared to naive controls. histological sections of livers from group a mice revealed markedly attenuated damage compared to sections from group c livers, in which massive hepatocyte necrosis was present. the beneficial effect of gc on immune mediated hepatitis was associated with a 20% decrease in the intrahepatic nkt lymphocyte number, and with significant lowering of serum ifny levels (3725 vs. 5620 pglml in groups a and c, respectively, p<0.05); administration of gc alone led to increased serum il-12 levels (573 pglml vs. 92 pglml in group d vs. group e, respectively, p<0.05). in vitro, administration of gc decreased nkt cell pro-liferation by 42% in the presence of dendritic cells, but not in their absence. conclusions: administration of gc led to significant amelioration of con a hepatitis that was associated with a dendritic cell-dependent decrease of nkt cell proliferation in vitro, and with decreased intrahepatic nkt lymphocytes and serum ifny levels in vivo. these results suggest that the effect of gc may be mediated by inhibition of intrahepatic nkt cells, resulting from competitive displacement of activating elements from the cdld molecule on antigen presenting cells. glucocerebroside, a naturally occurring glycolipid, holds promise as a new immunomodulatory agent, with a possible role in treatment of autoimmune hepatitis and other immune-mediated liver disorders. disclosures: background cd4+ cells constitutively expressing cd25 have a regulatory function, their experimental removal leading to spontaneous autoimmune disease in normal rodents. cd4+cd25+ regulatory t cells suppress both th1 and th2 responses, competing with effector cd4+ cells in recognizing the same peptide antigens. their ability of expansion is key to the maintenance of tolerance. autoimmune hepatitis type 2 (aih-2) is characterized by t cell immune responses against 6 well-defined regions on cytochrome p4502d6, the autoantigen of aih-2. aims: to investigate the ability of expansion of cd4+cd25+ after exposure to non-antigen-specific and cypzd6-specific stimuli in aih-2. methods: cd4+cd25+ t cells were analysed by triple colour flow-cytometry of freshlcryopreserved peripheral blood mononuclear cells (pbmcs) befare and after culture in the presence of a t cell expander capable of maintaining the original t cell function (cd3kd28 dynabeads t cell expander, dynal biotech, norway) and of 28 synthetic cyp2d6 peptides (10 pmol), spanning the 6 antigenic regions known to induce proliferative cd4+ responses in aih-2. patients and controls: nine patients with aih-2 (7 female, median age 11.4 yrs, range 2.5 to 21 yrs) were investigated, 3 at diagnosis and 6 while on sustained remission. nine healthy laboratory workers served as normal controls. results: before stimulation, the level of cd4+cd25+ t cells was significantly lower in aih-2 patients (3.59 2 1.08) than in normal controls (6.39 f 0.87; p=o.o2), a difference present both at diagnosis (2.24 ? 0.56, p<0.005) and during remission (4.25 2 0.35, p=o.ol). following exposure to the t cell expander, the level of cd4+cd25+ t cells increased 4.5 times (28.6 2 30.8) in normal controls, but only 1.85 times in am-2 patients (6.62 ? 1.85, p=o.ol). upon exposure to cymd6 peptides, cd4+cd25+ t cells remained numerically unchanged, in contrast to the pbmcs from the same patients giving a strong proliferative response (up to 6.9 times). summary & conclusion: our data show a numerical and functional impairment of regulatory t cells in am-2. this defect is likely to be key to the initiation and perpetuation of the autoaggressive process in this condition. orth, mark a mcniven, nicholas f larusso, mayo medical school, clinic and foundation, rochester, mn cryptosporidium parvum (cp) opportunistically infects intestinal and biliary epithelia causing worldwide morbidity, especially in patients with aids. epithelial invasion by cp involves host cell membrane alterations resulting in a parasitophorous vacuole that envelops the parasite and a dense-band within the host cell underlying the attachment site; both processes require host cell actin remodeling. since recent studies in other systems have demonstrated that cdc42, an actin-associated gtp-binding protein, plays a central role in microbial-induced actin remodeling, we tested the hypothesis that cp activates host cell cdc42 and its downstream effectors to induce actin rearrangement and microbial invasion. methods: we exposed cholangiocytes derived from normal human liver and immortalized by sv40 transformation to freshly excysted cp sporozoites and applied molecular and morphofogical approaches to test our hypothesis. results by immunofluorescent and immunoelectron microscopy, we found accumulation of cdc42 in cholangiocytes at the parasite-host cell interface during cp invasion. we confirmed activation of cdc42 in infected cholangiocytes by immunoprecipitation using an antibody to pak4, a downstream effector molecule that binds exclusively to the activated form of cdc42. phosphatidylinositol 3-kinase (pi-3k), a membrane-associated kinase associated with cdc42 activation, was also recruited to the site of attachment, as were n-wasp, pak4 and p34-arc, downstream effectors of cdc42. inhibition of pi-3k by wortmannin or ly294002 blocked cp-induced cdc42 accumulation. while overexpression in cholangiocytes of a constitutively active mutant of cdc42 promoted cp invasion (up to 50%), overexpression of function-deficient mutants of pi-3k, cdc42 or of the wa fragment of n-wasp inhibited cp invasion by 60 -80 %. moreover, inhibition of host cell cdc42 activation by dominant negative mutation inhibited cp-induced actin accumulation, and parasitophorous vacuole and dense-band formation at the attachment site. conclusions: these combined data suggest that cp invasion of cholangiocytes (and perhaps other target epithelia) results from the organism's ability to activate a complex host cell cdc42 signaling pathway that induces host cell actin remodeling at the attachment site. background: hepatitis b (hbv) and hepatitis c (hcv) infections are a world-wide health concern. studies of these infections have been hampered by a lack of a convenient animal model. we previously have shown that immunocompetent rats tolerized and transplanted with human hepatocytes can support hbv infection for at least 15 weeks. aim: to demonstrate that the immunocompetent rat which has been tolerized and transplanted with human hepatocytes can be used to sustain and study hcv infection. methods: sprague-dawley rats were injected in utero at 16-17 days gestation with one hundred thousand huh 7 cells (human hepatocyte cell line) to tolerized them to human hepatocytes. one week after birth, the tolerized newborns were intrasplenically transplanted with 5 million huh 7 cells. one week after transplantation, rodents were inoculated with one hundred thousand hcv rna copies, genotype l b human serum. animals were sacrificed at 6 and 12 weeks. the presence of human cells was determined by immunofluorescent staining of cryosectioned liver tissue using antibodies to human albumin. pcr and rt-pcr was used to confirm the presence of human albumin in liver tissue. the presence of hcv was assayed using an antibody to ns5a viral protein, and visualized using a rhodamine labeled secondary antibody. hcv infection in the liver was confirmed by the presence of negative strand rna using nested pcr. results: functional huh7 cells in the chimeric livers were confirmed by the presence of human albumin mrna and dna using rt-pcr and pcr. the presence of human albumin protein in the liver was further demonstrated by immunofluorescence of human albumin in frozen liver sections. eighty-one percent (n=57) of the tolerized, huh 7 transplanted rodents were positive for human albumin in the livers. seventy-two percent (n=36) of the chimeric animals infected with hcv were positive for the presence of ns5a hcv protein from immunofluorescence studies. livers of control rats that were only tolerized and control rats that were tolerized and transplanted with huh7 cells, but not inoculated with hcv were negative for ns5a (n=12). hcv viral replication could be demonstrated in livers of tolerized, transplanted and hcv infected rats by the presence of hcv rna bands of the correct size from nested pcr using negative strand specific primers. conclusion immunocompetent rats tolerized and transplanted with human hepatocytes can be a useful laboratory model to study hcv infection. ( we have previously observed that allogeneic hepatocellular transplants are highly immunogenic and are resistant to immunosuppressive therapy with a variety of agents. donor specific transfusion in combination with anti-cd40l mab is highly effective in inducing prolonged survival of skin and myoblasts and inducing indefinite and donor-specific tolerance to heart and islet allografts. the proposed mechanism of action for dst and anti-cd40l mab suggests peripheral deletion of alloreactive cd8+ t cells and induction of a regulatory cd4+ t cell population. resistance to induction of indefinite acceptance has been attributed to peripheral reappearance of alloreactive cd8+ t cells. in preliminary studies, we observed that dst and anti-cd40l mab prolonged fvbln hepatocyte (hc) survival in c57e3l/6 mice to median survival time (mst) of 88 days and induced indefinite acceptance (>90 days) in 43% of mice. hc rejection occurs by cd4-dependent and (cd4-independent) cd8-dependent pathways. this model permits evaluation on these two pathways separately. the current studies address the hypothesis that dst and anti-cd40l mab induces prolonged hc allograft acceptance by inducing immunoregulation of both alloreactive cd4+ and cd8+ t cells. methods: fvbln (halat-fvbin, h-24) hcs were transplanted into cd4 ko, cd8 ko, and c57bl/6 (all h-2b) mice. hc survival was monitored by detection of reporter halat protein in serum by elisa. for comparison, donor-matched islets were transplanted into streptozotocin-induced diabetic cd8 ko (h-2b) mice, and survival was monitored by blood glucose. recipient mice received peritransplant administration of dst ( 1 0~1 0~ fvbln splenocytes, day -7) and anti-cd4ol mab (1.0 mg, ip, d-7, -4, 0, 4) . some cd8 ko hosts were reconstituted cd8+ t cells (2x107 iv, d-10). results: when the cd8-dependent rejection was studied in isolation (cd4 ko mice), dst and anti-cd4ol mab prolonged hc survival to mst of 35 days (n=4) compared to 10 days in untreated cd4 ko (n=7); however, this was not significantly different from anti-cd40l mab alone. when cd4-dependent rejection was studied in isolation (cd8 ko mice), hcs were unexpectedly rejected with mst of 7 days (n=8) despite dst and anti-cd40l mab treatment. in contrast, islets of the same strain were accepted indefinitely in cd8 ko mice (n=5, mst>70 days). since this siraiegy effectively controlled hc rejection in c57bl16 mice which have both cd4-and cd8-dependent pathways available, the collective results suggested that perhaps host cd8+ t cells were necessary for the beneficial effects of the dst and anti-cd40l mab. to determine whether cd8+ t cells were required for induction of longterm hc survival with dst and anti-cd40l mab, cd8 ko hc recipients were reconstituted with cd8+ t cells prior to dst and anti-cd4ol mab treatment. cd8reconstituted cd8 kos accepted hcs for mst of 56 days. conclusion: the effects of dst and anti-cd40l mab on combined cd4-and cd8-initated hc rejection are more pronounced than on either pathway alone. cd8+ t cells appear to be required for induction of longterm hc survival under cover dst and anti-cd40l mab, which offers an apparently distinct mechanism compared to the existing paradigm for the mechanism of action of dst and anti-cd40l mab. engraftment of transplanted cells in the liver is affected by cellcell interactions involving hepatic endothelial cells and kupffer cells. the proximity of transplanted cells to hsc suggested that hsc could promote cell adhesion and extracellular matrix remodeling during cell engraftment. to investigate cell-cell interactions in the liver, we analyzed hsc activation in dppn-rats transplanted intrasplenically with f344 hepatocytes. analysis of tissues by immunostaining from animals 6h, 24h, 3d and 7d after cell transplantation showed appearance of desmin +ve hsc in periportal areas, reaching a peak on day 3 (4-10 fold increase in desmin f v e hsc in cell recipients, p<o.ool). using combined immunostaining and dppiv histochemistry, desmin +ve hsc were often observed in the immediate vicinity of transplanted cells. however, a-smooth muscle actin (sma) was not expressed in hsc perturbed by cell transplantation, whereas ccl&reatment in control animals induced sma expression in hsc, as shown by tissue immunostaining, as well as western blots of tissue extracts. after cell transplantation, perturbed hsc expressed desmin extensively, especially in areas with cell transplantation-induced ischemia and consequential ggt expression. to examine whether desmin +ve hsc were perturbed following activation of kupffer cells by cell transplantation, we administered carbon particles to hepatocyte recipients intrasplenically. these studies established that the majority (up to 72%) of desmin +ve hsc were located directly adjacent to carbon containing activated kupffer cells. as kupffer cells are activated within 6 hrs after cell transplantation, whereas hsc were perturbed maximally at a later time, we reasoned that these cell-cell interactions were likely mediated by soluble factors. to demonstrate whether tnf-alpha plays a role in the stellate cell activation process, we transplanted hepatocytes into animals treated with the tnf-alpha-blocker etanercept. however, etanercept did not prevent perturbation of hsc, suggesting that factors other than tnf-alpha must be involved. to eliminate the component of ischemia-mediated cell activations, we treated animals with intrasplenic nitroglycerine infusion before and during cell transplantation, which resulted in marked attenuation of hepatic ggt expression. interestingly, desmin +ve hsc were now distributed throughout the entire liver lobule, with 3-5 fold greater prevalence, compared to rats treated with cells alone without nitroglycerine, p<o.ool, suggesting that circulating soluble factors reached hsc more effectively. in contrast, treatment of animals with nitroglycerine alone did not perturb hsc. to determine effects on cell integration, bile canalicular reconstitution was analyzed with dppiv and atpase co-stainings. nitroglycerine pretreatment resulted in superior parenchymal integration of transplanted cells, in proximity with desmin +ve hsc. conclusions: transplantation of cells in liver sinusoids activates desmin, but not sma, expression in hsc. activated kupffer cells are likely mediators of hsc perturbation. improved cell engraftment following perturbation of hsc indicates that extracellular matrix remodeling could facilitate this process. these findings offer ways to further optimize liver repopulation with transplanted cells. knudsen, timothy b hummer, kim a buffers, kiyoshi ogata, cody a koch, jefiey l platt, mayo clinic, rochester, m n background hepatocyte transplantation has been of much interest as a biological liver support. how to treat hepatocytes to achieve optimum function has been uncertain. cultured, cryopreserved and hypothermically preserved hepatocytes have been employed for clinical hepatocyte transplantation, but the efficacy was rarely compared between those cell sources. using a xenogeneic hepatocyte transplantation model, the function of transplanted hepatocytes was quantified in vivo. methods: porcine hepatocytes were isolated by a two-step collagenase technique. the hepatocytes were: (1) immediately used; (2) kept in uw solution at 4°c; (3) cultured in supplemented williams e medium; or (4) cryopreserved in ljw solution with 10% dmso before use. the viability of hepatocytes was determined by trypan blue exclusion, and 1 x lo6 viable hepatocytes were injected into the spleens of rag-2-defficient mice. to evaluate the function of transplanted hepatocytes, the porcine albumin level in the recipient plasma was determined by a sandwich elisa. the number of surviving porcine cells in the recipients' spleen was estimated by a quantification of porcine genomic dna sequences. results: the viability of hepatocytes was as follows: freshly isolated hepatocytes, 92.2 2 1.9%; 24 hours, 48 hours, and 72 hours at 4"c, 85.8 t 3.1, 74.3 i 1.9, 71.0 i 3.2%; cryopreserved hepatocytes after thaw, 65.0 2 2.6%; cultured hepatocytes after dissociation, 78.4 t 6.3% (means t se). after transplanting comparable numbers of viable hepatocytes, the porcine albumin concentration was found to be lower in recipients of hypothermically-stored (group 2) and cultured hepatocytes (group 3) than recipients of fresh hepatocytes (group 1). cryopreserved hepatocytes (group 4) failed to secrete any albumin. because the half-life of porcine albumin in rag-2deficient mice was 13.5 hours, the porcine albumin concentration in the recipient plasma reflected real-time albumin secretion from the transplanted hepatocytes. the porcine genomic dna in the recipient spleen was quantified 4 weeks after transplantation. fewer cells were found in the hypothermically preserved hepatocyte-recipients (group 2). therefore, the lower level of the porcine albumin secretion in the group 2 was mainly attributable to the failure of the transplanted hepatocytes to survive. the amount of porcine genomic dna was comparable between cultured hepatocyte-recipients and freshly isolated hepatocyte-recipients, although the plasma of cultured hepatocyte-recipients included less porcine albumin. it suggested that the hepatocytes had dedifferentiated during culture and secreted less albumin thereafter in vivo. the cultured hepatocytes did not seem to differentiate again in murine spleen. conclusions: both the survival and differentiation of hepatocytes were important to maintenance of good function in vivo. the hypothermically preserved and cryopreserved hepatocytes did not survive long after transplantation contrary to their good viable look. cultured hepatocytes showed relatively good survival, although their dedifferentiation was not reversible resulting in poor outcome. freshly isolated hepatocytes are recommended for cell transplantation. medicine, ube-yamaguchi, japan; hiroshi nishina, university of tokyo, tokyo, japan; kiwamu okita, yamaguchi university school of medicine, ube-yamaguchi, japan obiective; we had developed an in vivo model to monitor the trans-differentiation and proliferation of bmcs into functional hepatocytes via hepatoblast phenotype (gfpicc14 model). in this model, transplanted gfp-positive bmcs via tail vein efficiently migrated to the peri-portal area of liver lobules under cc14induced chronic liver damage condition. transplanted bmcs gradually proliferated and spread from the peri-portal area (zone 1) into the central area (zone 2). under persistent liver damage condition, bmcs trans-differentiated into functional hepatocytes. previous analysis had shown that inflammatory signals had been important for the trans-differentiatin of bmcs. in this study, we focused on growth factors and analyzed which affected the transdifferentiation and proliferation of bmcs into hepatocytes in gfpl cc14 model. materials and methods; immunohistochemical staining and immunofluorescent double staining with antibody of gfp, several growth factors and their receptors were performed in gfpiccl4 model. hepatocyte growth factor (hgf), epidermal growth factor (egf), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), and transforming growth factor (tgf) were analyzed. the percentage of stained area was quantified in the sections at thirty random fields (n=6 in each group), and statistical analysis was performed using plsd test (anova). results; the most significant result was shown in fibroblast growth factors (fgfi, fgf2) and their receptors (flg, bek). in immunohistochemical staining, the stained region migrated and increased from zone1 into zone2 of liver lobules with the time after bmt. in immunofluorescent double staining, co-expressions both gfp and fgfs, fgf-receptors were detected at the same distribution. the percentage of stained area of fgfs and fgfreceptors gradually increased with significant difference (the data was shown in table 1 ). although expressions of other growth factors and their receptors were detected, the significant differences were not shown. conclusions; fgfs are the most important growth factor to transdifferentiate and proliferate bmcs into hepatocytes. cholangiocytes are the target cells of chronic cholestatic liver diseases (i.e., cholangiopathies), characterized by the dysregulation of the balance between apoptosis and proliferation that leads to changes in ductal bile secretion. in rats with bile duct ligation (bdl), there is increased cholangiocyte proliferation and enhanced basal and secretin-stimulated ductal secretion. we have shown that both cholinergic (by vagotomy) and adrenergic b y administration of a single intraportal injection of 6-hydroxydopamine (60hda)i denervation impairs the proliferative and secretory responses of intrahepatic bile ducts to bdl through an increase in cholangiocyte apoptosis. we have shown that bile acids and nerves co-ordinately regulate the balance between cholangiocyte apoptosislproliieration in hyperplastic rat livers. in bdl rats, while ursodeoxycholate and tauroursodeoxycholate feeding prevents the loss of bile ducts caused by vagotomy in a caz+/pkc-dependent manner, taurocholate feeding prevents vagotomy-induced duct damage in a pi3k dependent manner. no information exists regarding the interaction between bile acids and adrenergic innervation in the regulation of cholangiocyte function. thus, we tested the hypothesis that taurocholate feeding protects from bile duct damage induced by adrenergic denervation by 6-ohda. methods: immediately after bdl, rats received a single intraportal injection of 6-ohda (which induces degeneration of adrenergic terminal fibers, 50 mglkglbody weight) or vehicle, and subsequently were fed 1% taurocholate or control diet in the presence of daily injections of wortmannin (a pi3k inhibitor, 0.7 mgl kg body weight) or a control solution. we used 1 week bdl rats as control animals. seven days later, we evaluated cholangiocyte apoptosis by : (i) tunel assay in liver sections; and (ii) measurement of caspase 3 activity and protein expression for cleaved pro-caspase 3, and the number of purified cholangiocytes positive for annexin-v. cholangiocyte proliferation was evaluated by measurement of the number of pcna-and ck-19-positive cholangiocytes in liver sections, and pcna immunoblots in pure isolated cholangiocytes. the changes in ductal functional activity were evaluated by measurement of (i) secretinstimulated camp levels and cl-ihcos exchanger activity in pure cholangiocytes; and (ii) secretin-stimulated bile flow and bicarbonate secretion in bile fistula rats. results: taurocholate feeding prevented the increase in the number of apoptotic cholangiocytes and the enhancement of caspase 3 activity and protein expression for cleaved pro-caspase 3 induced by 6-ohda. taurocholate feeding prevented 6-ohda-induced decrease in the number of pcna-and ck-19 positive cholangiocytes, and the loss of pcna protein expression. at the functional level, taurocholate feeding prevented the decrease in secretin-stimulated camp levels, c1-l hco3 exchanger activity and bile and bicarbonate secretion. consistent with the concept that pi3k regulates the interaction of bile acids with adrenergic nerves in cholangiocytes, we found that administration of wortmannin blocks 6-ohda-induced activation of cholangiocyte apoptosis, and inhibition of cholangiocyte proliferation and ductal secretion. summarylconclusion: taurocholate feeding prevents the increase in cholangiocyte apoptosis and the loss of cholangiocyte proliferation and ductal functional activity induced by adrenergic denervation by 6-ohda. taurocholate effects are mediated by the pi3k pathway, since the simultaneous administration of wortmannin reverses such effects. these novel findings suggest that bile acids play a major role in sustaining the growth and functional activity of the intrahepatic biliary tree following liver transplantation. hepatocyte apoptosis by death receptors is emerging as a critical determinant in the initiation of hepatic inflammation and fibrosis during cholestatic liver diseases. however, the cellular sources of death ligands to activate the death receptors is unknown. we postulated that kupffer cells, which contribute to hepatic inflammation, may also contribute to hepatocyte apoptosis and liver fibrosis by generating death ligands. thus, our aim was to ascertain whether kupffer cells express death ligands and contribute to hepatocyte apoptosis and liver fibrosis in the bile duct ligated mouse, an animal model of cholestasis. methods: mice were subjected to bile duct ligation (bdl) or a sham operation and treated with gadolinium chloride, a kupffer cell intoxicant, or saline for three days. hepatocyte apoptosis in liver sections was assessed using the tunel assay. serum alt values were quantitated using commercially available kits. kupffer cells were isolated using arabinogalactan gradient centrifugation. real time pcr was used to measure expression of death ligands and fibrosis indicators. results: hepatocyte tunel positive cells, and serum alt values were 3.3-fold, and 5.2-fold, respectively, greater in 3 day-(bdl) saline-treated vs. gadolinium chloride-treated mice. kupffer cell expression of the death ligands tnf-alpha and fas ligand was 14 and 20-fold greater when kupffer cells were obtained from bdl vs. sham operated mice. the enhanced expression of fas ligand was verified by immunoblot analysis and tnf-alpha by an elisa. these data confirm that kupffer cells generate death ligands and contribute to hepatocyte apoptosis in cholestatic liver injury. consistent with a role for kupffer cellassociated hepatocyte apoptosis in liver inflammation, neutrophil infiltration into the bdl liver was abrogated by gadolinium chloride. hepatic mrna transcripts for a -smooth muscle actin, and collagen a 1(i), markers for stellate activation were all significantly attenuated gadolinium chloride vs. vehicle-treated animals. finally, the mechanisms for kupffer cell activation were explored in cell culture. isolated kupffer cells phagocytosed apoptotic bodies, which induced death ligand expression. in contrast, bile acids had no effect on kupffer cell gene expression. infection is associated with vigorous, durable cd8+ t cell responses against nonstructural hcv proteins. hcv persistence has been attributed to poor priming of cellular immune responses. one of the factors that contributes to poor priming of cellular immune responses may be the noncytopathic nature of hcv and to the subsequent absence or scarcity of virus-infected apoptotic or dead cells as source of exogenous antigens for crosspresentation. because crosspriming contributes to the induction of cd8+ t cells in vivo (nature 1999; 39877-80), we hypothesized that cross-priming with dendritic cells (dcs) containing self-replicating rna might be useful to induce strong, hcv-specific cellular immune responses. methods: choosing hcv ns3 as a model antigen, we generated a recombinant cytopathic pestivirus self-replicating rna to amplify hcv ns3 in dcs. the principal characteristic of this vector is its ability to replicate in transfected cells which in turn enhances production, processing and presentation of the encoded hcv ns3 antigen. moreover, the availability of cytopathic and noncytopathic forms of the same replicon enabled us to study the immunologic implications of dc apoptosis and antigen reprocessing, leading to crosspriming in vivo. results: the murine dendritic cell lines dc2.4 was transfected with cytopathic and noncytopathic recombinant replicon rna, respectively. hcv ns3 expression was detectable in more than 95% of the transfected cells by immunofluorescence. the time kinetics of apoptosis induction was monitored by flow cytometry using annexin v and propidium iodide staining. in contrast to the noncytopathic replicon, the cytopathic replicon led to dc apoptosis 12 hours after transfection. dc2.4 transfected with the cytopathic recombinant replicon rna were then used to immunize hla-a2 transgenic mice subcutaneously. ifn-positive, proliferative cd4+ t cells and ifn--y positive, cytotoxic cd8+ t cell responses were detectable after a single subcutaneous immunization with replicon-transfected dendritic cells and significantly stronger than after conventional, intramuscular dna immunization, the previous gold standard. crosspriming was demonstrated when t cells, primed by injection of h-2b+ dcs into the h-2b+ hla-a2+ mice, were tested with hla-a2+ antigen-presenting cells. transfer of cellular material from the vaccine dcs to the endogenous apcs, a phenomenon that underlies crosspriming, was directly visualized in vivo when fragments of csfe-labeled, injected h2b+ dcs were demonstrated in hla-a2+ host cells in lymph nodes and spleens by flow cytometry and if microscopy. finally, the protective effect and the in vivo function of the induced hcv-specific t cells were evaluated in a surrorgate challenge model with recombinant, hcv ns3 encoding vaccinia virus. whereas lo4 to lo7 pfu were detected in nonvaccinated control mice, no vaccinia virus was detected in mice vaccinated with replicon-transfected dcs, indicating complete protection. summary and conclusion: these findings demonstrate and explain the immunologic potential of the proposed vaccination strategy. moreover, they support the hypothesis that experimental forcing of exogenous antigens into the mhc class i pathway and subsequent promotion of cross-priming is particularly important to generate t cell responses against noncytopathic and tissue tropic viruses such as hcv. disclosures: introduction: hepatitis c virus (hcv) infection leads to chronic liver disease in about 85% of infected individuals while only a minority achieves spontaneous viral elimination in the initial phase of acute hepatitis c. anti viral mechanisms clearing the infection in these patients, however, could contribute to the genera-tion of therapeutic or prophylactic vaccines. the strong association of a broad, hla restricted, vigorous and long lived anti viral cd4+/ th1 t-cell response with spontaneously resolving acute hepatitis c has been demonstarted in the past. only few epitopes recognized by anti hcv specific cd4+ t cells that are associated with spontaneous viral clearance have been characterized so far. the aim of the present study was to identify and characterize hcv specific cd4+ t cell epitopes and their hla restriction during acute self limited hcv infection andlor viral control. methods: proliferative cd4+ t cell responses (3h-thymidin incorporation) against viral proteins (hcv-ns3, -ns4) and against overlapping 20-mer pep-tides covering the entire ns3 and ns4 (aa 1027-1972) region were performed in 25 patients with acute self limited hcv infection and in two patients during temporary viral control (hcv rna negative). ten patients with chronic hcv infection served as control group. cd4+ t cell clones were generated by limit-ing dilution of hcv protein specific t cell lines. hcv specific cd4+ t cell clones were further characterized fine specificity was assessed by proliferation assay after stimulation with overlapping peptides of the corresponding protein region, then the minimal epitope was defined with n-and c-terminal truncated peptides for each epitope. hla restriction was assessed by inhibition experi-ments (anti-dp, -dq, -dr) and cd25+ expression in the presence of a panel of ebv blasts by flowcytometry. results: we studied 25 patients with acute self limited hcv infection. all pa-tients displayed significant proliferative t cell responses against hcv-ns3 or -ns4 protein, that was strongly associated with self-limited disease and absence of hcv rna. proliferative responses against 20-mer peptides showed multiple responses within the ns3 and ns4 region, possibly reflecting multiple epitopes in this region. in contrast no virus specific t cell responses were detectable in a control group of patients with chronic hcv. for further characterization of t cell epitopes hcv specific t cell clones were generated from 6 patients against eight different hcv epitopes located within the ns3 (aa1027-1657, two epi-topes) and ns4alb (aa1658-1972, six epitopes) region. the minimal epitopes consist of 8 to 12 amino acids. all t cell clones were hla class i1 restricted and could be stimulated by protein or peptide pulsed ebv blasts expressing the cor-responding hla-dr or -dq. conclusion: the hcv ns3 and ns4 proteins contain several immunodominant cd4+ t cell epitopes which are associated with spontaneous viral clearance. the detailed characterization of minimal epitopes and hla restriction are a pre-requisite for the synthesis of hla class i1 tetramers and for the development of prophylactic and therapeutic t cell vaccines. background a brief period of tissue ischemia followed by reperfusion renders tissue resistant against subsequent prolonged ischemia and reperfusion, a phenomenon called ischemic preconditioning. hepatic ischemic preconditioning protects sinusoidal endothelial cells against subsequent cold storagelwarm reperfusion injury and improves graft survival after liver transplantation in rats. aim ischemic preconditioning also reduces liver injury after warm ischemia and reperfusion, in which hepatocyte damage and kupffer cell activation are dominant features. however, it is incompletely elucidated which cell type ischemic preconditioning affects and how it works. therefore, our aim was to investigate the mechanisms of ischemic preconditioning against warm ischemia and reperfusion injury. method male sprague-dawley rats were injected intravenously with 20 mglkg gadolinium chloride (gdcls), 10000 ulkg superoxide dismutase (sod) or 300 mglkg n-acethyl-l-cyctein (nac) to suppress kupffer cells, superoxide formation or oxygen radical formation, respectively. livers were then preconditioned by clamping the hepatic artery and portal vein to the median and left lobes for 10 min followed by 10 min reperfusion. subsequently, livers were subjected to warm ischemialreperfusion injury in in vivo experiments and in liver perfusion experiments as follows. in vivo experiments: the blood flow to the median and left lobes was occluded with a vascular clamp for 40 min. one hour after removal of the clamp, blood was collected for determination of alt activity and hyaluronic acid (ha) concentration. liver perfusion experiments: livers were perfused through the portal vein for 10 min with physiological buffer and stored at 37°c in the same buffer. after 40 min, livers were reperfused with the buffer containing 500 ng/ml ha for 60 min in a recirculating system for determination of alt activity in the perfusate and hepatic ha uptake as a marker of sinusoidal endothelial cell injury. livers of other non-treated rats were preconditioned by perfusion with the buffer containing h202 instead of ischemic preconditioning, and then subjected to warm ischemialreperfusion similarly. in other experiments, livers were subjected to ischemic preconditioning and perfused with the buffer containing 0.5 mglml nitro blue tetrazolium (nbt) for 10 min and fixed with formalin. since nbt reacts with superoxide to form insoluble blue formazan, kupffer cell superoxide formation was evaluated in histological sections. results ischemic preconditioning decreased serum alt activity compared to control (168 ? 34 [mean 5 semi vs 333 ? 52, p < 0.05), but did not change serum ha concentration. pretreatment with gdc13 reversed the effect of ischemic preconditioning (262 2 41), though gdc13 itself did not change alt activity after ischemialreperfusion (318 t_ 33). similar results were obtained in liver perfusion experiments, suggesting that ischemic preconditioning protected hepatocytes, but did not change sinusoidal endothelial cell injury. it is also suggested that kupffer cells mediate hepatocytes protection by ischemic preconditioning. in rats treated with sod or nac, decrease in alt activity in the perfusate by ischbackground: living donor liver transplantation (ldlt) and split livers have emerged as a solution to ease the shortage of liver transplants and have achieved remarkable success in pediatric populations. in adults, ldlt is limited by the size of the graft that can be safely harvested and transplanted. similar limitations apply when extensive liver resections are needed for the treatment of hepatic tumors. we have previously demonstrated that the anti-apoptotic gene a20 is part of the physiologic response of hepatocytes to injury, protecting them from apoptosis and limiting inflammation by inhibiting nf-kb activation. our objective was to check whether a20 expression would help decrease the minimal liver mass required for mice survival. methods and results: two-thirds partial hepatectomy is well tolerated and is an accepted model of liver regeneration in rodents. we performed an extended (78%) liver resection (n=12/group) in non-infected (ni) mice (10-12 week balb/c) and mice infected with recombinant a20 adenovirus (rad.a20) or a control adenovirus (rad.p-gal). surviving mice suffer a delay in regeneration as assessed by the number of proliferation cell nuclear antigen (pcna) positive nuclei 48 h following resection (5-10 positive cellslhpf in mice with 78% liver resection vs. 60-70 positive cells/ hpf in mice with a 2/3 liver resection). interestingly, such a delay was not noted in mice expressing a20 (70-80 pcna positive cells/ hpf were detected 48 h following 78% resection). a20 also prevented the development of coagulopathy as assessed by prothrombin time. we extended our resection to a radical hepatectomy comprising 87% of the liver (n=12lgroup). we observed 100% lethality in ni and rad.p-gal infected mice. expression of a20 resulted in the survival of 60% of the animals and correlated with increased pcna positive hepatocytes as an indicator of increased cell proliferation. a20 mediated increase in hepatocyte proliferation was not merely dependent upon its anti-apoptotic function. expression of a20 in hepatocyte cultures, in a nonapoptotic milieu, increased their proliferative response to serum and hgf as compared to ni and rad.p-gal infected cells. conclusion: these results demonstrate a novel pro-proliferatif function for a20 in hepatocytes and argue for its critical role in accelerating liver regeneration and promoting hepatocyte survival and function, even when facing extreme metabolic demands. gene therapy with a20 may allow for successful transplantation with smaller ldlt, split liver allografts and even hepatocellular grafts. additionally, a20 based therapy to the liver may allow for extensive liver resections in the setting of cancer therapy. employee graeme currie -gilead sciences, inc.: employee samuel didier -gilead sciences, inc.: investigator stefanos hadziyannis -gilead sciences, inc.: investigator craig james -gilead sciences, inc.: employee ching-lung lai -gilead sciences, inc.: investigator nicole lama -gilead sciences, inc.: employee pietro lampertico -gilead sciences, inc.: investigator peter neuhaus -gilead sciences, inc.: investigator eugene schiff -gilead sciences, inc.: investigator norah terrault -gilead sciences, inc.: investigator hans tillmann -gilead sciences, inc.: investigator 24 prolonged survival of porcine hepatocytes in cynomolgus monkeys. h nagata aliberti -no relationships to disclose sven erik behrens -no relationships to disclose vito racanelli -no relationships to disclose barbara rehermann -no relationships to disclose 35 characterization of novel cd4+ t cell epitopes in acute selflimited hcv infection previous studies from our lab show that interleukin-6 (il-6) is a complete biliary epithelial cell (bec) mitogen that is normally produced by bec at low levels and upregulated by mechanical, infectious, or immunologic biliary tract injury. bile duct ligation (bdl) in il-6-deficient (il-6-/-) mice results in a reproducible phenotype that includes rapidly decompensating biliary cirrhosis, which is at least partially attributable to impaired biliary tree integrity. methods and results: oligonucleotide array analysis of primary mouse il-64-and wild type il-6+/+ bec cultures was used to search for possible molecular mechanisms that might account for impaired biliary tree integrity in il-64-mice. the results showed upregulation of trefoil factor family (tff) messenger rna (mrna) in the il-6+/+ bec compared to the il-64-bec. tff are mucin-associated protease-resistant peptides that greatly contribute to barrier and epithelial repair in the gastrointestinal tract. various molecular analyses in vitro using bec cultures confirmed that il-6-mediated gp130 signaling through the signal transducer and activator of transcription 3 (stat3) importantly contributes to tff3 mrna and protein expression. in vivo, tff3 mrna and protein was expressed predominantly in the medium and large bile ducts and peribfiary glands in normal mouse liver in il-6+/+ mice, but weakly present or absent in il-64-mice. within several weeks after bdl, tff3 mrna and protein levels decreased transiently then increased to near baseline levels by twelve (12) weeks after bdl in the il-6+/+. in contrast, the il-64-mice failed to show the secondary recovery of tff3 by 12 weeks when mrna and protein levels were significantly lower than il-6+/+ (figure) . the retarded recovery of tff3 mrna and protein in the il-6-imice was reversed by daily treatment of recombinant il-6. similar results were obtained in analysis of normal human liver and various biliary tract diseases. conclusions: tff3 is the predominant trefoil factor expressed in the mouse and human biliary tree; its expression appears to be upregulated by stat3 signaling and suppressed by the mitogen-activated protein kinases (mapk) signaling in the bec. tff expression in the biliary tree importantly contributes to biliary tree integrity and repair reactions. blackard, carolynne zander, massachusetts general hospital, harvard medical school, boston, ma; gregory beck, university of massachusetts, boston, ma; emmett v schmidt, raymond t chung, massachusetts general hospital, harvard medical school, boston, ma backgroundlaims: the innate immune response (type i ifn system) is postulated to play a critical role in control of viral infection.the role of the ifn system in controlling hcv replication has been inferred but not proven. in addition, hcv protein expression has been shown to antagonize the ifn response in vitro. until recently, it has been impossible to assess the interaction between hcv and the type i ifn system in vivo without a model that successfully supports viral replication. we have developed a cell-based binary hcv replication model that successfully recapitulates the early stages of the hcv life cycle (pnas 98 9847,2001) . we therefore used this system (1) to assess the role of type i ifn in controlling hcv replication; and (2) to assess the effect of hcv expression or replication on ifn-induced intracellular signaling. methods: full-length hcv cdna corresponding to the ph77 strain previously shown to be infectious in the presence of ?7 was transfected into huh-t7 cells (a t7 stably-transfected huh7 be), as well as mutant m g h human fibroblast cell lines null for the ifn signaling proteins tyk2 (ul), irf9 (u2), statl (u3), jakl (u4), and stat2 (us) respectively. to test the effects of known ifn antagoflists on hcv replication, the ifn signaling antagonist sendai virus y2 protein was cotransfected into huh-t7 cells. in addition, neutralizing antibodies (nab) to type i ifn were added to selected huh-ti cells. to study the effect of hcv on ifn signaling, huh-ti cells were cotransfected with the reporter plasmids pisre-luc and prl-tk. ifna (1000 iu/ml) was added to selected cells after transfection. reporter gene expression was monitored by the dual-luciferase reporter assay system and measured as relative luciferase activity. hcv core protein was measured using the trak-c hcv core ag elisa. results ifna significantly reduced hcv core ag production from 203.5 pg/ml (ph77+/ifn-) to 41.3 pglml (ph77+/ifn t) (p=o.ool). in contrast, y2 protein enhanced hcv core ag production to 407.6 pglml in ph771y2 cotransfected cells (p=o.oool). control experiments confirmed that y2 protein inhibited ifna-induced isre-mediated signaling in huh-t7 cells; relative luciferase activity was reduced from 653 (ph771 ifnp nab increased hcv core ag replication by 42% and 23% compared to no treatment (p=o.o1). the combination of anti-1fna nab and anti-ifnp nab significantly enhanced hcv core ag production by 73% (p=o.004). the u3 cell line enhanced hcv core ag replication by over two-fold compared to the parental m g h and other mutant cell lines. hcv (ph77) transfection suppressed ifn-induced relative luciferase activity by 50% compared to ifn-treated pgem transfected cells (p= 0.w). transfection with a replication defective h77 mutant, subgenomic hcv core, or hcv structural protein mutant constructs had effects on suppression of isre-luc similar to h77.conclusions: using a binary, full length hcv replication model, we found that (i) hcv replication is enhanced in the presence of the ifn antagonist y2 as well as by ifn neutralizing antibodies, confirming the criticality of the type i ifn system in innate control of hcv replication.(2) statl null cell lines enhanced hcv replication, indicating that this protein is the critical signaling component in innate antiviral immunity to hcv. (3) hcv expression inhibits ifn signaling. hcv protein expression and specifically hcv core expression appears to be su€ficient to suppress ifn signaling. these findings demonstrate that the innate immune response exerts direct antiviral effects on hcv, and that hcv protein expression subverts type i ifn signaling. our replication model has the potential to provide new insights into the mechanisms of host control of hcv replication as well as the mechanism of hcv persistence. disclosures:gregory beck -no relationships to disclose jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose yoshitaka kamegaya -no relationships to disclose wenyu lin -no relationships to disclose emmett v schmidt -no relationships to disclose carolynne zander -no relationships to disclose ifna) to 21.2 (ph77/ ifndy2) (p=o.o001). anti-ifna nab and anti-background cytotoxic t lymphocyte activator 4 downregulates t cell activation by ligating b7. single nucleotide polymorphisms (snps) at positions -318 in the promoter and +49 in exon 1 of the ctla4 gene have been associated with an increased risk for autoimmune diseases. the +49g allele has also been associated with a greater likelihood of response to interferon-alpha in patients treated for chronic hepatitis c. since some of the ctla4 snps either individually or linked together (haplotypes) may alter ctla4 expression or activity, we hypothesized that they may be associated with natural hepatitis c virus (hcv) clearance or persistence. methods: utilizing a nested case-control study design from an injection drug using cohort and a hemophiliac cohorts, we matched subjects with hcv clearance (anti-hcv positive and hcv rna negative on two occasions separated by a minimum of 6 months) to two persistently hcv-infected individuals (hcv rna positive on two occasions) based on h n status, race, and gender. five ctla4 snps, which define all common haplotypes, were genotyped using either sequence specific primers or a single base extension method. conditional logistic regression was used to determine odds ratios and p-values for these snps. haplotypes were constructed using the phase program and analyzed using the snpem program. results: included in this study were 190 and 370 subjects who either cleared or had a persistent infection with hcv, respectively. the study cohort was 47% black, 46% white, and 7% other. blacks who were homozygous for the mutant allele 49g were more likely to experience viral clearance (or 0.45, p=0.03). this association was not found in white subjects (or 0.92, p=0.81). since, hla-cw"o4 is associated with viral persistence in these cohorts (thio et a1 j virol2002), we stratified the 49g homozygous individuals by c w w . both blacks and whites who did not have cww4 were more likely to have viral clearance if they were homozygous for 49g (or 0.45, p=0.13 and or 0.49, p=o.11, respectively) . stratification by other hla alleles associated with hcv clearance or persistence did not reveal additional relationships. the 49g formed haplotypes with the wild type alleles at three of the four other snps examined, and those haplotypes did not have a stronger association than 49g alone. 49g was found in haplotypes with both the mutant and wild type alleles at the -1722 promoter position, but neither of these haplotypes had a stronger association with clearance. conclusions: homozygosity for the ctla4 49g snp is associated with clearance of hcv in blacks regardless of hla genotype, whereas in whites, homozygosity at this position trends toward an association with clearance in hla-cw*m-negative persons. since ctla4 49g has been reported to augment t ceil activation by decreasing ctla4 expression, these results point to the importance of t cell activation in hcv clearance. disclosures:jacquie astemborski -no relationships to disclose james g goedert -no relationships to disclose recurrence of hcv infection post liver transplant is universal. high levels of viraemia characterize the post transplant course. in this setting, acute rejection in association with hcv re-infection of the graft (ar (hcv)) and recurrent chronic hepatitis (chi) are recognized clinical entities. we aimed to 1) determine the intrahepatic gene profile in ar (hcv) and chi. 2) examine whether the gene profile in ar (hcv) is different to that of acute rejection in non-hcv infected allografts (ar non-hcv). 3) examine whether the gene profile of chi is different to chronic hepatitis c pre-transplant (ch). methods: genechip arrays were utilized to study pooled rna from 34 liver tissue; 6 patients with ar (hcv), 6 patients with ar (non-hcv), 8 patients with chi, 8 patients with ch and 6 normal livers. probe level data was analysed using the bioconductor affy library and genes with more than 2 fold changes were considered to be upregulated. results: ar (hcv) us ar (non-hcv): compared to normal tissue, both groups were similarly characterized by upregulation of several t cell dependent immune activation genes such as the lymphocyte antigen campath-1, mac-2 binding protein and ifn related genes. however, compared to ar (non-hcv), ar (hcv) was specifically characterized by upregulation of several ifn-alpha associated genes including 2-50as, and p27. chi vs c h compared to normal, both groups were characterized by upregulation of genes involved in antigen presentation (proteasome subunit 42, mdw), t cell activation (t cell receptor and ctl-17), ifn-gamma inducible genes (ifn-56kd, ip-10, humig and class i1 mhc) and ifn-alpha associated genes (p27 and 2-5 oas). however, the ifn-alpha and gamma genes were more upregulated in chi compared to ch. furthermore, compared to ch, the chi group displayed increased expression of genes involved in cellular proliferation (pcna, cyclin, beta integrin), apoptosis (cytochrome c, fas-l, caspase 4), inflammation (macrophage mannose receptor, complement factor h) and fibrosis (angiotensin i1 receptor, proteoglycan core protein). conclusions: at the transcriptome level, the pathological process in hcv infected allografts with acute rejection or recurrent chronic hepatitis is characterized by upregulation of several ifn-alpha and gamma related genes compared to other livers. moreover, chronic hcv post transplant is further characterized by an increased process of cellular proliferation, apoptosis and fibrosis compared to chronic hepatitis in the pretransplant setting. collectively the data suggest that the high levels of viraemia which characterize the post transplant setting drive the immune response to act at a higher level. in addition, the upregulation of several interferon related antiviral genes supports the notion that hcv is a strong inducer of intrahepatic interferons, but is relatively resistant to their antiviral activity. (hcv) infection is thought to be mediated by a multispecific immune response. hcv-specific t cells have been described in hcv-seronegative virus-exposed individuals such as i.v.-drug addicts, family members of chronic hcv-patients and lab-personnel. however, it is not known whether these individuals had recovered from previous asymptomatic acute hepatitis c. in this study, we prospectively followed 10 individuals who experienced an injury with an hcv-contaminated needle. methods: between january 2001 and march 2003 we collected pbmc from 10 individuals (medical health professionals at our institution; 3 females, 7 males; age 30-45 years) who experienced an injury with an hcv contaminated needle. blood samples were taken on the day or the day after the event and at different time points during follow-up for up to 12 months. low levels of hcv-rna were examined in serum by the highly sensitive transcription-mediated tma-assay (detection limit 5-50 iuiml) and in rna of pbmc. t cell-cytokine secretion (elispot-assays for ifn-gamma and il-10, flow-cytometry-based ifn-g capture assays), t cell-proliferation (3-thymidin incorporation, cfse-staining), and t cell-cytotoxicity (51-chromium release assays) were investigated directly ex vivo and in t cell lines. results: none of the individuals became positive for hcv-rna in serum (tma-assay) or pbmc and all of them remained anti-hcvnegative throughout follow-up. at the time of the needle stick injury, hcv-specific cd4+ t cell responses were already detectable in 2 of the individuals and became detectable thereafter in 2 additional persons. hcv-specific cd8+ t cell responses could be investigated in one hla-a2-positive individual in more detail at several time points. he was negative for hcv-specific interferon gamma-and il-10-producing cells on the day of the injury as determined by elispot assays. however, after 18 weeks, we detected for core-178-specific ifn-gamma producing cd8+ t cells. out of 7 mhc-class i-restricted hcv epitopes tested, one additional peptide (ns3-1406) became positive 8 weeks later. the presence of core-178 and ns3-1406-specific cd8+ t cells was confirmed by a second flow-cytometry-based assay. hcv-specific inf-gamma positive cells were cd8-dim and hla-dr-negative. the frequency of ns3-1406 and core-178-specific cd8+ t cells was about 114500 and 118500 pbmc, respectively, in the elispot assay and remained constant in this subject until month 11 of followup. the increase of cd8+ t cell responses was accompanied by the development of an hcv-specific cd4+ response targeting predominantly the hcv-core and hcv-helicase antigens. conclusions: we here demonstrate in a prospective study the development of hcv-specific t cells in hcv-exposed individuals after needle stick i n j q in the absence of viremia. surprisingly, these hcvspecific t cells became detectable rather late after the potential exposure. our data are in line with previous studies demonstrating hcvspecific t cell responses in chimpanzees inoculated with subinfectious doses of hcv. t cell immunity in medical health professionals against hcv may contribute to the low prevalence of hcv among doctors and nurses since hcv prevalence in medical health personnel has been shown to be equal or even lower as compared to the general population in most studies. aims: laparoscopic cholecystectomy is the treatment of choice for symptomatic gallstone disease at present. despite its wide spread application in the past decade, the incidence of iatrogenic bile duct injuries related to this procedure has remained unchanged around 0.3-0.6%. we review the investigation, management and outcomes of such injuries referred to a tertiary referral center in the united kingdom. methods: prospectively collected data on iatrogenic bile duct injuries following elective laparoscopic cholecystectomy referred to our center was analyzed. the strasberg classification was used for defining the types of injury. results: a series of 125 patients were referred for management of iatrogenic bile duct injuries between january 1991 and june 2003.median follow up period was 55 months. 87 patients had type e injuries (to%), followed by 20 with type a (16%) 14 with type d (11%) and 2 each of types b (1.6%) and c (1.6%). an uncomplicated primary operation was described in 74 patients (60%). despite the operation being converted to an open procedure in 27 patients only 18 injuries were recognized at the time of surgery.(l4.4%) although the median time to recognition of biliary tract injury was 2 days (1-21) the median time to referral increased to 10 days (0-99) for a type a injury, and 16 days (0-2200) for a type e injury. a total of 29 patients underwent surgical intervention prior to referral of which 20 had biliary reconstruction. nine of these patients required subsequent revision surgery. after referral 15(13%) patients were managed conservatively while 6(5%) had ercp and biliary stenting and 24(29%) had interventional radiology as the main procedure. surgical intervention was required in 79 patients (53%) at our center following referral. this included t-tube insertion (7), primary common bile duct repair (lo), biliary reconstruction (60) and liver resection (2) with three patients requiring subsequent revision surgery. major associated vascular injuries were present in eight patients. one patient suffers from recurrent cholangitis while five remain asymptomatic on follow up. two required liver resection due to severe ischaemic necrosis. bowel perforations were associated in three patients. both vascular and bowel injuries occurred in patients with type e bile duct injuries. nine patients (7%) had recurrent cholangitis following treatment. three patients developed end-stage liver disease due to secondary biliary cirrhosis with one undergoing orthotopic liver transplantation. there were five deaths related to bile duct injuries. two had associated vascular injuries and one had bowel perforation. a further two patients with type a injuries died following multiorgan failure due to sepsis. conclusions: iatrogenic bile duct injuries are associated with significant morbidity and mortality. delay in recognition and appropriate referral remain a continuing problem. a multi disciplinary approach in a specialized institution has a significantly better long term outcome. background radio-frequency ablation (rfa) is widely used as a treatment of inoperable tumor leasons in the liver. preliminary studies of a vx2 hepatoma model in rabbits showed tumor specific t lymphocytes after rfa treatment and a significantly prolonged survival of these treated animals compared to a control group. aim: the aim of the study was to assess whether the observed tumor specific t cell response after rfa treatment, found in the vx2 tumor model, is transferable to humans. methods: 12 patients were included into a pilot study. 6 patients had metastases of colonic cancer and 6 patients were suffering from hepatocellular carcinoma (hcc) with underlying cirrhosis. all patients were treated with rfa (elektrotom him 106, berchtold, germany).blood samples were taken before and 4 weeks after rfa. an interferon gamma cytokine secretion assay (miltenyi, germany) was carried out on whole blood and measured by a flowcytometer (facs calibur, bd science, germany). as test antigens served autologous liver tissue and tumor tissue received by biopsy. t cells were doublestained with cd 8 antibody to detect cytotoxic t cells. the basic ifn gamma secretion was also measured unstimulated. the activated/non activated cd8 positive t cell ratio (anr) was calculated and analysed statistically with spss. only rfa naive were included. results: before rfa patients had a mean stimulation of 0,022 anr (sd=0,001) against liver tissue and 0,027 anr (sd=0,002) against tumor tissue (see tab.1). 4 weeks after rfa a strong increase of anr was observed with 0,11 anr (hcc) and 0,15 anr (colorectal carcinoma) against tumor tissue. this increase was also detectable 8 weeks after rfa (0,14 anr (sd=0,003) (hcc) and 0,14 anr (0,003) (colorectal carcinoma)) (see fig. 1 and tab.1) . the anr levels were allways low against liver tissue with 0,03 anr (sd=0,002). discussion: rfa shows significant tumor specific t-cell stimulation in patients with primary and secondary tumors of the liver. rfa seems to overcome immune tolerance or presents alterated and/or cryptic tumor antigens, and causes a significant tumor specific cytotoxic tcell response. this effect could be exploided in multimodal antitumoral strategies. the strategy of primary hepatic resection followed by secondary liver transplantation (slt)was not evaluated in-term of operative risks and survival. between 1991-2001, among 88 cirrhotic patients transplanted for hcc with mazzafero's criteria on analysis of the specimen, 18 had liver resection prior to slt : for recurrence (n=11), deterioration of liver function (n=4) or high risk for recurrence (n=3) with a mean time between liver resection and listing for lt was 20 months .the comparison between slt group and patients who underwent primary liver transplantation (plt) showed similar median age (55 vs. 53 years), sex and etiology of liver disease (alcohol/viral biclother). overall time on lt waiting list of the two groups was similar (5 vs. 3 months). pathologic analysis of the specimen revealed similar number (1.7 vs. 1.6) and tumor size (2.3 vs. 2.2 cm). pen-and post-operative course were not different in terms of operative time (551 vs. 530 min), blood loss (1282 vs. 1191 ml), transfusion (3 vs. 2 units), icu (10 vs. 9 days) or hospital stay (32 vs. 31 days), morbidity (56% vs. 51%) or 30-day mortality (5.7% vs. 5.6%). during a median follow-up of 32 months (3 to 158 months), 3 patients recurred after plt and one after slt. after transplantation, 3 and 5-year overall survivals were not different between groups (82 vs. 82% and 59 vs. 61%). results of our study showed that liver resection for hcc prior to transplantation did not increased the morbidity or impair long-term survival following lt. therefore liver resection prior to transplantation can be integrated in the treatment strategy for hcc. aims: ct-1 is a member of the il-6 family of cytokines which protects myocardiocytes against thermal and ischemic insults. in this work we analyzed the expression of ct-1 by normal and diseased liver and whether this cytokine might protect the liver against ischemia-reperfusion injury. methods: rat ct-1 cdna was cloned, recombinat rat ct-1 was produced and used for generation of poly and monoclonal antibodies which were employed in immunochemistry, western blot and elisa. the recombinant cytokine was assayed for therapeutic use in rat models of ischemia-reperfusion injury. wistar rats were subjected to 60 min ischemia of 70% of the liver by clamping the corresponding branches of the hepatic and portal vein. after this period the clamp was released and serum and tissue samples were obtained at 6 hours. rats were divided into three groups: group 1 received 100 mcg ct-1 10 minutes prior to ischemia (n=8), group 2 received saline instead of ct-1 (n=8) and group 3 were sham operated (n=4). results: immunohistochemistry of normal livers demonstrated ct-1 expression selectively in hepatocytes of the centrolobular area. six hours after reperfusion of ischemic livers the values of serum alt were 10 times higher in group 2 than in group 1 (pco.01). severe focal hepatocellular necrosis was found in animals from group 2 while no apparent morpholgical changes were observed in rats from group 1. serum ct-1 levels were raised after reperfusion in untreated animals as compared with sham operated rats. western blot of liver samples showed an increase of ct-1 expression in the non-ischemic liver lobule as compared with the damaged part of the liver. conclusions: ct-1 is a hepatoprotective cytokine produced by hepatocytes in centrolobular areas. ct-1 synthesis increases after ischemic insults. administration of recombinant ct-1 protects very efficiently the liver against ischemia-reperfusion injury. ct-1 may be a valuable therapy to attenuate hepatic damage of the donor liver during orthotopic liver transplantation. disclosures: naiara beraza -no relationships to disclose matilde bustos -no relationships to disclose pun fortes -no relationships to disclose maria ifiiguez -no relationships to disclose eduardo martinez-ans6 -no relationships to disclose jesus prieto -no relationships to disclose cardiotrophin-1 (ct-1) is a potent background: in experimental and clinical acute liver failure, both a loss of cerebrovascular autoregulation and an increase in cerebral blood flow (cbf) have been implicated in the development of brain edema. it is unclear whether the pathogenesis of these two phenomena are linked. the accumulation of glutamine within astrocytes appears to be a critical first step in the development of cerebral hyperemia, as experimental inhibition of glutamine synthetase with methionine sulfoximine (mso) attenuates brain edema and corrects cerebral hyperemia. to study cerebrovascular autoregulation without the confounding variable of brain edema, we examined animals one day after portacaval anastomosis (pca), where a decreased cerebral blood flow has been noted in the setting of a hyperdynamic systemic circulation. material and methods: a pca or sham surgery was performed in male sld rats (300-400 gm). at 24 hrs, mean arterial pressure (map) was measured continuously in anesthetized rats. noradrenaline was infused at a dose of 1 pglkglmin for 60 minutes. mso or saline was given as an intraperitoneal injection (150 mglkg) three hours prior to na infusion. relative changes in cortical blood flow (cbf) were measured with a laser doppler probe placed over the parietal cortex. the animals were mechanically ventilated and arterial blood gases were monitored throughout the experiment. three groups of rats were studied (n= 18). results: the sham group had significantly higher map, in accordance with the systemic vasodilatation seen in pca rats. adding na to all groups significantly raised map and hr. the sham+na group did not exhibit an increase in cbf with a rise in map. the pca+na group had a significant increase in laser doppler flow (ldf) from baseline that coincided with an increase in map. pretreatment of the pca animals with mso prevented the rise in cbf despite a rise in map. there were no significant differences in pc02 among the three groups. as expected, arterial ammonia levels were higher in pca animals and increased further after inhibition of glutamine synthetase in the group that received mso. conclusions: a rise of map in rats after pca results in an increase of cbf, indicating a loss of cerebrovascular autoregulation. the restoration of autoregulation with mso, despite higher arterial ammonia levels, suggests that accumulation of glutamine within the brain is critical to the pathogenesis of altered cerebrovascular regulation in this model. as similar conclusions can be reached regarding the development of cerebral hyperemia in experimental acute liver failure, it appears that the mechanisms responsible for cerebral hyperemia and failed cerebrovascular autoregulation are linked. future studies should focus on the nature of the intracerebral signal that is responsible for the loss of cerebral autoregulation in this model. a decreased intra-hepatic production of nitric oxide (no) participates on the pathogenesis of portal hypertension in cirrhosis. although non-specific no donor substances, such as nitrates, are able to decrease the intra-hepatic vascular resistance to portal blood flow, the use of these vasodilators to treat portal hypertension in cirrhotic patients is limited by the occurrence of side effects (arterial hypotension, headaches etc). aim. to test the effect of a liver-specific no donor (ncx-1000), an ursodeoxycholic acid-derived compound, on both systemic and intra-hepatic hemodynamics in portal hypertensive cirrhotic rats. method. after the development of ascites, cirrhotic rats (cc4-induced) were treated with ncx-1000 (28 mglkg b.w.) or ursodeoxycholic acid (15 mglkg b.w., control group) by gavage once a day for 14 days and, then, used in 1 of 3 studies: 1) in vivo mean arterial pressure (map), portal pressure (pp), and superior mesenteric artery (sma) blood flow measurements before and during progressive blood volume expansion (blood infusion); 2) in situ liver perfusion to obtain doselresponse curves to methoxamine (alpha*-adrenergic agonist) or flowlpressure curves; and 3) cgmp concentration measurement in liver tissue. results. both map and heart rate were similar in both groups, indicating that ncx-1000 had no effect on systemic hemodynamics. pp was also similar in both groups. during blood infusion, ncx-1000 treated rats and control rats showed similar map (p=0.134) and sma blood flow (p=0.449) increases; however, the pp increase observed in control rats was blunted in ncx-1000 treated rats (p=0.015). flowlpressure curves obtained during liver perfusion were similar in both groups; however, livers from ncx-1000-treated rats showed a decreased response to methoxamine (p=0.016) than controls. the concentration of cgmp in ncx-1000-treated livers was significantly higher (p=0.015; ) than in ursodeoxycholic acid-treated livers (27.65 t 7.95 vs. 11.20 2 6.03 fmollmg of protein). conclusion. ncx-1000 improves the portal system adaptability to portal blood flow increase and decreases the intra-hepatic hyper-reactivity to methoxamine observed in cirrhotic rat livers without causing systemic side effects. these beneficial effects are probably due to increase in the intra-hepatic no bioavailability. the mechanisms behind the upregulation of endothelial nitric oxide synthase (enos) in portal hypertensive animals remain unknown. the aim of this study was to investigate the mechanism that initiates enos upregulation in the superior mesenteric artery (sma) in portal hypertension. it has been shown that the vasoconstriction of the sma with the consequent increase in the sma cyclic strain is the earliest event that occurs within hours after portal vein ligation (pvl). thus, we tested the hypothesis that this early vasoconstriction of the sma may initiate enos upregulation in pvl animals. methods: portal hypertension was induced by partial ligation of the portal vein (n=99). in a separate group of rats, mesenteric vasoconstriction was achieved also by renal artery ligation (ral, n=26). corresponding sham-operated rats were used as control groups (n=50). effects of vasoconstriction of the sma in pvl and ral rats were evaluated by measuring perfusion pressure changes in the isolated sma beds in response to methoxamine, nos activity and enos protein expression. data were compared to the corresponding sham group. hemodynamic features were also determined. mean arterial pressure (map), portal pressure ( i " ) and sma blood flow (qsma) were measured by catheterization and pulsed doppler flowmetry. sma vascular resistance was calculated from map, pp and qsma. results: there was a significant increase in the sma vascular resistance in pvl (2.33%0.13 vs 1.2220.03 rnmhgl %flow, k0.05) and ral rats (23220.18 vs 1.18%0.02 mmhgf%flow, p<0.05) compared to their corresponding sham-operated rats, demonstrating the presence of sma vasoconstriction in both pvl and ral groups. the mesenteric vasculature of pvl and ral animals showed hyporeactivity to methoxamine compared to their respective sham groups (p< 0.01). whiie pvl rats were portal hypertensive (p<o.ol), ral rats were not. the sma vascular hyporeactivity of pvl and ral were corrected by l-nmma and while there was not significant difference in enos protein expression nos enzyme activity was significantly higher in the pvl and ral rats compared to the corresponding sham-operated groups (see figure) . conclusions: mesenteric arterial vasoconstriction in itself leads to enos upregulation and therefore, plays a triggering role in the initial increase of enos catalyhc activity in sma of pvl rats. helen hendrickson, vijay shah, mayo clinic, rochester, m n background. diminished enos derived no production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in cirrhotic portal hypertension. unfortunately, prior studies aimed at delivering a constitutively active form of enos (s1179denos) using an adenoviral vector (ads1179denos) were unsuccessful in correcting hepatic vasoconstriction in the bile duct ligated (bdl) rat liver. aim. to better understand the mechanism of s1179denos dysfunction in bdl liver by examining the influence of the enos inhibitory protein caveolin on recombinant s1179denos function in transduced hepatic stellate cells, which do not express enos endogenously but are a prominent cell target of systemically delivered adenoviral vectors. methods. cellular localization of caveolin was determined from fixed sham and bdl liver sections by immunogold electron microscopy. in vitro gene transfer was performed in the lx2 hsc line with ads1179denos and an adenoviral vector encoding caveolin (ad-cav). protein interactions were determined by immunoprecipita-tion and western blot analysis. nos activity was assessed by arginine to citrulline conversion assay. results. caveolin immunogold analysis in bdl liver demonstrated prominent expression not only in liver endothelial cells, but also in stellate cells. western blot analysis from lx2 cell lysates confirmed caveolin protein expression in vitro. in lx2 cells transduced with ads1179denos, immunoprecipitation of caveolin co-precipitated recombinant s1179denos and conversely, immunoprecipitation of s1179denos coprecipitated caveolin. to examine the influence of excess caveolin protein levels on s1179denos, lx2 cells were co-transduced with ads1179deno.s and adcav. co-transduction of adcav and ads1179d promotes the enhanced binding between s1179denos and caveolin as well as a decrease in the activity of recombinant s1179denos by 25% (p<0.05; ads1179denos vs ads1179denos +adcav; n=5). conclusion. these studies demonstrate a functional effect of enhanced caveolin expression and binding with s1179denos in stellate cells and indicate that the lack of function of recombinant s1179denos protein delivered in vivo to cirrhotic liver may be due to inhibition of s1179denos by caveolin-1 in stellate cells. background and aims: the presence of actin-like microfilaments in the neighborhood of sinusoidal endothelial fenestrae (sef) indicates that the cytoskeleton of sinusoidal endothelial cells (sec)s plays an important role in the modulation of sef. we have reported that a potent vasoconstrictor endothelin (et) causes contractions of sef as well as hepatic stellate cells via the eta and etb receptors, leading to the elevations of sinusoidal microvascular resistance supposed to be one the essential factors in the pathogenesis of portal hypertension. rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. the present study aimed to examine how a rho stimulator; lysophophatidic acid (lpa), and a rho inhibitor; y-27632 rho-kinase inhibitor, affect the morphology of sef in vitro. methods: monolayers of cultured sec were obtained by collagenase infusion of a rat liver and then subjected to primary culture for 24 hours. the cells were separated into three groups; control, lpa-treated (lopm), and y-27632-treated (10pm) groups (treatment duration, 30 min). sef morphology was observed by scanning electron microscopy. formation of f-actin stress fibers was observed by confocal microscopy. actin cytoskeleton around sef was investigated by permealizing cells with streptolysin-0 (1.5 u) for 30 min and examined by transmission electron microscopy. rho a and phosphorylated myosin light chain kinase were analyzed by western blotting. rho activation was measured by rhotekin binding assay. results: following lpa treatment, f-actin stress fiber and active rho increased concomitant with a decrease in diameter of sef. however, following y-27632 treatment, f-actin stress fiber and active rho were reduced, concomitant with an increase in diameter of sef. actin cytoskeleton around sef increased in lpa-treated cell, but decreased in y-27632-treated cell. rho a levels were similar in control, lpa-treated, and y-27632treated secs. phosphorylated myosin light chain kinase levels were diminished gradually after 5 min in y-27632-treated secs and increased after 5 min in lpa-treated cells. lpa treatment increased the amount of active rho, compared to control and y-27632 treatment. conclusion: these results indicate that rho may play an essential role in the regulation of contraction and dilation of sef through the actomyosin system. disclosures: analysis of visual-evoked response patterns suggests that gabaergic tone is increased in brain in liver failure and that this is a major cause of hepatic encephalopathy (schafer et al., troenterolow 86:540-545,1984) . however, the precise mechanisms responsible have eluded researchers in this area for the last two decades. studies in brain tissue from both human and experimental animals with liver failure have consistently shown that gaba synthesis, gaba receptors and their associated benzodiazepine modulatory sites are intact and that expression of the subunit proteins of the gaba-a receptor complex are unchanged (zwingmann et al., hevatologv 37420-428,2003; ahboucha et al., neurochem. int., 2003) . on the other hand, there is emerging evidence to suggest that neurosteroids (ns), a novel class of compounds with potent gaba agonist properties are increased in brain in endstage chronic liver failure in humans (ahboucha et al., hepatola 36: 318a, 2002) . in order to further assess this possibility, ns concentrations were measured using a sensitive radioassay in samples of frontal cortex obtained at autopsy from 15 cirrhotic patients who died in hepatic coma compared to an equal number of controls matched for age, gender, and autopsy delay intervals. patient material contained up to 12-fold increases of ns and subsequent analysis by gas chromatography-mass spectrometry showed that the major component ns with positive allosteric properties at the gaba-a receptor was allopregnanolone. patient material did not contain significantly increased concentrations of either gaba or benzodiazepines. in a separate series of experiments, ns concentrations were found elevated up to 2-fold (p<0.02) in brains of rats following end-to-side portacaval anastomosis (pca) and 5.2-fold (p<o.ol) in brains of rats following hepatic devascularization (pca followed by hepatic artery ligation) compared to sham-operated controls. administration of inhibitors of the ns synthesis enzyme 3-hydroxysteroid dehydrogenase (trilostane or indomethacin) resulted in dose-dependent reductions in brain concentrations of allopregnanolone and a concomitant improvement in locomotor activity scores in pca rats. together, these findings offer unequivocal evidence that ns with positive allosteric modulatory properties are generated in brain in both human and experimental liver failure and that these substances contribute to the "increased gabaergic tone" characteristic of hepatic encephalopathy. pharmacological agents aimed at reduction of ns synthesis or modulation of the ns site on the gaba-a receptor complex could afford effective novel therapeutic strategies in the treatment of hepatic encephalopathy [funded by cihr canada] abstract primary hepatocellular carcinoma (hcc) is one of the most common malignancies, ranks fi&h in frequency among all cancers worldwide, and causes over 1 million deaths annually. although a number of oncogenes and alterations in signaling pathways have been identified in hcc, current treatment for this liver tumor is largely ineffective and has poor patient prognosis. one of the potentially curative approaches in hcc therapy is based on interfering with hcc progression by blocking cell division. in regenerating liver, expression of the proliferation-specific forkhead box m l b (foxmlb) transcription factor is reactivated prior to hepatocyte dna replication and its levels are sustained throughout the period of hepatocyte proliferation. deletion of the foxmlb fllfl (loxp targeted) allele using albumin promoterlenhancer driven-cre recombinase (alb-cre) transgene resulted in significant reduction in regenerating hepatocyte proliferation, which was associated with diminished expression of cell-cycle promoting cdc25a and cdc25b phosphatases and increased nuclear levels of cyclin dependent kinase (cdk) inhibitor p21cipl protein. in order to test the hypothesis that foxmlb is required for the development of hcc, we subjected alb-cre foxmlb -/-mice and foxmlb fllfl (control) mice to a diethylnitrosamine (den)lphenobarbital (w) liver tumor induction protocol. our data demonstrated that alb-cre foxmlb -imice are highly resistant to liver tumor formation as evidenced by their failure to develop either hepatic adenomas or hcc following 33 weeks of denlpb tumor induction protocol. in contrast, at 23 weeks following den/pb tumor induction protocol, foxmlb fllfl livers displayed a large number of adenomas that progressed to hcc by 33 weeks following den/pb treatment. the alb-cre foxmlb -iliver displayed enzyme-altered foci as evidenced by protein expression of the glutathione-s-transferase placental isoform. however, alb-cre foxmlb -/hepatocytes failed to develop hyper-proliferative foci, whereas abundant brdu labeling was found in hepatic adenomas and hcc of denlpb treated foxmlb fllfl mice. this reduced dna replication of foxmlb -/-hepatocytes was associated with increased nuclear levels of the cdk inhibitor p27kipl protein. furthermore, foxmlb fllfl tumors expressed high nuclear levels of m-phase promoting cdc25b protein, while undetectable nuclear levels of cdc25b were found in foxmlb -ihepatocytes after den/pb treatment. the cdc25b phosphatase is required for mitosis because it activates the m-phase promoting cyclin b-cdkl complex, and its increased expression is a prognostic marker for a variety of aggressive tumors. consistent with this finding, foxmlb -/-hepatocytes failed to enter mitosis following denlph tumor induction protocol and this was associated with accumulation of large polyploid hepatocytes. these studies demonstrate that foxmlb is required for the proliferative expansion of hepatic tumors, suggesting that foxmlb can be used as a potential target in treatment of patients with hcc. disclosures: robert h costa -no relationships to disclose vladimir v kalinichenko -no relationships to disclose joseph kuechle -no relationships to disclose brian shin -no relationships to disclose xinhe wang -no relationships to disclose yoder yoder -no relationships to disclose key: cord-340325-0oh40b6r authors: witzigmann, dominik; kulkarni, jayesh a.; leung, jerry; chen, sam; cullis, pieter r.; van der meel, roy title: lipid nanoparticle technology for therapeutic gene regulation in the liver date: 2020-07-02 journal: adv drug deliv rev doi: 10.1016/j.addr.2020.06.026 sha: doc_id: 340325 cord_uid: 0oh40b6r hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs s holds great promise as a therapeutic approach. however, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. lipid nanoparticle (lnp) delivery technology is a revolutionary development that has enabled clinical translation of gene (editing) therapies. lnps can deliver sirna, mrna, dna, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. here we discuss the state-of-the-art lnp technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation. "survival rates have improved for almost every disease of every organ in the last few decades, with one notable exception: liver disease" [1] . this statement by the lancet commission clearly illustrates the global burden of liver disorders and the need for more effective therapeutic strategies [2] . the most frequently occurring liver diseases include hepatitis, liver cancer, alcoholic liver disease, fatty liver disease, and hereditary diseases. in addition to direct harmful effects, these diseases can significantly affect the liver's carbohydrate, fat, and protein metabolism. the increase in lifestyle-related incidence rates and the limited therapeutic efficacy of currently available treatments have resulted in substantial drug development efforts targeting the liver [2] . our ability to treat (hepatic) diseases by targeting their genetic background is increasingly becoming a clinical reality owing to the development of nucleic acidbased therapeutics. in contrast to small molecule drugs and biologics which target gene products (i.e. proteins), nucleic acid therapeutics have the potential to therapeutically regulate essentially any gene of interest at the dna or rna level. their versatility in treating inherited or acquired disorders originating in the liver stems from the ability to induce efficient gene silencing (inhibiting pathological/mutant protein production), gene expression (producing therapeutic proteins) or gene editing (correcting dysfunctional/mutated genes). several nucleic acid therapeutics have been approved by the u.s. food and drug administration (fda) and the european medicines agency (ema) with many more in various stages of clinical evaluation. these therapeutics include antisense oligonucleotides (aso) [3] , small interfering rna (sirna) [4, 5] , plasmid dna (pdna) [6, 7] , messenger rna (mrna) [8, 9] , and complexes containing guide rna (grna) as part of gene editing approaches [10, 11] . using nucleic acids therapeutically in vivo is challenging because of their unfavorable physicochemical characteristics, such as negative charge and relatively large size, which prevents their efficient uptake into cells [12] . in addition, nucleic acids are susceptible to degradation by nucleases in the circulation, suffer from rapid renal clearance, and induce immunostimulatory effects via pattern recognition receptors, resulting in adverse effects [13] . therefore, the clinical translation of nucleic acid therapeutics has been dependent on chemical modifications and advanced (nanocarrier) delivery technologies to improve nucleic acids' stability, promote their target tissue accumulation, enable their kupffer cells > hepatocytes. higher lnp doses corresponding to 1 mg/kg mrna shifted expression slightly towards hepatocytes while keeping the same pattern. transfection of all major liver cell types with equal potency was recently demonstrated for lnp-mrna systems (composed of branched-tail 306o i10 ) at a dose of 2 mg rna/kg by hajj et al. [51] . indeed, targeting the right cell type with the right dose is crucial to developing effective therapeutics. it should be noted that the lnp compositions described in these preclinical studies deviate from those used in the clinic (except for mc3-based lnps). systematic studies are therefore needed to improve our fundamental understanding of lnps' in vivo behavior. rigorous control of physicochemical lnp characteristics such as size distribution, zeta potential, and entrapment will be crucial to assess the intrahepatic distribution of a single lnp composition with different payloads. following intravenous injection, liver-resident macrophages, i.e. kupffer cells, are the first hepatic cells to interact with lnps (figure 1) . these phagocytic cells are part of the mononuclear phagocyte system (mps), also known as the reticuloendothelial system (res). they comprise 80% of the entire macrophage population within the body, illustrating their importance in host defense and lnp elimination [52] . [54] . (c) kupffer cell (kc) located within the hepatic sinusoid in close proximity to endothelial cells. adapted with permission from ucsf office of medical education [55] . three major elimination pathways have been described [56] . first, negatively charged lnp systems are recognized by class a scavenger receptors (sr-a) expressed primarily on kupffer cells resulting in rapid clearance [41, 57, 58] . second, mannose-and fucose-type receptors can be leveraged to selectively target lnp systems to kupffer cells. third, lnp opsonization by serum proteins results in mps sequestration. complement factors (e.g. c3b or c1q) and serum opsonins such as fibrinogen can coat lnps with unfavourable characteristics including large size, high surface charge, or lack of pegylation j o u r n a l p r e -p r o o f [59] [60] [61] . several research groups have explored strategies to prevent kupffer cell clearance in order to redirect lnps to hepatocytes. transient kupffer cell depletion using clodronate-loaded liposomes or by knocking out the endocytic caveolin1 gene are efficient methods in a research setting [41, 43] . however, the clinical utility of such approaches is limited. lsecs are located in close proximity to kupffer cells and play important roles in sequestering lnps and restricting access to hepatocytes [62] . many structural and functional features have been elucidated by braet and wisse [54, [63] [64] [65] [66] [67] . lsecs line the hepatic sinusoids and form pores, so-called fenestrations, that are clustered in sieve plates (figure 2) . endothelial fenestrae range from 50 to 200 nm in diameter and differ between species (table 1) . therefore, liver fenestrae physically restrict circulating lnps' access to the perisinusoidal space and thus limit cellular interactions with hepatocytes according to size. several research groups have investigated using pore-opening substances to modulate fenestrae size with limited success [64] . in addition to their structural characteristics, lsecs have high endocytic activity. a number of scavenger receptors, including stabilin-2, can efficiently sequester anionic nanoparticles [68] . hepatocytes, comprising 70-80% of the total liver cell population, are the most relevant hepatic target cell type for nucleic acid therapeutics (figure 1 ). owing to their broad range of functions, hepatocytes play a key pathogenic role in many disorders ( table 2) . hepatocytes are highly differentiated with a sinusoidal (basolateral) membrane towards the blood circulation and an apical membrane towards bile canaliculi. the sinusoidal membrane with its microvilli exhibits surface receptors important for lnp recognition. the most important receptors for lnp-nucleic acid are the low-density lipoprotein receptor (ldlr) and asialoglycoprotein receptor (asgpr) [76] . within a healthy liver, hepatocytes are postmitotic (i.e. nondividing cells) with an average life span of up to 6 months. many factors can alter lnp accumulation and clearance. the following sections detail important (patho)physiological factors affecting intrahepatic lnp distribution. an often-overlooked challenge in hepatic gene therapy is metabolic and cellular liver zonation, a phenomenon that separates various pathways along the porto-central axis of a liver lobule. first, some genetic disorders manifest in periportal or perivenous hepatocytes [110, 111] . second, metabolic zonation can vary among species and during development (infant versus adult). third, different non-parenchymal cell subtypes within the liver microenvironment can affect lnp clearance [45] . all these factors impact lnp development and gene therapy outcomes. figure 1 details the liver microarchitecture and its major metabolic pathways. metabolic liver zonation for glucose homeostasis, urea synthesis, carbohydrates, bile acids, or lipid metabolism has been discussed in several excellent reviews [110] [111] [112] [113] . advancements in omics and single-cell techniques are continuously elucidating new cell subtypes and improve our understanding of liver zonation [45, 110] [118] . these results demonstrate that whole-tissue (entire liver) analysis should be replaced by dissociated single cell-based techniques considering the metabolic liver zonation. liver disease progression results in pathological remodelling including microanatomical or target receptor alterations that could affect nanoparticle delivery and sequestration. firstly, liver infections or metabolic disorders can lead to chronic cell damage and cell activation. this can result in liver fenestrae rearrangement or fibrotic material deposition by activated stellate cells within the perisinusoidal space. thus, lnp transport to hepatocytes is inhibited, as is access to the key target cell for most gene therapies [119] . hepatic inflammatory processes can also enhance hepatic nanoparticle sequestration by kupffer cell activation [116] . secondly, downregulation of surface receptors crucial for lnp binding decreases gene delivery efficiency. for example, two independent studies have demonstrated lower asgpr expression with increasing stage of hcc (according to the barcelona clinic liver cancer staging) [120, 121] . this has serious implications for liver cancer interventions using asgpr-targeting approaches. thirdly, variations in serum proteins, such as apolipoprotein e (apoe), are known to mediate specific lnp binding and might affect efficacy. a recent study investigated the effect of apoe polymorphisms in therapeutic outcomes. diagnostic tools to stratify patients for lnp-based gene therapy therefore offer interesting possibilities [123] . the fundamental lnp design parameters for nucleic acid delivery are based on those established for small molecule liposomal formulations. these parameters include appropriate particle size (for efficient terminal sterile filtration and hepatic delivery), long-term stability in storage, optimized payload release rates to produce a therapeutic effect, robust and scalable manufacturing processes, and efficient entrapment. in applying these requisites to nucleic acid delivery systems, it became obvious that additional lipid components and functionalities were required beyond those used to compose smallmolecule carriers. the very first nucleic acid formulations, containing only phosphatidylcholine and cholesterol, demonstrated that nucleic acid entrapment within a particle was feasible, but the entrapment efficiency was poor [124, 125] . subsequent development of the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane (dotap) showed that ionic interactions between the lipids and payload can dramatically increase entrapment efficiencies and intracellular delivery. toxicity issues, resulting from the cationic lipids' permanent positive charge and non-biodegradable nature, plagued these initial lipoplexlike formulations [126] . through additional formulation development, and manufacturing process optimization, it was determined that lnp systems required four components: ionizable cationic lipids, phospholipids (typically phosphatidylcholine), cholesterol, and peg-lipids. the role of each component, the evolution of the composition, and the manufacturing processes are discussed in the following sections. to date, a vast number of ionizable cationic lipids covering a wide range of structures have been developed (figure 3 ), yet they all share a few aspects: (1) the headgroups contain tertiary amines that become protonated under acidic ph and typically are uncharged (or zwitterionic) at neutral ph; (2) the lipid tails contribute to making the molecule sufficiently hydrophobic to promote incorporation into a nanoparticle during formation; and (3) the protonated lipids generate structures that help elevate propensity for membrane fusion in acidified endosomes following internalization by the target cell. in addition to these similarities, the various lipids' performed functions are essentially identical. as the ph of the environment dictates the headgroup protonation, lnp are prepared in an acidic aqueous buffer (e.g. ph 4) that promotes the charge interaction between the ionizable cationic lipid and the anionic nucleic acid. subsequent buffer exchange into isotonic and ph-neutral buffer generates the final lnp suspension with a near net-neutral surface charge. this uncharged state is critical to preventing immune responses upon intravenous administration and facilitates delivery to hepatocytes [126] . the next function is to maintain a positive charge in the acidified endosome and promote membrane fusion to allow cytosolic delivery of the nucleic acid. this fine balance of positive charge at acidic ph and neutral charge at j o u r n a l p r e -p r o o f physiological ph is the result of substantial efforts towards optimizing the ionizable lipid for in vivo nucleic acid delivery. one of the first tested ionizable lipids, known as dioleyl-dimethylaminopropane (dodma), contained oleyl lipid tails (c18:1) conjugated to the dimethylamino-propyl headgroup through ether linkers. using the molecular shape hypothesis as a guiding principle [127] , the three components of these lipids (headgroup, linker, and tails) were systematically studied to determine optimal characteristics for each. the molecular shape hypothesis describes the macrostructure obtained upon hydration of a lipid with specific geometries. more specifically, lipids containing tails with larger cross-sectional areas than the lipid headgroups result in h ii phases or inverted micelles; comparatively, when the cross-sectional area of the tails is similar to that of the head group (resulting in a cylindrical geometry), the lipids tend to from bilayers. comparing different lipid tail-unsaturation suggested that the linoleyl chains (dlindma) provide optimal particle internalization and potential to generate membrane-destabilizing h ii phases [128] . dlindma with ester bonds resulted in a lipid, dlin-dap, with substantially reduced potency [30] . further studies suggested that degrading the ester bond within the acidified endosome contributed to efficacy loss [129] . simultaneously, a series of headgroup modified lipids were tested, and dlin-kc2-dma was designed with vastly higher potency than dlindap and dlindma (figure 3 ) [30] . further modifications and screening led to the development of dlin-mc3-dma (figure 3 ) [31] , used in the clinical formulation, onpattro ® , and now considered the gold-standard for ionizable cationic lipids. although several screening methods for ionizable lipids have been devised, the critical potency test for hepatic targets was the in vivo model for hepatic gene knocking down; the factor vii (fvii) model provided a modestly high-throughput approach [29] . fvii is a serum protein produced by hepatocytes in the liver and secreted into the blood circulation. its short half-life enables gene silencing assessment on the protein level within a short timeframe. it is important to stress that fvii-knockdown screens specifically identify lnps that target hepatocytes and ignore all other hepatic cell types. lnp containing sirna against murine fvii were intravenously administered over a dose range of 0.001-10 mg sirna per kg body weight and circulating fvii levels were determined by elisa 24 hours later. the metric used to compare formulations was the effective dose required to achieve 50% gene silencing (ed 50 ), and dlin-mc3-dma (mc3) was determined to be the most potent ionizable cationic lipid for lnp-based gene silencing. the potency improvements cover the range of dlindap with an ed 50 of ~20 mg/kg, while that for mc3 was 0.005 mg/kg in mice [30, 31] . further developments focused on lipid biodegradability to reduce potential toxicity, immunogenicity, and other adverse effects [130] . the design parameters for these lipids included high in vivo transfection efficiency, increased ability to be metabolized, and no generation or accumulation of toxic metabolites. one approach incorporates an ester linkage, which can be easily hydrolyzed by intracellular esterases or dlindma [133] , dlin-kc2-dma [30] , and dlin-mc3-dma [31] ; (ii) lipidoids like ckk-e12 [134] and c12-200 [29] ; and (iii) next-generation lipids including the biodegradable molecules l319 [130] , tt3 [135] , and sspalme [136] as well as lipids from proprietary libraries belonging to acuitas (a9) [137] and moderna (l5) [138] . two lnp componentsphospholipids and cholesterolhave generally been seen to promote formulation stability [139] . although that evidence is largely anecdotal in the lnp context, phospholipids such as dspc, with strong bilayer-forming properties and high phase transition temperatures, help increasing membrane rigidity and reducing membrane permeability. while the role of cholesterol remains j o u r n a l p r e -p r o o f largely unclear in the context of nucleic acid delivery systems, cholesterol-deficient particles can sequester cholesterol while in circulation, leading to potentially destabilizing effects [140] . this sequestration process is largely driven by the exchange of cholesterol away from the plasma membrane of peripheral tissues into lipoproteins in circulation followed by equilibration into circulating liposomes. recently, harashima and colleagues studied cholesterol-free lnp-sirna systems (only composed of the ionizable cationic lipid cl15h6, phospholipid, and peg-lipid) and they observed decreased potencies in the presence of serum likely due to particle instability as a result of cholesterol accumulation [141] . two studies suggested that the cholesterol amount typically formulated into an lnp is larger than what can be stably retained in lnps. more recently, it was determined that ~30-40 mol% helper lipid is required to efficiently entrap sirna within lnps, providing additional insight into the role of these helper lipids [27] . the helper lipids serve as a mechanism for spacing out ionizable lipids to achieve a membrane surface charge of approximately +1 per nm 2 (sirna has a surface charge of approximately -1 per nm 2 ). limited information is available on the role of helper lipids for lnp activity. however, some evidence has suggested that the replacement of dspc with dope in lipidoid-based lnps improves mrna delivery in vivo [142] . for lnp-pdna formulations, certain unsaturated phosphatidylcholines (i.e., sopc and dopc) improved the lnp activity over dspc in the presence of fbs in vitro [28] . dope-containing lnp-pdna systems showed best activity in murine serum suggesting a potential role of helper lipids in modifying the lnp surface affinity to distinct apolipoprotein subtypes. an additional role of cholesterol in lnp systems was recently investigated [143] . incorporating oxidized cholesterols such as 20α-oh redirected lnp-mrna systems from hepatocytes to hepatic endothelial cells and kupffer cells. although the mechanism of modifying lnp tropism remained elusive, formation of different protein coronas and/or recognition by scavenger receptors expressed on hepatic res (such as scavenger receptor class b type i as binding site for oxidized ldl) might have resulted in redirection of lnps [144, 145] . the final lnp component, the peg-lipid, is engineered to perform two specific functions. first, peg-lipids incorporate into the emerging nanoparticle during lnp formation. as lnp systems do not contain an aqueous core, peg-lipids reside almost exclusively on the lnp surface, and their concentrations control particle size [146] . both the peg molecular weight as well as the molar percentage of peg-lipid affect the characteristics of lipid-based particles [147] [148] [149] . specifically, as the peg-lipid is increased from 0.25 mol% to 5 mol%, a reduction in lnp size is observed from ~120 nm to 25 nm, but further increases to peg-content do not modify particle size [147, 24] . second, they improve the shelf-stability by creating a steric barrier that extends away from the surface of the lnp, thereby preventing particle aggregation and improving in vivo circulation lifetimes. however, for transfection purposes, peg-lipids have an established inhibitory effect [150, 151] . based on the hypothesized mechanism of lnp function, the nanoparticle requires an intricate balance between stability in storage and circulation, and instability within the cell to support intracellular delivery [152] . diffusible peg-lipids helped stabilize particles while enabling j o u r n a l p r e -p r o o f journal pre-proof intracellular delivery [25, 151] . these lipids are composed of acyl chains that are 14-carbons in length and can dissociate rapidly from the lnp in the circulation [153] . two hours post administration, only 20% of the injected peg-lipid is associated with the lnp. in contrast, peg-lipids with 18-carbon acyl chains, incorporate into the lnp and do not dissociate from the particle in the circulation. at high concentrations, these peg-lipids can contribute to extending circulation half-life (from < 30 minutes for diffusible peglipid to > 2 hours) [23, 153] . however, lnps designed to target hepatic disorders do not require a prolonged circulation lifetime due to the liver's natural ability to sequester nanoparticles. therefore, diffusible peg-lipids are ideal for such applications. lnp production methods have evolved over time with certain processes gaining prominence. rapidmixing methods have gained favor for their decreased labour requirements as they combine nanoparticle formation and nucleic acid entrapment into a single step [154] , and provide more homogenous nanoparticles. the first report of rapid-mixing was by batzri and korn, where an ethanolic lipid solution was rapidly injected into an aqueous solution to form liposomes [155] . applying this method to nucleic acids involved combining pre-formed cationic liposomes with nucleic acids to produce lipoplexes [156] . more recently, a t-junction mixing chamber was used for two separate mixing steps [154] . the first mixing step brought together an ethanolic lipid stream with an acidic aqueous buffer containing nucleic acid at an equal flow rate (1:1 v/v mixing). this created metastable particles that were combined with aqueous buffer in a second mixing step (through the t-mixer) to dilute the ethanol content and stabilize the nanoparticles. to simplify this process into a single step, the mixing ratio was modified to 1 part ethanol and 3 parts aqueous. these rapid-mixing methods produce homogenous nanoparticles with entrapment efficiencies > 90% and, importantly, have been proven to be fully scalable [33, 146, 157, 158] . a key advance during the development of onpattro ® for hepatocyte gene silencing was identifying an optimized ionizable cationic lipid with an apparent pka between 6.2 and 6.5 [17, 31] . further increasing the pka value to 7.15, resulted in improved gene silencing in lsecs [159, 160] . incorporating ionizable cationic lipids exhibiting higher pka values increased accumulation in the mps, most likely due to scavenger receptor recognition [159] . the lipid sensitivity to phospholipase is another important factor modulating intrahepatic lnp distribution and activity. three different lipases have been described including the lipoprotein and endothelial lipase in lsecs and the hepatic lipase in hepatocytes [161] . lnp-sirna systems that incorporate ionizable cationic lipids that are sensitive to endothelial lipase (e.g. ester linkages between head and tail functions) have enhanced gene silencing in hepatocytes but exhibit significantly reduced activity in lsecs [159] . co-treatment with lipase inhibitors or incorporating lipase-resistant ionizable cationic lipids can recover gene silencing in lsecs [159] . based on micro-anatomical, subcellular, and (patho)physiological considerations, an ideal lnp for gene regulation in hepatocytes must satisfy the following design criteria: nanoparticle size < 80 nm to efficiently pass through liver fenestrae and improve lnp stability, apparent ionizable cationic lipid pka value around 6.4, near neutral surface charge to prevent sequestration by the mps, and lack of immune stimulation and toxic effects. achieving these and other criteria facilitating efficient nucleic acid entrapment and lnp formulation are detailed in the following section. it is important to mention that upon intravenous administration lnps adsorb serum proteins on their surface. many, if not all, of the abovementioned physiochemical characteristics impart distinct properties to the lnps which ultimately influence protein adsorption. this "biomolecular corona" covering nanoparticles significantly impacts systemic circulation and nano-bio interactions [162] [163] [164] . efficient targeting and gene regulation in hepatocytes stems from the presence of apoe in the corona of lnps and enabled the success of onpattro ® [33, 76] . a recent publication suggested that the ionizable lipid composition plays a major role in the corona formed [165] . how the biomolecular corona can be leveraged to optimize targeting of different cell types within the liver microenvironment needs to be investigated. research in the late 1980s focusing on in vivo pdna delivery showed that in the absence of a delivery system, naked nucleic acid injected into the circulation rapidly broke down and the products accumulated in hepatic tissue [166] . as interest towards asos and sirna grew, lnp compositions and production methods simply translated from plasmids to these shorter nucleic acids [167] . more recently, formulations have become sufficiently potent to support mrna therapeutics' discovery and translation [168] . figure 4 illustrates the different lnp-based treatments for hepatic diseases by silencing pathogenic genes, j o u r n a l p r e -p r o o f expressing therapeutic proteins, or correcting genetic defects. table 3 highlights preclinical lnp-based hepatic gene therapy approaches. refining lipid-dna complexes to more advanced formulations required additional lipids, and such nanoparticles were termed stabilized plasmid lipid particles (splp) [169, 170] . the composition of these formulations largely drew from those used for small molecules therapeutics and included about 6-8 mol% the ability to transfect dividing (liver cancer) cells was most recently highlighted in a study where lnp systems optimized for pdna were found to yield potent transfection [28] . while most dna delivery applications have focused on gene therapy, a highly interesting application is employing dna as barcodes for diagnostic and screening purposes [148] . utilized as short fragments and each with a unique sequence, dna barcodes allow for high-throughput, multiplexed in vivo screening to determine the biodistribution, uptake, and functional activity within the liver microenvironment (as outlined in section 2.2. and 3.5) [50, 143] . notably, as a diagnostic tool, dna barcodes have also been used for developing personalized cancer nanomedicines by co-loading them together with anticancer drugs into lipid nanocarriers. utilizing this strategy, multiple anticancer medicines can be administered at sub-therapeutic doses and the most effective drug can subsequently be identified in the biopsies according to their barcode [175] . as only limited therapies are available for liver cancer, this methodology could well be used towards identifying effective and novel treatments for liver cancer. although lipid calcium phosphate nanoparticles (lcps) are beyond the scope of this review, in the context of liver cancer, it is relevant to note the work by leaf huang and colleagues demonstrating that lcp-based dna delivery enables mitigation of liver metastasis [176] [177] [178] . all procedures and compositions developed for dna delivery readily translated into effective delivery systems for other nucleic acids [167] . sirna only requires cytoplasmic delivery as all rna-induced silencing complex (risc)-related machinery is located in the cytosol. this quick translation resulted in demonstrating the first robust gene silencing in non-human primates (nhp) using nanoparticles known as stable nucleic-acid lipid particles (snalps) containing sirna against apolipoprotein b (apob) [133] . only twelve years later, onpattro ® was approved by the fda for treating attrv [179] . in the early 2000s, the concept of modifying nucleic acids was largely applied to improving their cytoplasmic persistence to enable long term knockdown (decreased sirna turnover). as such, modified sirnas were entrapped into lnp systems with the rationale that a delivery system was specifically required to increase sirna's liver accumulation and intracellular quantity. this had to be achieved in a manner where the cost of raw materials and processing was offset by a potent formulation, i.e. a drastic reduction in material requirement made the formulation commercially viable. with lnp formulations containing dlin-mc3-dma, murine data suggested that as a little as 0.005 mg sirna/kg body weight was required to achieve 50% gene silencing, with no observable toxicities. while alternative technologies such as sirna-conjugates are also gaining prominence [4] , the applicability of lnp technology for hepatic targets is quite clear. it should be noted that sirna-conjugates require substantially higher doses (~1 mg/kg, weekly subcutaneous administration) in order to achieve gene knockdown [180] . lnp-sirna systems have shown utility in decreasing viral loads and virulence, various applications in hepatic oncology, and in metabolic liver disease treatment. rnai finds strong support in anti-viral applications where strict adherence to treatment regimens is critical to success. lnp-sirna treatments can provide sustained knockdown for months leading to longterm viral gene suppression with a potential to eliminate certain viruses. one example is using lnp-sirna as a therapeutic intervention for the ebola outbreak in 2013, which resulted in almost 28,000 cases and 11,300 deaths [181] . lnp-sirna formulations could be rapidly adapted to provide sirna complementarity to the specific strain and showed that a combination of three sirnas against the viral rna synthesis genes suppressed the infection in non-human primates (nhp) [181] . similarly, lnp-sirna modification with galnac-conjugated peg-lipids to specifically accumulate in hepatocytes (of chimeric mice with humanized livers) reduced hepatitis b virus (hbv) genomic dna and antigens [182] . other anti-viral lnp system examples include those for hepatitis delta virus (co-infected with hbv) and hepatitis c virus [183, 184] . lnp-mediated sirna delivery for hepatic oncology applications has largely focused on downregulating genes critical for cell cycle regulation, thereby inducing apoptosis. one example is lnp-sirna against polo-like-kinase 1 (plk1), which regulates multiple cell cycle progression stages. plk1 is over expressed in multiple tumors including liver cancer and down-regulation has been shown to perform well as an intervention [185] . similarly, simultaneous vascular endothelial growth factor (vegf) and kinesin spindle protein (ksp) knockdown has been shown to inhibit proliferation in hepatocellular carcinoma and induce apoptosis [186] . zhou et al. demonstrated that delivery of the small rna let-7g j o u r n a l p r e -p r o o f inhibited tumor growth and dramatically extended survival in a myc-driven genetic liver cancer tumor model [187] . examples of lnp-sirna delivery for liver-related metabolic disorders are plentiful. an interesting clinical observation was that loss-of-function mutations in proprotein convertase subtilisin/kexin type 9 (pcsk9) resulted in low cholesterol levels in circulation. this finding prompted the investigation into using sirna to downregulate pcsk9 as a treatment for hypercholesterolemia. murine and nhp studies showed that specific pcsk9 transcript lowering resulted in reversible and durable knockdown of pcsk9, apolipoprotein b (apob), and low-density lipoprotein associated cholesterol [188] . similarly, in the first demonstration of rnai in higher-order mammals, apob knockdown resulted in reduction of apob levels, serum cholesterol content, and ldl particle concentration in nhps [133] . other lipid-trafficking related targets include apolipoprotein c3 knockdown for hyperlipidemia [189] , and angiopoietin-like 3 protein inhibition for hypertriglyceridemia [190] . lastly, we discuss the specific case of onpattro ® (patisiran), an lnp-sirna formulation targeting the ttr gene. ttr is a homotetrameric serum protein that is synthesized in hepatocytes and secreted into the systemic circulation (note similarity to fvii) [191] . when mutated, ttr deposits as amyloid fibrils in cardiac or peripheral nervous tissue resulting in multi-system failure including ocular, cardiovascular, nephropathy, gastrointestinal, and neuropathy (autonomic and peripheral sensorimotor) manifestations. ttr downregulation with lnp-sirna is a powerful approach to treat this disease. murine data suggested that at doses of 0.1 mg/kg sirna, > 85% liver ttr mrna knockdown and ttr protein serum concentrations could be achieved [192] . further testing in nhps showed that an intravenous dose of 0.3 mg/kg every 4 weeks resulted in rapid and reversible knockdown although serum levels increased two weeks after each administration. increasing the dosing frequency to once every three weeks resulted in sustained and robust knockdown (> 90%) following the third dose. introducing exogenous mrna to induce a therapeutic effect has great potential for a variety of applications. the true benefits of lnp technology for gene regulation in the liver are best highlighted with mrna. specifically, mrna requires a delivery system as modifications to the nucleotides alone have not proven successful in meeting the potency requirements for clinical translation. in addition to this, the exorbitant costs of mrna production imply that lower doses and less frequent dosing regimens are more likely to gain favourable reception. as such, dramatic advances are seen for mrna formulations as vaccines, in protein replacement therapies, and gene editing. reader is referred to several recent articles [193] [194] [195] [196] [197] [198] [199] [200] . however, several recent studies have used the "liver as a bioreactor" to produce relevant neutralizing antibodies. pardi et al. showed that intravenous delivery of lnp-mrna encoding a broadly neutralizing antibody against hiv-1 resulted in sufficient expression to protect from hiv-1 challenge [201] . similarly, another study showed that an lnp-mrna system as prophylactic and therapeutic anti-rabies intervention protected mice from a rabies virus challenge [202] . and elevated hematocrit in porcine and non-human primate (nhp) models [203] . similarly, delivering mrna encoding human clotting factor ix (fix) to fix-knockout mice displayed a reduction in hematocrit loss following injury, indicating fix expression can rescue hemophilia b phenotypes [204] . gene editing is the next major application of mrna therapeutics. various approaches have been explored including crispr/cas9 and zinc-finger nucleases (zfn). an initial gene editing demonstration used a combination of viral delivery (sgrna and repair template) combined with lnp-mrna encoding cas9 to correct a mutation in the fumarylacetoacetate hydrolase gene [205] . the study showed approximately 6% of hepatocytes were edited and it is assumed that the limitation was the viral delivery. comparatively, finn et al. used lnp-mrna formulations encoding for cas9 protein, co-delivered with sgrna targeting ttr. they showed sustained 12-month circulating ttr knockdown (97%) following a single administration of 3 mg/kg rna body weight in a murine model with ~70% editing in the liver (~70% liver cells are hepatocytes) [206] . similarly, lnp-mediated delivery of mrna encoding zinc-finger nucleases (zfn) targeting ttr and pcsk9 resulted in > 90% knockout at mrna doses 10-fold lower than reported previously [137] . in the same study, co-delivery of lnp-mrna encoding zfn targeting the albumin gene and a viral vector for templates of promotor-less human ids or fix resulted in integration of those templates at the albumin locus and generated therapeutically relevant levels of those proteins in murine models. in addition to continuous efforts in optimizing ionizable cationic lipids for enhanced genome editing in the liver, a recent study by cheng et al. demonstrated that bioengineering lnp formulations with additional lipids, so-called selective organ targeting (sort) molecules, can tune the j o u r n a l p r e -p r o o f the rapid translation from lab bench to patients was primarily driven by a holistic design of lnp composition and processes to support scalability while maintaining potency. onpattro ® paved the way for the next generation of lipid-based therapeutics and its success in phase 2 trials spurred development of mrna therapeutics. gene therapies enabled by lnps are under clinical development for a broad range of applications (table 4 ) [211] . in this section we discuss the clinical data for onpattro ® and some mrna therapeutics currently under development. the onpattro ® story, while heavily reviewed in literature, makes for a compelling case to support the development of other lnp nucleic acid formulations [33] . initial efforts laid the foundations to support further clinical development, although it was clear that improved potency was required. dlindma-based lnp-sirna against ttr (aln-ttr01) was administered once to 24 healthy subjects at doses ranging from 0.01 to 1.0 mg sirna per kg body weight, with another eight subjects receiving placebo [212, 213] . over the period of 30 days, 38% serum ttr reduction was observed with persistent reduction for approximately one week. while the knockdown was arguably insufficient for therapeutic efficacy at the highest dose, the study validated the rnai approach in humans. subsequent clinical development used mc3-based lnp, named aln-ttr02 or onpattro ® (patisiran). another phase 1 study included 13 healthy subjects receiving onpattro ® , four subjects receiving placebo, and another six receiving a control sirna [214] . the onpattro ® doses ranged from 0.01 to 0.5 mg/kg sirna and ttr serum levels were measured over 70 days. at sirna doses of 0.3 mg/kg, rapid and robust ttr knockdown was observed; this was sustained over two weeks for a period of 21 days following administration. at these doses and with promising results, further development was warranted. in a subsequent phase 2 study, the dosing regimen for onpattro ® was established [86] . attrv patients received two onpattro ® infusions at doses 0.01-0.3 mg/kg every four weeks or 0.3 mg/kg every three weeks. the q3w dosing regimen resulted in a mean 85% knockdown after the second dose. only few mild-to-moderate infusion-related reactions were observed and one patient reported three serious adverse events. the similarity to preclinical data is quite astonishing; in nhp studies, increasing dosing frequency to q3w (from q4w) resulted in 96% maximal knockdown, and ~85% mean knockdown following the initial dose [192] . in the phase 3 apollo study, 148 patients received onpattro ® at a dose of 0.3 mg/kg once every three weeks, with 77 patients receiving placebo [32] . the primary endpoint was the modified neuropathy impairment score + 7 (mnis + 7), which is used to measure the level polyneuropathy in attrv patients. the test uses highly standardized, quantitative methods to measure muscle weakness, muscle stretch reflexes, sensory loss, and autonomic impairment with higher scores corresponding to disease worsening [215] . over a period of 18 months, attrv patients on placebo showed a linear increase in their mnis+7 from 0 to 28.0. onpattro ® , with an mnis+7 of -6.0, is the only attrv treatment that has been able to halt and even reverse disease progression in patients [32] . in addition to this, onpattro ® also met all secondary endpoints. this led to ema and fda approval in august 2018 [33] . with lnp technology validated as a safe approach for gene modulation in the liver, a wide range of applications have emerged. a substantial effort is focusing on vaccine applications without necessarily transfecting the liver. however, the potential for treating liver diseases is also clear. translate bio was developing a formulation for treating otc deficiency, however disappointing preclinical toxicology data resulted in the termination of the program [216] . moving forward, they chose to focus on developing their cystic fibrosis mrna therapeutic. moderna therapeutics is advancing an lnp candidate formulation for treating methylmalonic acidemia [217] . the focus of this review is on the hepatic applications of lnp formulations, and indications for extrahepatic targets have been summarized elsewhere [218] . highlighted lnp-based nucleic acid therapeutics in the clinic are summarized in table 4 . therapeutic development, and gene therapy in particular, requires concerted efforts from formulation developers, process developers, and clinical sponsors to allow for successful clinical translation. specifically, the therapeutic has to be safe and effective, be producible at a large scale, and meet all regulatory requirements for the corresponding drug class. onpattro ® has shown that this is possible for systemic nucleic acid therapeutics, as it overcame barriers that typically halt the clinical translation of such nanocarrier-based therapeutics. intravenous administration of nanoparticulate formulations can potentially result in infusion-related reactions such as hypersensitivity manifesting as mild flu-like symptoms, or more severe cardiac anaphylaxis [219] . both complement activation as well as complement-independent phagocytosis are involved in such reactions. the reader is referred to excellent articles on complement activation-related pseudoallergy (carpa) and complement independent pseudoallergy (cipa) [220, 219] . several physiochemical properties such as lamellarity, surface charge, and cholesterol content may influence hypersensitivity reactions [221] . infusion-related reactions can be managed by pre-dosing patients with a combination of anti-histamines (h1/h2 blockers), corticosteroid immunosuppressants (e.g., dexamethasone), and oral acetaminophen in addition to reducing the rate of infusion [222] . onpattro ® 's phase 3 trial suggested that the most frequent reactions included flushing, backpain, abdominal pain, and nausea described as mild-to-moderate. the severity and frequency of these reactions decreased with repeated administration and exposure of onpattro ® . it should be noted that asos and galnac-sirna conjugates do not require pre-medication and can be administered subcutaneously (by healthcare professionals), but the doses required to achieve equivalent gene silencing are a few orders of magnitude higher than required for lnps and can only be limited to gene silencing applications [192] . another substantial barrier to clinical translation is producing formulations at commercial scales. as following this, the next processing steps introduce shear as buffer exchange is not done by dialysis, but rather by tangential flow filtration. given the inherent instability of these formulations, particle size increases are observed during this step. these processes use terminal, redundant sterile filtration rather than complete aseptic processing. the impact that buffer exchange has on particle size also affects the ability to sterile filter the formulation and the yield of material. robust process design is critical for successful and timely clinical translation of such formulations. developing lnp delivery technology has enabled the clinical translation and approval of the first sirna drug for inhibiting pathogenic protein production in hepatocytes [32, 33] . importantly, onpattro ® provides a valuable treatment for attrv amyloidosis patients, whose options were previously limited to ttr stabilizers or a liver transplant [34] . at the same time, lnp-sirna development has yielded fundamental insights into optimally designing formulations for hepatocyte gene silencing, (large scale) production methods, in vivo behaviour, immunostimulatory effects, and cost-effectiveness. as these criteria and parameters are now firmly established, it is anticipated that other hepatocyte-targeted lnp-sirna treatments will be developed, such as to knockdown proprotein convertase subtilisin/kexin type 9 for hypercholesteremia treatment [234] . while these advances in lnp development are ground-breaking, other liver-targeted nucleic acid therapeutics, such as asos [3] and galnac-sirna conjugates [4] , are also gaining momentum. for example, the aso tegsedi ® (inotersen) was recently approved for the same indication as onpattro ® [235] . with both onpattro ® and tegsedi ® set at the same list price ($450,000 per year), it remains to be seen which treatment will prove to be most beneficial and cost-effective. tegsedi's ® major advantage is its subcutaneous administration (versus onpattro's ® intravenous infusion), this advantage could be outweighed by its less favorable toxicity profile; patients require monitoring of platelet count, renal and hepatic impairment. subcutaneous administration (and a less complex production process) is also the j o u r n a l p r e -p r o o f main advantage of galnac-sirna conjugates although currently approved conjugates have to be administered by healthcare professionals. most recently, the galnac-sirna conjugate givlaari™ (givosiran, $575,000 per year) was approved for treating acute hepatic porphyria [236, 237] , while new drug applications were filed for lumasiran for treating primary hyperoxaluria type 1, [238, 239] and inclisiran for treating hypercholesteremia [240] [241] [242] . vutrisiran, a galnac-sirna conjugate for treating attrv amyloidosis, is currently undergoing phase 3 trials and has been granted orphan drug designation in the u.s. and the european union [243] . although there is preclinical evidence that lnp-sirna can induce hepatic gene silencing following subcutaneous administration, the dose needed for effective gene silencing is considerably higher than for intravenously administered formulations [147] . of note, while lnp-sirna systems have been optimized for hepatic gene silencing, preclinical studies have also demonstrated their ability to induce effective gene silencing in extrahepatic target sites including the bone [244] and tumors [245, 246] . a major area of interest is applying lnp-sirna for immunotherapy, by silencing target genes in lymphocytes following intravenous administration for immunotherapy [247] [248] [249] [250] [251] (covered by peer et al. in this issue [252] ). as mentioned before, lnp technology's true benefits are currently proving to be of significant value for gene regulation approaches using large nucleic acid-based therapeutics, such as mrna and gene editing complexes, which cannot be accomplished by nucleic acid modification or galnac conjugation. intravenously administered lnp-mrna effectively transfect hepatocytes and induce protein expression in the liver, providing opportunities for protein replacement therapy without affecting the genome. for example, an et al. demonstrated that treatment with lnp containing mrna encoding human methylmalonyl-coa mutase (hmut) had sustained functional benefits in mouse models of methylmalonic acidemia, a rare, inherited, pediatric metabolic disorder [217] . other examples include using the liver to produce coagulation factors [253] , or therapeutic antibodies against hiv [201] and chikungunya virus [254] . although this review focuses on gene therapy for diseases originating in the liver, it is worth mentioning that as with lnp-sirna, intravenously injecting lnp-mrna to induce protein expression in immune cells is gaining considerable traction [255, 256] , especially for developing (personalized) cancer immunotherapies [257, 258] . in addition, lnp-mrna systems have revealed their potential for ex vivo car t cell engineering [259] . moreover, lnp-mrna-based vaccinations following subcutaneous, intradermal, or intramuscular administration have demonstrated to effectively protect from viral challenge [260] [261] [262] [263] [264] [265] . given mrna's relatively short optimization time from target identification to therapeutic, several companies including moderna, biontech, and curevac as well as universities around the globe have initiated lnp-mrna vaccine programs to combat the recent sars-cov-2 pandemic [266] [267] [268] [269] . typical vaccine production relies on isolation and large-scale virus propagation with subsequent processing to purify material (e.g. inactive virus or specific surface protein) that raises a response against a specific viral antigen. with mrna delivery, these timelines can be dramatically reduced, and the breadth of immune coverage expanded. additionally, mrna vaccines leverage several aspects of lnp technology: (1) lnp systems j o u r n a l p r e -p r o o f are not completely immune-silent and can act as adjuvants [270, 271] , (2) few doses are required (i.e. prime and booster), and (3) the mrna dosage is relatively low (compared to protein replacement therapies). lnp technology, and ionizable cationic lipid development in particular, have been instrumental for translating therapeutic gene regulation in hepatocytes from bench to bedside. as lnps are a multicomponent and modular platform, they represent a versatile toolbox with many opportunities to develop future gene therapies with more potent therapeutic effects and improved toxicity profiles. for example, incorporating lipophilic prodrugs in lnp systems has shown to be an attractive approach for reducing nucleic acid therapeutics' immunostimulatory effects [272] or for designing combination therapies with additive therapeutic effects [273] . generating more potent (ionizable cationic) lipids and improved understanding of nano-bio interactions in vivo are continuously fueling the optimization of lnp systems for delivering nucleic acid therapeutics. deciphering intracellular trafficking pathways and mechanism(s) of endosomal escape will facilitate efforts to boost lnp potency [274] [275] [276] [277] . elucidating the nature and dynamic of the biomolecular corona formed on lnps (following intravenous injection) and understanding its implications for biodistribution will be crucial to develop gene therapies beyond the liver [162] [163] [164] 278, 279] . therefore, we expect that lnp-based gene therapies will be developed for indications beyond (ultra) rare diseases in the near future and increasingly become integrated in mainstream medicine. prc is a co-founder of acuitas 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disease gene therapy hepatocellular carcinoma: epidemiology and molecular carcinogenesis hepatocellular carcinoma hepatitis b and c virus hepatocarcinogenesis: lessons learned and future challenges pathogenesis of hepatitis b virus infection hepatitis b virus infection hepatitis b virus infection evolving epidemiology of hepatitis c virus liver zonation: novel aspects of its regulation and its impact on homeostasis metabolic zonation of the liver: the oxygen gradient revisited functional specialization of different hepatocyte populations metabolic zonation of the liver: regulation and implications for liver function inverse zonation of hepatocyte transduction with aav vectors between mice and non-human primates gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects phenotype determines nanoparticle uptake by human macrophages from liver and blood liver macrophages: old dogmas and new insights relation between localization and function of rat liver kupffer cells therapeutic targeting of liver inflammation and fibrosis by nanomedicine expression of asialoglycoprotein receptor 1 in human hepatocellular carcinoma variable asialoglycoprotein receptor 1 expression in liver disease: implications for therapeutic intervention apoe polymorphism in attr amyloidosis patients treated with lipid nanoparticle sirna entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene transfer introduction of liposome-encapsulated sv40 dna into cells transfection reagent lipofectamine triggers type i interferon signaling activation in macrophages the molecular basis of nonbilayer phases cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations j o u r n a l p r e -p r o o f journal pre-proof of sirna biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of rnai therapeutics synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery molecular tuning of a vitamin e-scaffold ph-sensitive and reductive cleavable lipid-like material for accelerated in vivo hepatic sirna delivery rnai-mediated gene silencing in non-human primates lipopeptide nanoparticles for potent and selective sirna delivery in rodents and nonhuman primates an orthogonal array optimization of lipid-like nanoparticles for mrna delivery in vivo a neutral lipid envelope-type nanoparticle composed of a ph-activated and vitamin e-scaffold lipid-like material as a platform for a gene carrier targeting renal cell carcinoma nonviral delivery of zinc finger nuclease mrna enables highly efficient in vivo genome editing of multiple therapeutic gene targets a novel amino lipid series for mrna delivery: improved endosomal escape and sustained pharmacology and safety in nonhuman primates the role of helper lipids in lipid nanoparticles (lnps) designed for oligonucleotide delivery transbilayer movement and net flux of cholesterol and cholesterol sulfate between liposomal membranes hydrophobic scaffolds of ph-sensitive cationic lipids contribute to miscibility with phospholipids and improve the efficiency of delivering short interfering rna by small-sized lipid nanoparticles optimization of lipid nanoparticle formulations for mrna delivery in vivo with fractional factorial and definitive screening designs nanoparticles containing oxidized cholesterol deliver mrna to the liver microenvironment at clinically relevant doses binding characteristics of scavenger receptors on liver endothelial and kupffer cells for modified low-density lipoproteins scavenger receptor class b type i as a receptor for oxidized low density lipoprotein microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of sirna development of lipid nanoparticle formulations of sirna for hepatocyte gene silencing following subcutaneous administration barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics zebrafish as a predictive screening model to assess macrophage clearance of liposomes in vivo poly(ethylene glycol)--lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-dna particles on the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids influence of particle size on the in vivo potency of lipid nanoparticle formulations of sirna a scalable, extrusion-free method for efficient liposomal encapsulation of plasmid dna single bilayer liposomes prepared without sonication simple mixing device to reproducibly prepare cationic lipid-dna complexes (lipoplexes) on the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and sirna rapid synthesis of lipid nanoparticles containing hydrophobic inorganic nanoparticles relationship between the physicochemical properties of lipid nanoparticles and the quality of sirna delivery to liver cells mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoe-deficient mice biomolecular coronas provide the biological identity of nanosized materials corona composition can affect the mechanisms cells use to internalize nanoparticles secreted biomolecules alter the biological identity and cellular interactions of nanoparticles synergistic lipid compositions for albumin receptor mediated delivery of mrna to the liver the fate of plasmid dna after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanoldestabilized cationic liposomes lipid-based dna therapeutics: hallmarks of non-viral gene delivery stabilized plasmid-lipid particles: construction and characterization stabilized plasmid-lipid particles for systemic gene therapy nuclear localization signal peptides enhance cationic liposomemediated gene therapy synergism between a cell penetrating peptide and a ph-sensitive cationic lipid in efficient gene delivery based on double-coated nanoparticles improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of gala and maltotriose theranostic barcoded nanoparticles for personalized cancer medicine delivery of oligonucleotides with lipid nanoparticles liver specific gene immunotherapies resolve immune suppressive ectopic lymphoid structures of liver metastases and prolong survival relaxin gene delivery mitigates liver metastasis and synergizes with check point therapy fda approves first-of-its kind targeted rna-based therapy to treat a rare disease advanced sirna designs further improve in vivo performance of galnac-sirna conjugates lipid nanoparticle sirna treatment of ebolavirus-makona-infected nonhuman primates highly specific delivery of sirna to hepatocytes circumvents endothelial cell-mediated lipid nanoparticleassociated toxicity leading to the safe and efficacious decrease in the hepatitis b virus hepatitis b virus therapeutic agent arb-1740 has inhibitory effect on hepatitis delta virus in a new dually-infected humanized mouse model inhibition of hepatitis c virus in mouse models by lipidoid nanoparticle-mediated systemic delivery of sirna against prk2 abstract 2829: preclinical characterization of tkm-080301, a lipid nanoparticle formulation of a small interfering rna directed against polo-like kinase 1 simultaneous silencing of vegf and ksp by sirna cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma hep3b cells modular degradable dendrimers enable small rnas to extend survival in an aggressive liver cancer model therapeutic rnai targeting pcsk9 acutely lowers plasma cholesterol in rodents and ldl cholesterol in nonhuman primates loss-of-function mutations in apoc3 and risk of ischemic vascular disease silencing of angptl 3 (angiopoietin-like protein 3) in human hepatocytes results in decreased expression of gluconeogenic genes and reduced triacylglycerol-rich vldl secretion upon insulin stimulation transthyretin and familial amyloidotic polyneuropathy. recent progress in understanding the molecular mechanism of neurodegeneration preclinical evaluation of rnai as a treatment for transthyretin-mediated amyloidosis characterization of hiv-1 nucleoside-modified mrna vaccines in rabbits and rhesus macaques mrna vaccines-a new era in vaccinology nucleoside modified mrna vaccines for infectious diseases nucleoside-modified mrna immunization elicits influenza virus hemagglutinin stalk-specific antibodies nucleoside-modified mrna vaccines induce potent t follicular helper and germinal center b cell responses zika virus protection by a single low-dose nucleoside-modified mrna vaccination optimization of lipid nanoparticles for intramuscular administration of mrna vaccines blankschtein, mrna vaccine delivery using lipid nanoparticles administration of nucleoside-modified mrna encoding broadly neutralizing antibody protects humanized mice from hiv-1 challenge schlake, mrna mediates passive vaccination against infectious agents, toxins, and tumors sequenceengineered mrna without chemical nucleoside modifications enables an effective protein therapy in large animals therapeutic efficacy in a hemophilia b model using a biosynthetic mrna liver depot system genome editing with cas9 in adult mice corrects a disease mutation and phenotype a single administration of crispr/cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing selective organ targeting (sort) nanoparticles for tissue-specific mrna delivery and crispr-cas gene editing nanotechnology for organ-tunable gene editing monitoring translation activity of mrna-loaded nanoparticles in mice fast and efficient crispr/cas9 genome editing in vivo enabled by bioreducible lipid and messenger rna nanoparticles delivering the messenger: advances in technologies for therapeutic mrna delivery trial to evaluate safety and tolerability of aln-ttr01 in transthyretin (ttr) amyloidosis -full text view -clinicaltrials.gov safety and efficacy of rnai therapy for transthyretin amyloidosis trial to evaluate safety, tolerability, and parmacokinetics of aln-ttr02 in healthy volunteer subjects -full text view -clinicaltrials.gov development of measures of polyneuropathy impairment in hattr amyloidosis: from nis to mnis + 7 translate bio announces fda clearance to proceed with a single-ascending dose (sad) phase 1/2 clinical trial for ornithine transcarbamylase (otc) deficiency, globenewswire news room long-term efficacy and safety of mrna therapy in two murine models of methylmalonic acidemia current issues of rnai therapeutics delivery and development mechanism of nanoparticle-induced hypersensitivity in pigs: complement or not complement? activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: prediction and prevention liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of doxil and ambisome trial design and rationale for apollo, a phase 3, placebo-controlled study of patisiran in patients with hereditary attr amyloidosis with polyneuropathy patisiran, an rnai therapeutic, for hereditary transthyretin amyloidosis 3rd, first-in-humans trial of an rna interference therapeutic targeting vegf and ksp in cancer patients with liver involvement pharmacokinetics and exploratory exposure-response of sirnas administered monthly as arb-001467 (arb-1467) in a phase 2a study in hbeag positive and negative virally suppressed subjects with chronic hepatitis b a phase i/ii study of tkm-080301, a plk1-targeted rnai in patients with adrenocortical cancer (acc) first-in-human phase i study of the liposomal rna interference therapeutic atu027 in patients with advanced solid tumors safety, tolerability, and pharmacokinetics of bms-986263/nd-l02-s0201, a novel targeted lipid nanoparticle delivering hsp47 sirna, in healthy participants: a randomised, placebo-controlled, double-blind, phase 1 study preclinical mammalian safety studies of epharna (dopc nanoliposomal epha2-targeted sirna) abstract ct156: a first-in-human phase i/ii clinical trial assessing novel mrna-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma safety and efficacy in advanced solid tumors of a targeted nanocomplex carrying the p53 gene used in combination with docetaxel: a phase 1b study 455pdfirst-in-human, first-in-class phase i study of mtl-cebpa, a rna oligonucleotide targeting the myeloid cell master regulator c/ebp-α, in patients with advanced hepatocellular cancer (hcc) intellia therapeutics announces second quarter 2019 financial results and company update, intellia ther effect of an rna interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (pcsk9) and the concentration of serum ldl cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled inotersen treatment for patients with hereditary transthyretin amyloidosis phase 1 trial of an rna interference therapy for acute intermittent porphyria gs-14-envision, a phase 3 study to evaluate efficacy and safety of givosiran, an investigational rnai therapeutic targeting aminolevulinic acid synthase 1, in acute hepatic porphyria patients sun-325 safety and efficacy of lumasiran, an investigational rna interference (rnai) therapeutic, in adult and pediatric patients with primary hyperoxaluria type 1 mp12-14 safety and efficacy study of lumasiran, an investigational rna interference (rnai) therapeutic, in adult and pediatric patients with primary hyperoxaluria type 1 (ph1) inclisiran: uk to roll out new cholesterol lowering drug from next year inclisiran in patients at high cardiovascular risk with elevated ldl cholesterol effects of renal impairment on the pharmacokinetics, efficacy, and safety of inclisiran: an analysis of the orion-7 and orion-1 studies phase 1 study of aln-ttrsc02, a subcutaneously administered investigational rnai therapeutic for the treatment of transthyretin-mediated amyloidosis lipid nanoparticle delivery of sirna to osteocytes leads to effective silencing of sost and inhibition of sclerostin in vivo a glu-urea-lys ligand-conjugated lipid nanoparticle/sirna system inhibits androgen receptor expression in vivo sirna lipid nanoparticle potently silences clusterin and delays progression when combined with androgen receptor cotargeting in enzalutamide-resistant prostate cancer systemic gene silencing in primary t lymphocytes using targeted lipid nanoparticles harnessing rnai-based nanomedicines for therapeutic gene silencing in b-cell malignancies a modular platform for targeted rnai therapeutics leukocyte-specific sirna delivery revealing irf8 as a potential anti-inflammatory target systemic rnai-mediated gene silencing in nonhuman primate and rodent myeloid cells targeted lipid nanoparticles for rna therapeutics and immunomodulation in leukocytes systemic delivery of factor ix messenger rna for protein replacement therapy a lipidencapsulated mrna encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection cell specific delivery of modified mrna expressing therapeutic proteins to leukocytes synthesis and biological evaluation of ionizable lipid materials for the in vivo delivery of messenger rna to b lymphocytes lipid nanoparticle assisted mrna delivery for potent cancer immunotherapy systemic rna delivery to dendritic cells exploits antiviral defence for cancer immunotherapy ionizable lipid nanoparticle mediated mrna delivery for human car t cell engineering characterization of hiv-1 nucleoside-modified mrna vaccines in rabbits and rhesus macaques nucleoside-modified mrna immunization elicits influenza virus hemagglutinin stalk-specific antibodies nucleoside-modified mrna vaccines induce potent t follicular helper and germinal center b cell responses zika virus protection by a single low-dose nucleoside-modified mrna vaccination expression kinetics of nucleoside-modified mrna delivered in lipid nanoparticles to mice by various routes ciaramella, mrna vaccines against h10n8 and h7n9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials self-amplifying rna sars-cov-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered covid-19 patients work on a covid-19 vaccine candidate | moderna biontech and pfizer announce completion of dosing for first cohort of phase 1/2 trial of covid-19 vaccine candidates in germany curevac´s optimized mrna platform provides positive pre-clinical results at low dose for coronavirus vaccine candidate | english com%2fnews%2fcurevac-s-optimized-mrna-platform-providespositive-pre-clinical-results-at-low-dose-for-coronavirus-vaccine-candidate unmodified mrna in lnps constitutes a competitive technology for prophylactic vaccines lipid-derived nanoparticles for immunostimulatory rna adjuvant delivery dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids modular lipid nanoparticle platform technology for sirna and lipophilic prodrug delivery | biorxiv efficiency of sirna delivery by lipid nanoparticles is limited by endocytic recycling brief update on endocytosis of nanomedicines boosting intracellular delivery of lipid nanoparticle-encapsulated mrna endocytic profiling of cancer cell models reveals critical factors influencing lnp-mediated mrna delivery and protein expression isolation methods for particle protein corona complexes from protein-rich matrices an analysis of the binding function and structural organization of the protein corona key: cord-018414-6ffhm895 authors: kang, yoogoo; elia, elia title: anesthesia management of liver transplantation date: 2016-07-22 journal: contemporary liver transplantation doi: 10.1007/978-3-319-07209-8_9 sha: doc_id: 18414 cord_uid: 6ffhm895 anesthesia for liver transplantation pertains to a continuum of critical care of patients with end-stage liver disease. hence, anesthesiologists, armed with a comprehensive understanding of pathophysiology and physiologic effects of liver transplantation on recipients, are expected to maintain homeostasis of all organ function. specifically, patients with fulminant hepatic failure develop significant changes in cerebral function, and cerebral perfusion is maintained by monitoring cerebral blood flow and cerebral metabolic rate of oxygen, and intracranial pressure. hyperdynamic circulation is challenged by the postreperfusion syndrome, which may lead to cardiovascular collapse. the goal of circulatory support is to maintain tissue perfusion via optimal preload, contractility, and heart rate using the guidance of right-heart catheterization and transesophageal echocardiography. portopulmonary hypertension and hepatopulmonary syndrome have high morbidity and mortality, and they should be properly evaluated preoperatively. major bleeding is a common occurrence, and euvolemia is maintained using a rapid infusion device. pre-existing coagulopathy is compounded by dilution, fibrinolysis, heparin effect, and excessive activation. it is treated using selective component or pharmacologic therapy based on the viscoelastic properties of whole blood. hypocalcemia and hyperkalemia from massive transfusion, lack of hepatic function, and the postreperfusion syndrome should be aggressively treated. close communication between all parties involved in liver transplantation is also equally valuable in achieving a successful outcome. dr. thomas starzl of denver, colorado, usa, who believed that "liver transplantation is an effective treatment providing exactly what is needed for patients with end-stage liver disease (esld)," performed the first successful orthotopic liver transplantation (olt) in a 3-year-old boy with biliary atresia in 1963 (starzl et al. 1963) . during the first two decades of the procedure's history, liver transplantation led by starzl and sir roy calne of cambridge encountered almost insurmountable challenges, including complexity of surgical technique, primitive anesthesia and intensive care, less-than-adequate immunosuppression and organ preservation, and devastating infection. the number of procedures performed was relatively few, and the success rate was low. however, their keen observations on these early clinical experiences laid the foundation of modern liver transplantation (starzl and putnam 1969; calne 1983) . breakthroughs were made in each decade following the first transplantation. in the 1980s, venovenous bypass was introduced to maintain better hemodynamic stability (shaw et al. 1984) , cyclosporine was found to be a superior immunosuppressant to azathioprine, and anesthesiologists answered important clinical questions, including those relating to the monitoring and treatment of coagulopathy, hemodynamic changes, and the role of the electrolyte imbalance. in the 1990s, fk506 (tacrolimus) became the immunosuppressant of choice ), university of wisconsin solution was introduced to extend the safe cold ischemia time to 24 h (kalayoglu et al. 1988) , and the piggyback technique simplified surgery in select patients (tzakis et al. 1989 ). in the past 15 years, liver transplantation has been performed in most major medical centers with a 1-year survival rate of greater than 85 %, and living donor liver transplantation has become a valuable alternative. liver transplantation requires a true multidisciplinary approach, and anesthesiologists and intensivists have played a major role in the successful outcome of liver transplantation. in support of the important role of anesthesiologists in liver transplantation, the american society of anesthesiologists (asa) developed the guidelines for director of liver transplant anesthesia in 2001. the guidelines specified that the director should have fellowship training in critical care medicine, cardiac anesthesiology, or transplantation anesthesiology that includes the perioperative care of at least ten liver transplant recipients or experience in the perioperative care of at least 20 liver transplant recipients in the operating room. in addition, the director is expected to obtain a minimum of 8 h of accreditation council for continuing medical education (accme) category i continuing medical education (cme) credit in transplantation-related educational activities within the most recent 3-year period. in this chapter, physiology and pathophysiology of liver disease and anesthesia care of liver transplantation are described based on clinical experience at the university of pittsburgh (pittsburgh, pa, usa) and thomas jefferson university (philadelphia, pa, usa). the liver, which weighs 1200-1500 g in adults, is traditionally divided into the right and left lobe in reference to the location of the falciform ligament. couinaud, however, divided the liver into the right and left hemiliver using the cantlie's line, which extends from the inferior vena cava (ivc) to the gall bladder, and each hemiliver is further divided into four segments (couinaud 1954) . the left hemiliver is composed of the traditional left lobe along with the caudate and quadrate lobe. liver resections based on these segmental definitions are right hepatectomy (segments 5-8), right lobectomy (segments 4-8), left hepatectomy (segments 1-4), and left lobectomy (segments 1-3) ( fig. 1) (bismuth 1982) . the liver has a unique dual blood supply: arterial supply from the hepatic artery, a branch of the celiac axis, and venous supply from the portal vein formed by the union of the splenic and superior mesenteric vein. despite liver mass constituting only 2.5 % of the total body weight, the total hepatic blood flow is approximately 100 ml/ 100 g/min, or 25 % of cardiac output. the hepatic artery supplies approximately 25-30 % of hepatic blood flow and 45-50 % of the oxygen requirement, while the portal vein supplies 70-75 % of hepatic blood flow and 50-55 % of oxygen. the venous drainage is through the right, middle, and left hepatic veins, which merge into the ivc. the valveless portal vein is a low pressure/low resistance circuit, while the hepatic artery is a high pressure/high resistance system. hepatic 553-565, copyright (2000) , with permission from elsevier) blood flow is primarily regulated by local metabolic demand with an inverse relationship between portal venous and hepatic arterial flow: an increase in the hepatic adenosine level triggered by a reduced portal venous flow increases hepatic arterial blood flow (gelman and ernst 1977; lautt et al. 1985) . the hepatic artery buffer response appears to be functional even after liver transplantation (payen et al. 1990) , and this response may be responsible for the development of the small-for-size syndrome after living donor or split liver transplantation (kiuchi et al. 1999 ). small-for-size syndrome develops in a patient who received a donor graft that was less than 1 % of the recipient's body weight and is caused by decreased hepatic arterial flow in response to increased portal venous flow and pressure. subsequently, a prolonged postoperative reduction in hepatic arterial flow can lead to centrilobular tissue necrosis, biliary ischemia, and hepatic arterial thrombosis (smyrniotis et al. 2002) . there is no buffer response in the portal system because the portal vein cannot regulate its blood flow. therefore, alterations in the hepatic arterial blood flow do not induce compensatory changes in the portal blood flow (lautt 1983) . the mean pressure in the hepatic artery is similar to that in the aorta, while portal vein pressure ranges between 6 and 10 mmhg. the portal pressure depends primarily on the degree of constriction or dilatation of the splanchnic arterioles and on intrahepatic resistance. both afferent systems merge at the sinusoidal bed, where the pressure is estimated to be 2-4 mmhg higher than that in the ivc. the liver serves as a blood reservoir, and it replenishes blood volume of up to 25 % rapidly in the case of an acute bleeding episode (lautt 2007) . hepatic blood volume may expand considerably in cardiac failure by venous congestion. the liver is innervated by the left and right vagi, the right phrenic nerve, and fibers from the t7-t10 sympathetic ganglia. the hepatic artery is innervated mainly by sympathetic fibers, and hepatocytes, by the unmyelinated sympathetic fibers. the bile ducts are innervated by both sympathetic and parasympathetic fibers. the role of hepatic innervation is unclear, as denervation of the transplanted liver does not affect its function (kjaer et al. 1994) . bile flow begins from the bile canaliculi to the common bile duct. hepatic lymph forms in the space between the sinusoid and the hepatocyte (space of disse) and flows to lymph nodes in the hilum and ivc. the transdiaphragmatic lymphatic flow is the cause of pleural effusions in the presence of large ascites. the liver is made of parenchymal cells (hepatocytes) and non-parenchymal cells (sinusoidal endothelial, kupffer, stellate, dendritic, and lymphocyte) . hepatocytes make up 60-80 % of liver cells and carry out hepatic metabolic, synthetic, and detoxification functions. polyhedral hepatocytes are arranged in one-cell thick plates with endothelium-lined sinusoids on both sides. each hepatocyte cell membrane has three distinct membrane domains. the sinusoidal membrane is adjacent to the sinusoidal endothelium and has numerous microvilli abutting into the space of disse. fenestrae within the sinusoidal endothelium without the basement membrane permit intimate contact between sinusoidal blood and the hepatocytes to allow the passage of big molecules, including lipoproteins. liver sinusoidal endothelial cells make up 15-20 % of liver cells and release nitric oxide to regulate vascular resistance. they are, along with dendritic cells and lymphocytes, part of the innate immune system. the space of disse contains phagocytic kupffer cells that participate in the hepatic inflammatory process. the ito cells, also known as stellate cells, are the major site of vitamin a storage, and their activation results in hepatic fibrosis and cirrhosis. reticulin fibers in the space of disse support the sinusoidal framework, and weakening of these supporting fibers results in rupture of sinusoidal walls and formation of blood-filled cysts known as peliosis hepatis, a forerunner of cirrhosis. the apical membrane circumscribes the canaliculus, the earliest component of the biliary system. the lateral hepatic membrane is found between adjacent hepatocytes. the functional unit of the liver is the acinus. terminal portal veins communicate with terminal hepatic venules, with sinusoids bridging the gap between the two vessels ( fig. 2) . each sinus contains three zones with equal blood pressure and oxygen content. the periportal zone (zone 1) receives blood highest in oxygen content and the pericentral or perivenular zone (zone 3) receives blood lowest in oxygen content. as a result, the hepatocytes in the perivenular zone (zone 3) are more vulnerable to ischemic damage and nutrient depletion. oxidative and reductive functions are predominantly performed by hepatocytes at the periportal zone and glucuronidation is performed by those at the perivenular zone, although hepatocytes of the two different zones are functionally integrated (lamers et al. 1989 ). the unique structure of the liver acinus is well-suited for bidirectional transfer of nutrients. the low pressure in the portal venous system allows blood to flow slowly through the sinusoids. hepatic arterial blood flows mainly to the terminal bile canaliculi, although it augments sinusoidal flow to give a gentle pulsatility. in patients with liver cirrhosis, the sinusoids acquire features of systemic capillaries: the space of disse widens with collagen deposits at the basement membrane, endothelial fenestrations become smaller and fewer, and hepatic microvilli efface. all of these changes reduce transport across the sinusoidal walls and result in hepatic dysfunction. furthermore, widespread fibrosis and scarring reduce the number and size of the small portal and hepatic veins and increase intrahepatic vascular resistance to the development of portal hypertension (popper 1977) . the sluggish blood flow in the altered vascular architecture promotes thrombosis, causing further cell necrosis and fibrosis (wanless et al. 1995) . the liver undergoes rapid regeneration through proliferation of hepatocytes to maintain the critical mass necessary for normal liver function. for example, the newly transplanted hemiliver from a living related donor regenerates to about 85 % of its original whole liver size in 7-14 days. the major hepatic growth factors are epidermal growth factor and hepatocyte growth factor (michalopoulos 1990) . administration of the epidermal or hepatocyte growth factor to normal rats, however, does not cause hepatocyte replication. this negative response suggests that liver regeneration involves a two-step process: the initial signal generated by an acute increase in metabolic demand associated with the loss of hepatocytes triggers a set of early response genes that prime hepatocytes to respond to various growth factors. in apoptosis or programmed cell death, aging hepatocytes are removed and new cells are produced in a continuous manner (ellis et al. 1991 ). the liver has three major functions: metabolism, bile production and secretion, and filtration of harmful substances. the principal role of the liver is to provide the body with normal glucose levels, which are regulated by insulin, glucagon, growth hormone, and catecholamines (pilkis and granner 1992) . the liver converts glucose into glycogen (glycogenesis) and utilizes glucose for the synthesis of fatty acids. cirrhotic patients are frequently hyperglycemic although their insulin level is elevated (petrides and defronzo 1989) . this insulin resistance is caused by multiple mechanisms. cirrhotic patients have an increased basal metabolic rate and use preferentially fatty acids as an energy source. reduced glucose uptake and limited glucose storage in the liver and muscle lead to hyperglycemia. other contributing factors are increased serum fatty acids, which inhibit glucose uptake by muscle; altered second messenger activity after insulin binding to its receptors; an increased concentration of serum cytokines associated with elevated levels of endotoxins; and increased levels of glucagon and catecholamines. the liver is the major organ for protein synthesis, and albumin is the most important protein product. albumin is the major contributor to plasma oncotic pressure and binds and transports bilirubin, hormones, fatty acids, and other substances. hypoalbuminemia is commonly caused by decreased hepatic synthetic function, although it can be secondary to an enlarged volume of distribution, reduced level of amino acid precursors, and losses into the urine, peritoneum and pleural cavity, and leads to peripheral edema, ascites, and pleural effusions. the low serum oncotic pressure stimulates the hepatic albumin synthesis in healthy subjects, but this is impaired in patients with cirrhosis (pierrangelo et al. 1992) . the liver synthesizes all coagulation factors (except von willebrand factor) and protein c and s. factors ii, vii, ix, and x undergo a posttranslational vitamin k-dependent modification involving γ-carboxylation of specific glutamic acid residues in the liver. the liver is the primary site of interconversion of amino acids. anabolic processes synthesize proteins from amino acids, while catabolic processes convert amino acids either to keto acids by transamination or ammonia by oxidative deamination. ammonia, in turn, is converted to urea by the krebs-henseleit cycle. in patients with liver disease, derangement of both anabolic and catabolic processes results in decreased production of blood urea nitrogen (bun) and accumulation of ammonia, a contributing factor in the development of hepatic encephalopathy. the liver produces acute-phase reactants, such as α-fetoprotein, ceruloplasmin, fibrinogen, transferrin, complement, and ferritin. they are expressed during acute and chronic systemic inflammation, and their activation is mediated by interleukin-6, tumor necrosis factor, interferon-γ, and glucocorticoids. the liver takes up fatty acids and cholesterol from diet and peripheral tissues to produce and release lipoprotein complexes into circulation. fatty acids released from adipocytes are bound to serum albumin and transported to the liver for the synthesis of phospholipids and triglycerides. the liver produces fatty acids from small molecular weight precursors, and cholesterol synthesis is regulated by the ratelimiting enzyme 3-hydroxyl-3 methylglutaryl coenzyme a reductase (hmg-coa reductase). lipids are exported out of the liver by very low-density lipoprotein (vldl) particles, which are the major carriers of plasma triglycerides during non-absorptive states. lipids are temporarily stored in the liver as fat droplets, or as cholesteryl esters in the case of cholesterol, and are directly excreted into bile or metabolized into bile acids. the liver is the major site for sterol excretion and production of bile acids. various abnormalities in lipid metabolism are common in liver disease. hypertriglyceridemia (250-500 mg/dl) is the most common presentation and may be caused by decreased synthesis of lipoproteins, decreased hepatic clearance of lipoprotein complexes, or re-entry of biliary content into the serum. alcoholic liver injury results in increased fatty acid synthesis and steatosis (lieber 1993) . paradoxically, an increased high-density lipoprotein (hdl) 3 level has been noted with moderate alcohol consumption, which may explain the reduced risk of atherosclerosis in these patients (chait and brunzell 1990) . patients with cholestatic liver diseases have elevated total serum cholesterol and triglycerides because the bile is rich in cholesterol, phospholipids, and lecithin. the liver eliminates drugs through two types of reactions. the phase 1 reactions include oxidation, reduction, hydroxylation, sulfoxidation, deamination, dealkylation, and methylation of reactive substances. these reactions involve systems such as cytochrome p450 and typically occur in the periportal area of the liver. the phase 2 reactions, which transform lipophilic agents into more water-soluble compounds, take place in the pericentral area. in patients with liver disease, hepatic drug clearance is usually reduced due to the enlarged volume of distribution and decreased hepatic metabolism. as a result, a large initial dose of medications followed by small, titrated maintenance doses are required to achieve the desired pharmacologic effects. several hormones are deactivated or altered in the liver. the deactivated hormones are insulin, glucagon, steroid hormones, aldosterone, thyroxine, and triiodothyronine. the liver converts testosterone into androsterone and estrogen into estrone and estriol. abnormal levels of estrogen and testosterone in patients with liver disease lead to testicular atrophy, loss of pubic and axillary hair, spider angioma, and gynecomastia. the liver removes various substances from the body, and bile formation is one of the most important excretory functions. when membranes of old erythrocytes rupture, the released hemoglobin is taken up by the reticuloendothelial cells and is split into heme and globin. heme converts to biliverdin, which, in turn, is reduced to free bilirubin and released into the plasma. the free bilirubin-albumin complex is taken up by the hepatocytes. bilirubin conjugates primarily with glucuronic acid and is actively transported into the bile. a small portion of conjugated bilirubin returns to the plasma directly from the sinusoids or indirectly by absorption from the bile ducts and lymphatics. bilirubin is converted into urobilinogen by the intestinal bacterial flora. some urobilinogen is reabsorbed through the intestinal mucosa and is re-excreted into the intestine. bile acids, which enhance absorption of vitamin k, are also excreted into the bile by the liver. the liver, located between the splanchnic and systemic venous system, acts as a vascular filter. kupffer cells phagocytose immune complexes, endotoxins, and bacteria in the portal venous blood and process antigens for presentation to immunocompetent cells. the liver also removes activated coagulation elements from circulation to prevent excessive coagulation or fibrinolysis. liver cirrhosis is defined as progressive fibrosis and the formation of regenerative nodules, and is the final common pathway in which hepatocytes are replaced by connective tissue after various, repetitive insults. the amount of remaining functional hepatic mass and the degree of architectural distortion determine the functional state of the liver. portal hypertension is inevitable in advanced cirrhosis and leads to ascites, variceal bleeding, and encephalopathy. the severity of cirrhosis is frequently classified using the child-pugh score (table 1) , and a score of >6 suggests a short life expectancy. hepatic encephalopathy is a reversible neuropsychiatric condition in both acute and chronic liver failure. in chronic liver disease, hepatic encephalopathy develops in 28 % of patients within 10 years of compensated cirrhosis and is associated with spontaneously developed or surgically created portosystemic shunting (butterworth 2001) . the degree of encephalopathy is stratified by a coma scale: grade 1, subtle confusion; grade 2, somnolence; grade 3, unconsciousness with response to pain stimulation; and grade 4, deep coma. clinically, asterixis, flapping tremor, and fetor hepaticus (musty, sweet breath odor) are confirmatory of hepatic encephalopathy. the main cause of hepatic encephalopathy is the altered expression of several genes for various neurotransmitter proteins in the brain (butterworth 2001) . decreased expression of the glutamate transporter (glt-1) increases extracellular brain glutamate. an increased expression occurs in some receptors: monoamine oxidase increases degradation of monoamine transmitters, the peripheral-type benzodiazepine receptor increases inhibitory neurosteroids, and neuronal nitric oxide synthase increases nitric oxide production. although its plasma level is not closely related to the severity of encephalopathy, ammonia is still considered to be a major contributing factor. ammonia and manganese are known to alter the expression of the peripheraltype benzodiazepine receptor and neuronal nitric oxide synthase in exposed cells (warskulat et al. 2001) . magnetic resonance spectroscopy reveals brain edema and increased brain glutamine/glutamate in the frontal and parietal lobes; histologic findings are swelling and glycogen deposition in astrocytes. these changes in the brain coincide with impairment in the visuopractic capacity, visual scanning, and perceptual-motor speed on neuropsychiatric testing (tarter et al. 1984) . subclinical hepatic encephalopathy can be detected by having patients perform a simple timed connect-the-numbers test. treatment of hepatic encephalopathy is based on the ammonia-lowering strategy, such as protein restriction, oral non-absorbable antibiotics, and lactulose. rifaximin reduces the plasma ammonia level by destroying intestinal bacteria that produce urease. metronidazole (800 mg/day) is another antibiotic, although its adverse effects limit its use to 1 week at a time. lactulose is a substrate for gut bacteria and reduces the formation of ammonia by lowering intestinal ph. rifaximin class a = 5-6 points, b = 7-9 points, and c = 10-15 points inr international normalized ratio and lactulose are commonly used together, and the antibiotic is discontinued once eradication of disaccharide-metabolizing intestinal bacteria is indicated by an increase in stool ph. oral or parenteral ornithine aspartate, a substrate for the conversion of ammonia to urea and glutamine, has effects similar to those of lactulose, but with fewer adverse effects. in patients with severe encephalopathy, the molecular-absorbent recycling system (mars) may be utilized to remove small and middle molecular weight water-soluble substances (sorkine et al. 2001) . the system appears to increase blood pressure and systemic vascular resistance, possibly by removing nitric oxide. in fulminant hepatic failure, progressive hepatic coma is accompanied by a gradual increase in cerebral blood flow and intracranial pressure (icp) (see ▶ chap. 12, "fulminant hepatic failure: diagnosis and management"). subsequently, vasogenic cerebral edema and severe intracranial hypertension develop and approximately 30-50 % of patients die of brain herniation. monitoring of icp using a ladd epidural sensor is useful in detecting intracranial hypertension, monitoring the therapeutic effects, and identifying patients who would survive after transplantation without neurologic damage (lidorsky et al. 1992) . non-invasive neurologic assessment includes transcranial doppler (tcd) to measure cerebral bloodflow velocity, determination of the cerebral metabolic rate for oxygen by calculating the oxygen content difference between arterial and jugular bulb venous blood, evoked potentials, and serial computed tomography (ct) scans (aggarwal et al. 1994) . treatment includes osmotic and loop diuretics, barbiturateinduced coma, and hypothermia. the definitive treatment is usually transplantation. the presence of hyperdynamic circulation with a markedly increased cardiac output and decreased systemic vascular resistance was first described by kowalski and abelmann in the early 1950s (kowalski and abelmann 1953) . several hypotheses have been proposed to explain this phenomenon, including an overactive sympathetic nervous system, inadequate clearance of vasoactive substances by the diseased liver, the presence of arteriovenous shunts, nitric oxide-induced vasodilation, and relative hypoxia in peripheral tissues (benoit et al. 1984; yokoyama et al. 1989; kalb et al. 1993; d'souza et al. 1993) . although cardiac output is frequently two to three times normal, impaired systolic and diastolic function together with attenuated cardiac responsiveness to stimuli suggests that cardiomyopathy is present in cirrhotics (cirrhotic cardiomyopathy) (lee 1989) . caramelo et al. noted a 50 % decrease in cardiac output with volume expansion in a ccl 4 -induced cirrhotic rat model (caramelo et al. 1986) . in another rat model, the chronotropic response to isoproterenol was attenuated compared with that in control animals (lee et al. 1990 ). cardiac response to physical exercise is blunted in patients with cirrhosis, indicated by alterations in the pre-ejection period, isometric contraction time, and ratio of the pre-ejection period to left ventricular ejection time. in addition, abnormalities in myocardial diastolic indices suggest non-compliant ventricles. histologically, myocardial fibrosis, mild subendocardial edema, and vacuolation of myocyte nucleus and cytoplasms are observed. the development of cirrhotic cardiomyopathy is multifactorial. it appears that the β-receptor system, the main stimulant of the ventricle, is dysfunctional. in humans, lymphocyte β-receptor density, which reflects cardiac β-receptor status, is reduced in patients with severe ascites (gerbes et al. 1986) , and β-receptor density of the cardiomyocyte sarcolemmal plasma membrane is reduced in cirrhotic rats (liu and lee 1999) . further, the β-receptor signal transduction pathway is impaired at several levels (ma et al. 1996) . although cardiac contractile impairment may result from overactivity of the muscarinic m 2 receptor, the receptor density and binding affinity are unchanged, suggesting normal parasympathetic function (jaue et al. 1997) . high serum catecholamine levels, a result of desensitization and down-regulation of β-receptors, may lead to myocardial dysfunction in the presence of α-mediated coronary vasoconstriction. additionally, overproduction of nitric oxide inhibits β-receptor-stimulated cyclic adenosine monophosphate (camp) release, causing myocardial dysfunction and vasodilation (hare and colucci 1995) . coronary artery disease (cad) was previously believed to be relatively uncommon in patients with cirrhosis as a result of generalized vasodilation and elevated levels of hdl and estrogen. in addition, autopsy findings showed relatively fewer atherosclerotic changes and myocardial infarction. however, studies have shown that cad is not uncommon, and moderate-to-severe cad was found in approximately 27 % of patients who underwent coronary artery catheterization as a part of liver transplantation workup (carey et al. 1995) . in another study of 161 liver transplantation candidates who were at risk for cad and referred for coronary angiography, 25 % of patients had at least one moderate or severe (>50 %) coronary stenosis (tiukinhoy-laing et al. 2006) . endocarditis is three times more common in patients with liver disease (snyder et al. 1977) . this is attributed to translocation of intestinal bacteria through the intestinal wall and portosystemic collaterals, and reduced immune response. the incidence of pericardial effusion in cirrhotic patients is approximately 32-63 % and correlates with the degree of liver failure (shah and variyam 1988) . the effusion is usually small and may require drainage if it affects cardiac function. patients with liver disease exhibit three common cardiac electrophysiological disturbances: electromechanical dissociation, prolongation of ventricular repolarization (the q-t interval), and chronotropic incompetence (milani et al. 2007) . pulmonary hypertension associated with portal hypertension was first described in 1951 (mantz and craige 1951) . pulmonary hypertension defined as a mean pulmonary artery pressure of >25 mmhg and pulmonary vascular resistance of >240 dyn/s/cm à5 (3 wood units) is more common in patients with liver disease, with a prevalence of 0.25-0.73 % (lebrec and capron 1979; mcdonnell et al. 1983) . pulmonary artery pressure is a function of pulmonary venous pressure, pulmonary vascular resistance, and cardiac output [(pulmonary artery pressure = pulmonary venous pressure + (pulmonary vascular resistance â cardiac output)]. therefore, pulmonary hypertension is not uncommon in patients with liver disease because of their poor left ventricular compliance, increased pulmonary vascular resistance, and increased pulmonary blood flow from portosystemic shunting. the pathophysiology and management of pulmonary hypertension are well-described in ▶ chap. 10, "hepatopulmonary syndrome and portopulmonary hypertension". portal hypertension is caused by an increased intrahepatic vascular resistance and increased splanchnic blood flow. endothelin-1, a powerful vasoconstrictor produced by the sinusoidal endothelial cells, is known to increase intrahepatic vascular resistance and activates stellate cells, and its level increases as cirrhosis progresses (kojima et al. 2002; gandhi et al. 1996) . normally, vasodilatory compounds, such as nitric oxide, counterbalance the increased intrahepatic vascular resistance induced by endothelin. in liver cirrhosis, however, nitric oxide production is inhibited by caveolin-1, a hepatic membrane protein that binds with endothelial nitric oxide synthase. hypoxemia of varying severity is present in 45-69 % of patients with significant liver disease (krowka and cortese 1985) . the common causes are pleural effusions, impaired diffusion capacity, arteriovenous shunting, atelectasis caused by ascites or diaphragmatic dysfunction, aspiration secondary to encephalopathy, and deconditioning (hourani et al. 1991) . ventilation-perfusion mismatch, pulmonary vasodilation, and infection also contribute to hypoxemia. mild forms of hypoxemia are most common, although moderate-tosevere hypoxemia may be found in patients with advanced liver disease complicated by adult respiratory distress syndrome (ards), infection, and multiple organ failure. hepatopulmonary syndrome, first described by fluckiger in 1884 (fluckiger 1884) , may cause severe hypoxemia in a subset of patients with liver disease. the syndrome consists of a triad of liver dysfunction, severe hypoxemia (pao 2 < 70 mmhg in room air), and pulmonary vasodilation, and is characterized by dyspnea, cyanosis, clubbing of the digits, exercise desaturation, and orthodeoxia (hypoxemia in upright position). other concomitant clinical signs are a markedly increased alveolar-arterial oxygen gradient, portal hypertension, and vascular abnormality such as spider angioma and pulmonary vasodilation. the pulmonary vascular dilation (from 8-15 μ to 15-100 μ) at the precapillary level is believed to be the main pathology of the hepatopulmonary syndrome, which is caused by decreasing erythrocyte transit time and impairing diffusion of oxygen to the erythrocytes at the center of the bloodstream (genovesi et al. 1976 ). in contrast with other pulmonary diseases, oxygenation improves dramatically with a high inspired oxygen concentration (fio 2 ), because a high alveolar concentration of oxygen overcomes the diffusion barrier and oxygenates the erythrocytes in the center of the bloodstream. the pathophysiology and management of hepatopulmonary syndrome are described in ▶ chap. 10, "hepatopulmonary syndrome and portopulmonary hypertension". non-cardiogenic pulmonary edema occurs in 37-79 % of patients with advanced liver disease, particularly in those with fulminant hepatic failure, and appears to be associated with sepsis and a neurogenic mechanism. the presence of this complication is ominous: matuschak and shaw reported that all 29 patients who developed non-cardiogenic pulmonary edema died before liver transplantation (matuschak and shaw 1987) . in contrast, a rapid reversal of ards after liver transplantation has been reported (doyle et al. 1993) . pulmonary edema caused by fluid overload responds to diuretics and has a relatively benign course. pleural effusions are found on chest x-rays in about 10 % of patients. these are caused by the unidirectional passage of ascites via diaphragmatic defects into the pleural space. diagnostic thoracentesis is necessary to confirm the transudative nature and to exclude infection, malignancy, or embolic disease. optimal control of ascites may prevent symptomatic pleural effusions, and transjugular intrahepatic portosystemic shunt (tips) is effective in treating refractory hydrothorax in 84 % of patients (siegerstetter et al. 2001 ). approximately 10 % of hospitalized cirrhotic patients with ascites develop the hepatorenal syndrome, which is a form of acute pre-renal kidney injury caused by circulatory dysfunction secondary to an imbalance between circulating vasodilatory and vasoconstrictive substances. the primary contributing factor for the hepatorenal syndrome is nitric oxide-induced vasodilation of the splanchnic vascular bed causing systemic arterial underfilling and relative hypovolemia (arroyo et al. 1996) . this relative hypovolemia activates baroreceptor-mediated sympathetic and the renin-angiotensin system to constrict all vascular beds including the renal vasculature (guevara et al. 1998 ). the initial prostaglandin-mediated compensatory renal vasodilation is followed by renal arterial vasoconstriction and renal hypoperfusion. a striking feature of the hepatorenal syndrome is the lack of any histologic change and its reversibility: the affected kidneys resume their function after successful liver transplantation. the renal failure may be rapid (type 1) or insidious (type 2) and results in sodium and water retention and dilutional hyponatremia. since the hepatorenal syndrome is a functional renal failure, the urine is similar to that found in pre-renal azotemia: oliguria, low urinary sodium, and an increased urine osmolality and urine to plasma osmolality ratio. the major criteria for the diagnosis of the hepatorenal syndrome are as follows: (1) advanced hepatic disease and portal hypertension; (2) low glomerular filtration rate (serum creatinine >1.5 mg/dl or creatinine clearance <40 ml/ min); (3) absence of nephrotoxic drug use, shock, systemic infection, or recent fluid losses; (4) lack of sustained improvement after diuretic withdrawal and volume resuscitation with 1.5 l of normal saline; (5) proteinuria (<500 mg/dl); and (6) no ultrasound evidence of urinary obstruction or parenchymal disease. minor criteria include oliguria (<500 ml/day), urinary sodium <10 meq/l, urinary osmolality greater than plasma osmolality, urinary red blood cells (rbcs) <50/hpf, and serum sodium <130 meq/l. it is noteworthy that conventional renal function tests, such as bun and creatinine levels, overestimate renal function in patients with liver failure because malnutrition and muscle wasting contribute to a low creatinine level and liver dysfunction impairs urea synthesis. the hepatorenal syndrome is treated with the administration of vasopressin-1 agonists (i.e., terlipressin), tips, and, most reliably, liver transplantation. one uncontrolled trial using terlipressin with albumin for a median duration of 26 days (range 8-68 days) showed improvement in serum sodium as well as a decrease in the creatinine level below 2 mg/dl (mulkay et al. 2001) . hemodialysis is a temporary measure and its efficacy is not reliable. the only primary preventive measure showing some promise is the administration of albumin along with antibiotics as soon as the presence of spontaneous bacterial peritonitis is diagnosed; this possibly works by preventing hypovolemia and subsequent activation of vasoconstrictor systems. all phases of hemostasis are impaired in patients with liver disease, including clot formation, fibrinolysis, and their inhibitory processes. thrombocytopenia is found in 30-64 % of cirrhotic patients, and platelet count is commonly below 75,000/mm 3 . thrombocytopenia is primarily caused by splenomegaly associated with portal hypertension, which pools up to 90 % of platelets in the spleen. however, the degree of thrombocytopenia does not closely correlate with the size of the spleen. impaired hepatic synthesis of thrombopoietin also leads to thrombocytopenia. thrombopoietin is involved in the maturation and formation of platelets, and its return to a normal level coincides with a gradual increase in platelet count by the fifth day after liver transplantation (kawasaki et al. 1999) . other contributing factors are increased destruction of platelets by immune mechanisms, excessive activation of coagulation, and direct bone marrow suppression by toxins such as ethanol and folate deficiency. additionally, platelet dysfunction is common, as demonstrated by impaired platelet aggregation to adenosine diphosphate (adp), collagen, and thrombin (rubin et al. 1979) . the liver produces all coagulation factors except for von willebrand factor. therefore, plasma levels of clotting factors are directly related to the severity of liver disease, and prothrombin time (pt) is considered to be one of the most sensitive hepatic synthetic function tests. the plasma fibrinogen level, being an acutephase reactant, typically is normal or increased in chronic liver disease. a reduction in the fibrinogen level may indicate either a greatly reduced hepatic reserve or significant extravascular loss to ascites. markedly prolonged thrombin time indicates the presence of dysfibrinogenemia in some patients. dysfibrinogenemia is characterized by an excessive number of sialic acid residues in the fibrinogen molecule and abnormal polymerization of fibrin monomers. its clinical significance is unclear. patients with liver disease have a tendency to develop fibrinolysis due to decreased hepatic clearance of plasminogen activators, especially tissue plasminogen activator (tpa), and reduced production of α 2 -antiplasmin and thrombin activatable fibrinolysis inhibitors (van thiel et al. 2001) . elevated levels of d-dimers, fibrin degradation products, and plasminogen are present in ascitic fluid, indicating that absorption of ascitic fluid may contribute to the hyperfibrinolysis. on the other hand, excessive activation of coagulation is common in liver disease because of inadequate hepatic clearance of activated coagulation factors, reduced level of coagulation inhibitors, and enlarged vascular beds. the hypercoagulable state may lead to localized or disseminated intravascular coagulation (dic), particularly in the presence of sepsis, trauma, or major surgery. the diagnosis of excessive activation of coagulation is based on the presence of a known triggering factor and the progressively worsening of coagulation with thrombocytopenia. an anesthesia consultation is performed once a patient with esld is referred to the liver transplantation center. the type of liver disease is identified because patients with hepatocellular disease may have more pronounced hepatic dysfunction than those with cholestatic disease or hepatocellular cancer, and certain types of liver disease may affect other vital organ function (i.e., hemochromatosis, familiar amyloidosis, etc.). the anesthesia consultation is focused on evaluation of the functional reserve of extrahepatic organs, and various tests or specific consultations may be requested (table 2) . cardiovascular assessment is performed to determine two things: (1) whether a patient can be expected to survive the operation and immediate postoperative period; and (2) whether transplantation in patients with severe cardiopulmonary disease would be futile and an inappropriate use of a scarce donor organ (lentine et al. 2012) . a suggested strategy for cardiac assessment is shown in fig. 3 (raval et al. 2011) . overall cardiac performance is evaluated by transthoracic echocardiography to assess myocardial contractility, abnormality in cardiac anatomy, intracardiac or intrapulmonary shunting, and pulmonary artery pressure. most patients over age 50 years undergo non-invasive stress testing because they may have multiple cad risk factors (i.e., diabetes, hypertension, hyperlipidemia, and pre-existing cardiovascular disease), and limited physical activity masks underlying ischemic heart disease. an exercise stress test may not be feasible in many patients with advanced liver disease, and dobutamine stress echocardiography (dse) is commonly used, although adenosine or dipyridamole may be used when dobutamineinduced tachycardia is not desirable. dse, with its high sensitivity and specificity, appears to be the most reliable screening test (plotkin et al. 1998) , and dobutamine-induced tachycardia may mimic intraoperative stress on the cardiovascular system. on the contrary, dse has been reported to have poor sensitivity (as low as 13 %) and negative predictive value (as low as 75 %) (harinstein et al. 2008) , and its results may not correlate with adverse cardiac events within 30 days after transplantation (safadi et al. 2009 ). cardiac ct scan is a non-invasive technique measuring calcium deposits within the coronary vasculature. the total amount of calcium, adjusted to the age and gender of the patient, is reported as a calcium score. high scores suggest a greater potential for coronary artery stenosis (shaw et al. 2003; o'rourke et al. 2000) , and a calcium score of >400 has a predictive value of cardiac complications within 1 month after transplantation (kemmer et al. 2014 ). this test, however, may have limited predictive value as a single screening study for cad. cardiac ct angiography is an alternative to invasive coronary angiography. it does appear to have negative predicting value of 100 % for clinical coronary events in patients undergoing liver transplantation (cassagneau et al. 2012 ) but may not be suitable for the diagnosis of obstructive lesions at this time. because of the difficulty in diagnosing cad using non-invasive testing methods, coronary angiography is recommended for patients with a positive dse or multiple high-risk factors to identify the degree and type of obstruction. in addition, coronary angiography should be able to detect non-obstructive lesions (coronary artery stenosis <50 %), which are unlikely to be detected by stress tests but can be responsible for acute coronary syndromes (unstable angina, myocardial infarction, or sudden cardiac death) (rubin et al. 1994; gulati et al. 2009 ). cardiac catheterization, however, can be difficult in patients with severe liver disease due to bleeding complications and the increased risk of contrast-induced nephropathy (sharma et al. 2009 ). if significant coronary artery stenosis (>70 % stenosis) is detected, revascularization may be table 2 liver transplantation evaluation at the thomas jefferson university hospital attempted before liver transplantation. bare metal stents are favored over drug-eluting stents to avoid the need for long-term antiplatelet therapy (6 weeks vs. 1 year). when angioplasty is not amenable, coronary artery bypass grafting (cabg) is performed. it is clear that 1-year survival after cabg is greater in patients with child-pugh class a (80 %) than with child-pugh class b (45 %) and c (16 %) (filsoufi et al. 2007 ). therefore, patients with child-pugh class a can undergo cabg relatively safely while waiting for liver transplantation. on the other hand, patients with child-pugh class b and c may require simultaneous cabg and liver transplantation. patients with mild-to-moderate valvular disease undergo liver transplantation without excessive complications. similar to that of cabg, mortality after corrective valvular surgery depends on the severity of liver disease. therefore, child-pugh class c patients with severe aortic or mitral valve stenosis may undergo percutaneous balloon valvuloplasty or simultaneous valve replacement with cardiopulmonary bypass and liver transplantation. myocardial disease is commonly detected by transesophageal echocardiography (tee). patients with chronic cardiomyopathy may have attenuated systolic contraction and diastolic relaxation, altered repolarization, and reduced cardiac response to β stimulation (liu et al. 2002 for pulmonary evaluation, results of chest x-ray, arterial blood oxygen tension in 100 % oxygen, and spirometry are reviewed to identify the degree of pulmonary shunting, obstructive, or restrictive disease. when the hepatopulmonary syndrome is suspected, contrast tee or tc-99 m macro aggregated albumin scintigraphy may be performed for its definitive diagnosis (krowka et al. 2000) . for evaluation of renal function, results of bun, creatinine, glomerular filtration rate, levels of serum and urine electrolytes, urine output, and renal ultrasound are reviewed. the diagnosis criteria of the hepatorenal syndrome have been described earlier. in patients with chronic renal failure, simultaneous liver and kidney transplantation is performed, the criteria for which are end-stage renal disease with dialysis, no dialysis but a glomerular filtration rate <30 ml/min and proteinuria >3 g/day with a 24-h urine protein/creatinine ratio >3, and acute kidney injury requiring dialysis at least twice per week for more than 6 weeks (charlton et al. 2009 ). in patients with fulminant hepatic failure, reversibility of the neurologic function should be investigated using clinical signs, eeg, brain ct scan, the cerebral metabolic rate of oxygen, and tcd. in addition, icp monitoring is recommended when a high icp (>25 mmhg) is suspected, although its benefit should be weighed against potential complications (vaquero et al. 2005) . poor prognostic indicators of fulminant hepatic failure are progressive hepatic failure for 7-14 days, grade 3-4 encephalopathy, intracranial hypertension, cerebral swelling, severe coagulopathy, rapid shrinkage of the liver, metabolic acidosis, hemodynamic instability, and sepsis. for the coagulation system, pt, activated partial thromboplastin time (aptt), and platelet count are reviewed. in general, no specific coagulation therapy is requested because of the potential long waiting period and fluid overloading. abdominal magnetic resonance imaging (mri) is reviewed to assess the degree of portosystemic shunting and anatomy of hepatic vasculature. additional consultation may be requested from various specialists to identify the type and severity of the specific organ dysfunction. after the evaluation, all information of the potential recipient is compiled to stratify whether the patient's condition can be optimized or meet the criteria of contraindications. contraindications are diseases or conditions patients could have that may not improve survival after liver transplantation. they include malignancy with poor prognosis, active bacterial and viral infection, severe cardiopulmonary dysfunction, and technical difficulties. active alcoholism is a contraindication, although demonstrable abstinence for 6 months is considered acceptable. the presence of multiple organ dysfunction is a relative contraindication for liver transplantation as the 2-year survival is approximately 25 %. indications and contraindications of liver transplantation, however, have evolved over the past 50 years, and further modifications are expected to occur. anesthesiologists participate in the transplantation candidate selection committee for discussion of the hepatic disease, its complications, and the extrahepatic organ function of each patient. once the patient is placed on the active candidate list, the united network for organ sharing (unos) is notified and the patient is given a meld (model for end-stage liver disease) or peld (pediatric end-stage liver disease) score for fair distribution of donor livers. although surgical techniques are fully described elsewhere, a brief description of their physiologic effects is warranted here. in olt, after removal of the diseased liver, the donor liver is placed anatomically in the right upper quadrant. for the convenience of description, the procedure is divided into three stages: stage 1 (dissection stage), stage 2 (anhepatic stage), and stage 3 (neohepatic stage). the dissection stage begins with an inverted y-shaped bilateral subcostal skin incision and ends with the skeletonization of the diseased liver. the anhepatic stage begins with the occlusion of the hepatic artery, portal vein, and ivc for hepatectomy. however, the patient is virtually anhepatic once the hepatic artery or portal vein is occluded. three surgical techniques are used for hepatectomy and vascular reconstruction during the anhepatic stage: olt with simple venous crossclamping, olt with venovenous bypass, and the piggyback technique. in olt with simple venous cross-clamping, the diseased liver is removed together with the retrohepatic portion of the ivc after crossclamping of the suprahepatic and infrahepatic ivc, hepatic artery, and portal vein (fig. 4) . after surgical hemostasis of the hepatic bed, the donor liver is placed in the right upper quadrant, and sequential anastomoses of the suprahepatic ivcs, infrahepatic ivcs, portal veins, and hepatic arteries are performed. during the infrahepatic ivc anastomosis, the liver allograft is flushed with 1000 ml of cold lactated ringer's solution or 5 % albumin solution through a cannula in the portal vein. this flush technique allows preservation solution, metabolites, and air in the donor liver to escape through the incompletely anastomosed infrahepatic ivc. a second flush may be used by allowing 300-500 ml of blood to escape through the incompletely anastomosed portal vein by unclamping the infrahepatic ivc (back-bleeding technique). when the portal vein of the recipient is less than optimal, the superior mesenteric vein, collateral vein, or venous graft may be used for portal blood supply. the hepatic artery is reconstructed by end-to-end hepatic arterial anastomosis. however, an arterial graft is placed between the graft hepatic artery and the infrarenal aorta of the recipient with a side clamp on the aorta when the size or anatomy of the recipient hepatic artery is less than optimal. the liver is reperfused by the sequential unclamping of the infrahepatic ivc, portal vein, suprahepatic ivc, and hepatic artery. after hemostasis, choledochocholedochostomy is performed frequently with a t-tube. choledochojejunostomy using a roux-en-y loop is performed when the bile ducts are diseased or mismatched in size. the abdomen is closed once the absence of foreign bodies in the peritoneal cavity is confirmed. in patients with a large graft or swollen intestine, the abdomen may require secondary closure. olt with venovenous bypass was developed in 1983 to minimize reduction of venous return associated with the cross-clamping of the ivc and portal vein by diverting blood from the ivc and portal vein to the axillary vein using a centripetal magnetic pump (shaw et al. 1984) . once the hepatic hilum is dissected, cannulas are inserted into the left superficial femoral vein (7 mm) and portal vein (9 mm) for outflow from the patient and into the left axillary vein (7 mm) for venous inflow. the cannula site and size may vary depending on the preference of the surgical team or anatomic variations. the cannulas and heparin-bonded tubings are flushed with heparin solution (2000 u/l) to avoid thrombosis during preparation. systemic heparinization is not used because of the presence of pre-existing coagulopathy and the use of heparin-bonded tubings. the bypass run begins by unclamping all cannulas while the pump speed is gradually increased to achieve the maximal flow rate. hepatectomy and anastomoses of the suprahepatic and infrahepatic ivc are performed once full bypass is achieved. the removal of the portal cannula for portal venous anastomosis leads to a partial bypass, which reduces venous return. bypass is terminated after the engrafted liver is reperfused, and cannulas are removed. the advantages of venovenous bypass are (1) well-preserved cardiac output by uninterrupted venous return from the viscera and lower extremities; (2) effective decompression of the portal venous system, which decreases bleeding and intestinal congestion; (3) avoidance of renal congestion, oliguria, and hematuria; and (4) simplified anhepatic stage allowing meticulous hepatectomy and vascular anastomoses. long-term complications are neurovascular injury, thrombosis, infection, lymphocele, and seroma at the cannulation sites. as an alternative to the traditional venovenous bypass technique, percutaneous cannulation was introduced. in this technique, inflow to the patient is achieved by percutaneous cannulation of the right internal jugular vein (16-20 french) performed by the anesthesia team using a seldinger technique, and outflow from the patient by percutaneous cannulation of the left femoral vein (16-20 french) and a portal cannula by the surgical team. this technique is generally safe, but the inadvertent extravascular placement of an inflow cannula may cause a massive hemothorax (sakai et al. 2007 ). the piggyback technique was originally designed for patients with significant cardiovascular disease, portacaval shunt, superior vena caval syndrome, or small donor livers (tzakis et al. 1989) . in this technique, the diseased liver is removed without the retrohepatic portion of the ivc by peeling the diseased liver off the ivc after transaction of the hepatic veins, hepatic artery, and portal vein. therefore, systemic venous return can be relatively well preserved via the intact ivc during the anhepatic stage. vascular anastomoses are made between the reconstructed ostia of the recipient by combining hepatic veins and the suprahepatic ivc of the graft for the drainage of the hepatic venous blood. the portal vein of the recipient and the graft are anastomosed for portal blood supply, and the infrahepatic ivc of the graft is ligated. the neohepatic stage begins with reperfusion of the grafted liver by sequential unclamping of the infrahepatic ivc, portal vein, suprahepatic ivc, and hepatic artery, although the sequence of unclamping may vary depending on the surgical technique. reperfusion is followed by hepatic arterial anastomosis (if it has not been performed already), biliary reconstruction, and closure of the abdomen. immediate preoperative consultation is made when a donor organ is identified. the patient is re-evaluated to identify any interval changes during the waiting period. anesthetic and postoperative management and their risks are explained to the patient one more time. in general, pre-medication is withheld in most cases because of potential encephalopathy and hypovolemia, and narcotics (e.g., fentanyl 1-5 μg/kg) are commonly administered intravenously in the operating room. necessary medications and anesthesia equipment are listed in table 3 . a device that delivers fluids and blood rapidly on demand is considered standard equipment (i.e., fms2000 ® fluid warming system, belmont instrument corp., billerica, ma, usa) (elia and kang 2002 ). an autotransfusion system is helpful in minimizing the need for bank blood (dzik and jenkins 1985; kang et al. 1991) . a system that monitors coagulation, either a conventional coagulation profile, thromboelastography r with circle (teg; haemonetics, braintree, ma, usa), or (kang 1986 (kang , 1997 . teg and rotem provide similar physical properties of blood coagulation, although teg monitors shear elasticity and rotem monitors viscoelasticity. in general, 20 units each of cross-matched packed rbcs (prbcs) and fresh frozen plasma (ffp) are available at all times, and 10 units of each are prepared in the operating room. platelets (10-20 units) should be available on demand. two large-bore intravenous (iv) catheters (up to 8.5 or 9 french) are secured, typically in the right antecubital and right or left internal jugular vein. when the antecubital vein is unavailable, two catheters may be placed in the same internal jugular vein. catheter patency is confirmed by noting the line infusion pressure of <300 mmhg during fluid infusion at 500 ml/ min. sterile technique should be followed during catheterization, and antiseptic ointment or antiseptic patch is applied at the skin puncture site. a nasogastric tube is placed with copious lubrication and topical vasoconstrictor to avoid nasal or esophageal variceal bleeding. proper monitoring is prerequisite to a successful outcome because patients undergoing liver transplantation develop clinically significant hemodynamic, hematologic, metabolic, and other homeostatic abnormalities. non-invasive monitoring is similar to that for patients undergoing any major surgery. for invasive monitoring, two intra-arterial catheters (20 gauge in the left radial artery and 16-18 gauge in the right femoral artery) are used at the thomas jefferson university hospital. femoral arterial pressure monitoring is preferred because it reflects central arterial blood pressure more accurately in the presence of low systemic vascular resistance, particularly after reperfusion (lee et al. 2015) . radial arterial pressure monitoring is useful for blood sampling and backup pressure monitoring when the aorta is partially or completely clamped during aorta-tohepatic artery anastomosis. a pulmonary artery catheter (pa catheter) is inserted via the right internal jugular vein to monitor cardiac output, intracardiac pressures, and core temperature. carotid artery puncture should be assiduously avoided because of the presence of coagulopathy. an oximetric-type pa catheter provides additional information on mixed venous hemoglobin oxygen saturation (svo 2 ). the right ventricular ejection fraction-type pa catheter monitors the right ventricular ejection fraction and right ventricular end-diastolic volume. it has been shown that central venous pressure (cvp) and pulmonary capillary wedge pressure (pcwp) are not as sensitive as right ventricular end-diastolic volume in estimating preload, particularly during the anhepatic stage (dewolf et al. 1993b ). recently, non-invasive, continuous cardiac output monitoring was introduced; however, the technique is not reliable in monitoring cardiac output in hyperdynamic patients. in some centers, a cvp catheter is used instead of a pa catheter. this, of course, is justified if hemodynamic derangement is kept minimal during the entire surgical procedure. however, most centers use a pa catheter for three reasons: (1) hemodynamic instability can be unpredictable during liver transplantation; (2) determination of cardiac output and preload is more clinically significant than cvp monitoring; and (3) it is an important educational tool for trainees. tee is used in all patients at the thomas jefferson university hospital to monitor myocardial contractility, ventricular end-diastolic volume, wall motion abnormality, air or thromboembolism, intrapulmonary shunting, and patency of the reconstructed major veins. a tee probe may cause esophageal variceal bleeding (burger-klepp et al. 2012) and it should therefore be placed gently. various laboratory tests are performed, including arterial blood gas tension and acid-base state, and serum level of electrolytes, ionized calcium, glucose, lactate, and ionized magnesium if available. typical test times are before and after induction of anesthesia, every hour during the dissection stage, 5 min after the onset of the anhepatic stage, every 30 min during the anhepatic stage, 15 min before reperfusion, 5 and 30 min after reperfusion, and every hour thereafter. coagulation is monitored by conventional coagulation profile (pt, aptt, fibrinogen level, and platelet count) and teg or rotem at the following times: before induction of anesthesia, every hour during the dissection stage, 15 and 60 min after onset of the anhepatic stage, 15 min before reperfusion, 5 and 30 min after reperfusion, and every hour thereafter. monitoring of teg or rotem and the platelet count is preferable as a conventional coagulation profile has several drawbacks when used during liver transplantation (kang 1995) . pt is a very sensitive hepatic function test and is prolonged in most patients undergoing liver transplantation. administration of ffp to correct the pt may not be possible or desirable in the course of surgery. aptt follows a similar time course to pt, and its correction may not be practical. it is a sensitive test for the heparin effect, and its prolongation indicates the presence of heparin released from the bypass circuit or grafted liver. the fibrinogen level is frequently maintained within the acceptable range, although severe hypofibrinogenemia may indicate either active fibrinolysis or excessive activation of coagulation. the level of fibrin(ogen) degradation products is usually elevated in most patients due to excessive activation of coagulation and reabsorption of defibrinated blood from the abdominal cavity and does not have any immediate clinical significance. further, coagulation profile results may not be available in a timely manner. teg/rotem has several advantages over a conventional coagulation profile and has been accepted as a standard coagulation monitoring tool by the asa (american society of anesthesiologists 2015). it rapidly and reliably measures blood coagulability (quality) instead of the quantity of each coagulation component. an accurate differential diagnosis can be made for replacement therapy and pharmacologic therapy by comparing teg/rotem of untreated blood with that of blood treated with various blood components (ffp, platelets, cryoprecipitate) or pharmacologic agents (protamine sulfate, heparinase, ε-aminocaproic acid [eaca], aprotinin) (fig. 5 ) . lastly, circumferential identification tags around the wrists or ankles are removed to avoid the compartment syndrome. both arms are placed on padded arm boards in an abducted position, and excessive abduction should be avoided to prevent a plexus stretch injury. the extremities are protected with foam padding to avoid pressure injuries. a rapid-sequence induction is preferred because of uncertain gastric emptying. anesthesia is commonly induced with propofol (2-3 mg/kg) or etomidate (0.3 mg/kg), and fentanyl (2-5 μg/kg) is frequently added. succinylcholine (1-2 mg/kg) or rocuronium bromide (1.2 mg/kg) is used to facilitate intratracheal intubation. anesthesia is maintained using volatile inhalation agents and narcotics. isoflurane is the preferred inhalation agent because its effect includes less myocardial depression and biotransformation. nitrous oxide is avoided because it distends the bowel and increases the size of any entrained air. midazolam (1-4 mg) may be added for amnesia. for muscle relaxation, protamine-treated blood untreated blood 5 min after reperfusion fig. 5 effects of pharmacologic agents on pathologic coagulation immediately after reperfusion (from kang yg (1986) monitoring and treatment of coagulation. in: winter pm, kang yg (ed) hepatic transplantation, anesthetic and perioperative management. prager, new york, with the permission of the publisher) rocuronium bromide, vecuronium bromide, or cisatracurium besilate are commonly used. antibiotics and immunosuppressants administered during surgery may vary from center to center. at the thomas jefferson university hospital, unasyn ® (ampicillin/sulbactam 3 g) is given before incision and every 4 h thereafter. for patients allergic to cephalosporin or penicillin, vancomycin is administered within 2 h before skin incision (1 g for patients <80 kg and 1.5 g for those >80 kg). for immunosuppression, methylprednisolone (500 mg iv) and basiliximab (20 mg iv) are given during the anhepatic stage and tacrolimus is given in the postoperative period. liver transplantation imposes a great deal of physiologic stress on patients, and maintenance of physiologic homeostasis is essential to a successful outcome. the goal of hemodynamic management is to optimize tissue perfusion by maintaining the hyperdynamic state characteristic of esld. in general, there are two schools of thought about maintaining hemodynamic stability. the first endorses maintaining the hyperdynamic state to optimize cardiac output and tissue perfusion. patients with esld have generalized vasodilation and are known to have oxygen debt at the tissue level. therefore, maintaining the hyperdynamic state, instead of 'normal blood pressure,' ensures ample oxygen delivery to tissues and avoids tissue acidosis. this, in turn, optimizes tissue metabolism and hepatic blood flow. the second school of thought endorses maintaining arterial blood pressure within the normal range. this may include the use of various vasopressors (phenylephrine, norepinephrine, vasopressin, etc.) with or without hypovolemia. in extreme cases, blood is removed from the patient to induce hypovolemia, and blood pressure is supported by vasopressors (massicotte et al. 2010) ; it has been claimed that blood loss is minimal without increasing perioperative complications, although fluid restriction may lead to tissue ischemia, renal failure, and air embolism (melendez et al. 1998; schroeder et al. 2004 ). further, α-vasopressors (norepinephrine and phenylephrine) decrease hepatic blood flow by reducing portal venous flow dramatically in the presence of a limited hepatic arterial buffer response (mehrabi et al. 2005 ). hemodynamic instability represented by reduced cardiac output and hypotension is typically caused by hypovolemia associated with drainage of ascites, rapid third-space fluid loss, surgical bleeding, and inadvertent compression of major vessels (ivc, portal vein, hepatic veins, and aorta). intravascular volume is usually replenished by administration of a mixture of prbcs and ffp (typically, prbc:ffp: plasmalyte-a ® = 200:300:250 ml) using a rapid-infusion device. this mixture yields hematocrit of 26-28 vol.% and coagulation factor levels of 30-50 % of normal. a low hematocrit is chosen to optimize microcirculation and minimize the rbc wastage. calcium-containing fluid (i.e., lactated ringer's solution) should not be used to prevent clot formation in the reservoir of the rapid-infusion device. continuous administration of ffp is necessary to compensate for the loss of coagulation elements (procoagulants, prolysins, and their inhibitors) by surgical bleeding and excessive activation of coagulation. in patients with minimal blood loss, colloids (albumin or ffp) and crystalloids may be required to compensate for the third-space fluid loss and continuous production of ascites. close communication with the surgical team is essential to identify the cause of hemodynamic instability, as is communication with the blood bank to facilitate adequate supply of blood products. intraoperative autotransfusion has been shown to be effective and safe during liver transplantation, and its use may be considered when the prbc requirement is >5 units. its use is not recommended for patients with peritoneal infection or malignancy (liang et al. 2008 ). when lower cardiac output and/or hypotension persists even with adequate preload, dopamine or epinephrine may be infused for patients with hypotension, while dobutamine can be used when patients are normotensive. high venous pressures (cvp and pcwp) may be seen in patients with volume overload, large ascites, and pleural or pericardial effusion. drainage of ascites and effusion may decrease intrathoracic pressure and central venous pressures and improve cardiac performance. thoracentesis and pericardiocentesis can be performed after the abdomen is opened to minimize the risk of injury to the thoracoabdominal organs. unexpected pulmonary hypertension may be observed in some patients. because of the high perioperative mortality in patients with pulmonary hypertension, it deserves a thorough intraoperative investigation. the pa catheter should be able to differentiate between pulmonary hypertension with high pulmonary vascular resistance and pulmonary hypertension with fluid overloading. pulmonary hypertension caused by fluid overloading may dissipate gradually by intraoperative fluid loss, and phlebotomy may be required in severe hypervolemia. in portopulmonary hypertension with increased pulmonary vascular resistance, the presence of right ventricular function is investigated. a low cardiac output with a high cvp suggests the presence of right ventricular dysfunction. tee findings of right ventricle dysfunction are low fractional area change (fac), tricuspid annular plane excursion (tapse) of <16 mm, flattening of the ventricular septum, apicalization of the right ventricle, and right ventricular dilation. additionally, the pulmonary vascular response to various vasodilators (i.e., diltiazem, nitroglycerin, epoprostenol, and nitric oxide) may be evaluated. liver transplantation may continue when pulmonary hypertension is mild to moderate with normal right ventricular function. in such cases, right ventricular function is supported by maintaining optimal preload and improving myocardial contractility by inotropes. complications of massive blood transfusion (ionic hypocalcemia, ionic hypomagnesemia, hyperkalemia, and acidosis) may develop at this stage and should be treated aggressively. normothermia can be well-maintained even during massive transfusion when a rapid-infusion device is used. hemodynamic changes that occur during the anhepatic stage are caused primarily by interruption of venous return from the ivc and portal vein. in the simple cross-clamping technique, clamping of the ivc and portal vein reduces venous return by up to 40 %, leading to low cardiac output, hypotension, and compensatory tachycardia (pappas et al. 1971) . calculated systemic vascular resistance is frequently elevated, although this is a reflection of the exclusion of the vascular tree of the lower extremities and splanchnic bed. it is noteworthy that cross-clamping of the ivc and the portal vein decreases the central blood volume and pressure (cvp and pcwp) but progressively increases total intravascular blood volume as blood is sequestered in the vascular bed of the gastrointestinal and pelvic organs, kidneys, and lower extremities. a prolonged low output state, portal hypertension, and renal venous congestion may lead to acidosis, intestinal swelling, and hematuria. we, at thomas jefferson university hospital, prefer to treat the low output state by administration of fluid and/or inotropes (dopamine or dobutamine 2-5 μg/kg/min). venovenous bypass is more physiologic technique than a simple cross-clamping technique as it returns venous blood from the portal and ivc system (shaw et al. 1984) . hence, hemodynamic changes that occur during the anhepatic stage with venovenous bypass are minimal when the bypass flow rate is greater than 25 % of the baseline cardiac output and, therefore, the bypass flow should be monitored and adjusted as needed. improper positioning of the cannula tip in the femoral or portal vein, or a kinked bypass circuit, may not drain blood adequately and the surgical team should correct their positions. a low pump speed reduces venous return, while a high pump speed collapses the outflow venous wall and decreases the bypass flow. the perfusionist, therefore, should adjust the pump speed to maximize the bypass flow. in addition, hypovolemia decreases the bypass flow, and it should be corrected by the anesthesia team. the anesthesia and surgical teams should be prepared for potential acute complications of venovenous bypass. bleeding or air entry may result from venous laceration during cannulation or improperly secured cannulas. entry of a small volume of air (up to 50 ml) into the bypass pump may not cause immediate systemic air embolism because it is trapped in the cone-shaped pump head by centripetal force. thromboembolism may be caused by the migration of pre-existing thrombi or those developed during a low bypass flow rate (<1000 ml/min), particularly in hypercoagulable conditions (i.e., budd-chiari syndrome, neoplasms, and congenital protein c deficiency). most importantly, the bypass may have to be terminated unexpectedly when serious complications occur. therefore, the anesthesia team should be prepared for unexpected cross-clamping of the ivc and portal vein at all times. after completion of the ivc anastomosis, the portal cannula is removed to facilitate the portal venous anastomosis, resulting in partial bypass. low bypass flow and low cardiac output during this period can be improved by the administration of fluids or dopamine, but full correction of central hypovolemia, as reflected on cvp, pcwp, or tee, is avoided to prevent fluid overload on reperfusion. in the original description of the piggyback technique, adequate venous return is maintained through the intact ivc and portal vein using portoaxillary venovenous bypass (tzakis et al. 1989 ). currently, many transplantation centers do not incorporate the portoaxillary venovenous bypass in the piggyback technique, which makes patients vulnerable to significant hypovolemia. hepatectomy in the presence of portal hypertension can be difficult, and hypovolemia is not uncommon as a consequence of inadvertent compression of the ivc and portal vein, partial side-clamping of the ivc, and cross-clamping of the portal vein during portal anastomosis. hence, temporary portacaval shunt or portal-axillary venovenous bypass may be instituted in surgically challenging patients to maintain preload. as described earlier, 1000 ml of cold lactated ringer's solution or albumin (5 %) is flushed through the portal vein and drained via the incompletely anastomosed ivc to remove preservative solution, metabolites, and air from the allograft. additionally, approximately 300-500 ml of blood may be allowed to escape through the incompletely anastomosed portal vein to enhance the washout by partial unclamping of the infrahepatic ivc (back-bleeding technique) immediately before reperfusion of the grafted liver. in this case, blood should be administered simultaneously to avoid hypovolemia. other factors that affect circulation during the anhepatic stage are similar to those of the dissection stage, although lactic acidosis, citrate intoxication, hypomagnesemia, hyperkalemia, and coagulopathy are more pronounced. hyperkalemia is treated by dextrose (5-10 g) and insulin (5-10 units) to move potassium intracellularly (dewolf et al. 1993a ). in severe hyperkalemia, prbc or phlebotomized blood can be washed using an autotransfusion system to remove potassium before transfusion (ellis et al. 1987) . at the end of the anhepatic stage, all biochemical variables are normalized to prepare for reperfusion. significant hemodynamic changes occur on reperfusion of the grafted liver (fig. 6) . unclamping of the infrahepatic ivc and portal vein results in transient hypovolemia and hypotension due to acute sequestration of the blood in the engrafted liver. unclamping of the suprahepatic ivc increases preload by mobilizing blood from the low extremities and splanchnic circulation. this is followed by severe hemodynamic changes, the so-called postreperfusion syndrome (aggarwal et al. 1993) . the postreperfusion syndrome, which occurs in approximately 30 % of patients, is defined by abrupt hypotension (below 70 % of the baseline value) that develops within 5 min of reperfusion and lasts for more than 1 min. other associated hemodynamic changes are bradycardia, high cvp and pcwp, low systemic vascular resistance, and conduction defects. acute reduction in myocardial contractility is observed in tee. the postreperfusion syndrome appears to be caused by a combination of several factors. for example, an acute increase in preload may result in right ventricular strain and an acute decrease in blood temperature (2-3 c) by the systemic entry of the cold preservation solution may decrease cardiac conduction and contractility. other physical factors are air embolism and thromboembolism, which may cause right ventricular strain or right ventricular outflow tract obstruction (ellis et al. 1989; suriani et al. 1996) . chemical factors involved are acute hyperkalemia and acidosis. systemic entry of hyperkalemic preservation solution increases serum potassium level to a very high level (up to 12 mmol/l), causing severe bradycardia and conduction defects (martin 1986 ). return of the acidic blood from the viscera and lower extremities increases the base deficit by 5-10 mmol/l. in addition, unknown endogenous vasodilators or myocardial depressants (i.e., vasoactive intestinal polypeptide, nitric oxide, and eicosanoid) released from the allograft or congested viscera may decrease systemic vascular resistance and impair myocardial function. several measures may be taken to prevent the postreperfusion syndrome, although they are not always successful. at the end of the anhepatic stage, blood volume is adjusted to avoid fluid overloading on reperfusion, and ionic hypocalcemia, hyperkalemia, and metabolic acidosis are corrected. prophylactic administration of cac1 2 (15 mg/kg), nahco 3 (0.5-1 mmol/kg), regular insulin (10 units), 50 % dextrose (1 ml/kg), and epinephrine (5-10 μg) are recommended by some centers (ellis et al. 1989) . once the postreperfusion syndrome develops, severe hypotension and bradycardia are treated with small doses of epinephrine (5 μg increments) to support contractility, heart rate, and vasomotor tone, followed by a dopamine or epinephrine infusion, if necessary. symptomatic hyperkalemia (tall, peaked-t wave, and widening qrs complex with bradycardia) is treated by administration of cac1 2 (15 mg/kg) and nahco 3 (0.5-1 mmol/kg). arrhythmias are treated in the standard fashion. when pulmonary edema develops, positive end-expiratory pressure (peep) is applied and inotropes may be given. patients who develop intracardiac or pulmonary embolism are supported by inotropes. when severe fluid overloading is a concern, phlebotomy may be considered. the postreperfusion syndrome dissipates gradually over the next 5-15 min, although low (1987)) systemic vascular resistance and hypotension with a high cardiac output may persist for several hours. when hypotension is suspected to cause tissue and myocardial ischemia, it may be treated with ephedrine, dopamine, or epinephrine. overzealous administration of fluids may result in hepatic congestion, while norepinephrine may interfere with hepatic blood flow by decreasing portal venous flow. octreotide and vasopressin may increase arterial blood pressure by decreasing portal pressure and flow, although its effects on hepatic circulation and metabolism are unclear (fayed et al. 2013; wagener et al. 2008) . hemodynamic changes that occur during hepatic arterial and biliary reconstruction are relatively minor, except for intermittent fluctuation of the preload associated with continuous third-space fluid loss and compression of the liver and great vessels. gas exchange is maintained satisfactorily in most patients. minute volume is gradually decreased during the anhepatic stage to match the reduced oxygen consumption and carbon dioxide production, and is increased during the neohepatic stage. alveolar recruitment maneuvers are performed intermittently to avoid atelectasis caused by pleural effusions, cephalad traction of the rib cage, and compression of diaphragm. intermittent endotracheal suctioning, using a suction catheter or bronchoscope, may be required to remove secretions. drainage of pleural effusions and ascites decreases intrathoracic pressure and improves oxygenation within 2 h. patients with preoperative ards may require a high fio 2 and a high level of peep to ensure adequate gas exchange, and a volume ventilator may be necessary to overcome the high airway pressure. frank pulmonary edema can develop, particularly after reperfusion, from the increased pulmonary capillary permeability or fluid overload. in such cases, patients are ventilated with a high level of fio 2 and peep, while the underlying cause is treated. closure of the abdominal cavity may interfere with ventilation by increasing intrathoracic and airway pressures. primary closure with mesh or secondary closure may be necessary. in patients with fulminant hepatic failure, preoperative cerebral monitoring is continued as cerebral hyperemia and intracranial hypertension persist during surgery, although they are somewhat attenuated by general anesthetics. however, a sudden increase in preload may dramatically exacerbate intracranial hypertension on reperfusion of the grafted liver. hence, optimal preload should be maintained during the entire procedure. after reperfusion, cerebral hyperemia may gradually decrease as the liver begins to function. intraoperative changes in coagulation are summarized in table 4 . surgical bleeding is common due to numerous collateral vessels associated with portal hypertension, difficulty in dissection of the diseased liver, pre-existing coagulopathy, and pathologic changes in coagulation. the average blood loss in adults is 5-15 units each of prbc and ffp, although blood loss may reach more than 100 units each. during the dissection stage, dilutional coagulopathy develops as bleeding reduces the levels of coagulation factors and platelets (fig. 7) . fibrinolysis may develop, particularly in patients with hepatocellular disease, as a result of a low level of inhibitors of fibrinolysis and impaired hepatic clearance of tpa (lewis et al. 1989a) . excessive activation of coagulation, evidenced by a gradual increase in thrombin-antithrombin complex, develops at the end of the dissection stage (kratzer et al. 1991) . management of coagulation begins with normalization of physiologic variables, such as ionic hypocalcemia, hypothermia, and acidosis impair coagulation (rohrer and natale 1992) . this is followed by continuous infusion of coagulation factor-rich blood (rbc:ffp: plasmalyte-a ® or normal saline = 1 unit:1 unit:250 ml) to maintain coagulation factor levels above the critical level (30-50 % of normal). specific blood components may be administered based on teg/rotem. in general, platelets (5-10 units) are administered for a small maximum amplitude (ma) (<40 mm). platelet administration, in addition to increasing ma, improves reaction time (r) and clot formation rate (α), because the coagulation cascade leading to fibrin formation occurs on the surface of platelets. its administration, however, is withheld during the anhepatic stage to avoid potential thrombosis and during massive blood transfusion (>150 ml/min) to minimize wastage. two units of ffp may be administered when the reaction time is prolonged (r> 12 min) even after platelet administration cryoprecipitate (6 units) containing factors i and viii are rarely required unless severe fibrinolysis is left untreated because plasmin selectively destroys factors i, v, and viii. however, cryoprecipitate may be used for patients with severe hypofibrinogenemia (<100 mg/dl). pathologic coagulation superimposes on dilutional coagulopathy during the anhepatic stage. the heparin effect is seen as a prolonged aptt and reaction time on teg/rotem at the onset of the venovenous bypass as a small dose of heparin (2000-5000 units) in the bypass circuit enters systemic circulation. this heparin effect dissipates over the next 30-60 min. the effects of the absence of the hepatic synthetic and clearance function begin to develop during this stage. the absence of hepatic clearance of tpa promotes fibrinolysis in approximately 30 % of patients . similarly, the absence of hepatic clearance of activated coagulation factors results in excessive activation of coagulation evidenced by a progressive increase in thrombin-antithrombin complex and fibrin (ogen) degradation products. severe fibrinolysis (fibrinolysis time <60 min) may be treated by the administration of a single, small dose of eaca (250-500 mg) ). administration of a large or repeated dose of eaca is not recommended in order to avoid potential thromboembolism (gologorsky et al. 2001) . the postreperfusion syndrome occurs in coagulation at the onset of the neohepatic stage. a typical coagulation profile shows prolonged pt, aptt, reptilase time, and thrombin time. a generalized decrease in coagulation factors (i, v, vii, and viii) and platelets is accompanied by a sharp increase in the tpa level, a shortened euglobulin lysis time, and a moderate increase in fibrin(ogen) degradation products and thrombin-antithrombin complex. fibrinolysis is observed in up to 80 % of patients and is severe in about 40 % . fibrinolysis is caused by a 20-fold increase in tpa being released from the allograft and congested viscera, which overwhelms the activity of the plasminogen activator inhibitor (virji et al. 1989; porte et al. 1989 ). there are ample data to support the finding that fibrinolysis is primary in origin: a relatively steady antithrombin level, only moderate levels of fibrin(ogen) degradation products and d-dimers, selective decreases in factors i, v, and viii, and no known microthrombi formation (lewis et al. 1989a, b) . fibrinolysis resolves over the 120 min following reperfusion. the heparin effect occurs in approximately 30 % of patients, as heparin is released from the allograft and dissipates over the next 60-90 min. to identify the presence of fibrinolysis and the heparin effect, teg/rotems of untreated blood (native), blood treated with antifibrinolytic agent (eaca or aprotinin), and blood treated with an agent neutralizing heparin (protamine sulfate or heparinase) are compared 5 min after reperfusion. when fibrinolysis is present, early treatment using a single, small dose of eaca (250-500 mg) is recommended in order to reduce delayed oozing and to minimize the loss of factors i, v, and viii . prophylactic administration of eaca or tranexamic acid (amca) is a common practice in many centers, but has not shown scientific efficacy. fibrinolysis prophylaxis is not recommended by the authors, because the presence of fibrinolysis can easily be detected by teg/rotem and can be treated effectively with a small dose of eaca in most patients. when the heparin effect is present, a small dose of protamine sulfate (25-50 mg) may be given in severe cases. in addition, blood coagulability can be impaired by reperfusion hypothermia, acidosis, and ionic hypocalcemia. in contrast, excessive activation of coagulation leading to fatal intracardiac or pulmonary embolism may occur in some patients (gologorsky et al. 2001; warnaar et al. 2008 ). this complication appears to be associated with a massive transfusion, release of a large quantity of tissue thromboplastin from the less than optimal allograft, impaired tissue perfusion, and possibly antifibrinolytic therapy. intracardiac thrombosis can be treated by infusion of tpa (40-100 mg over 2 h) while observing resolution of thrombi using tee (boone et al. 2011; jackson et al. 2006) . coagulopathy improves gradually after reperfusion. generalized oozing, however, may occur even in the presence of acceptable coagulation profiles and teg/rotem, possibly due to delayed bleeding caused by the loss of a poorly formed clot or by the residual effects of reperfusion fibrinolysis. several other pharmacologic agents are reported to improve coagulation. aprotinin (2,000,000 kiu followed by 500,000 kiu/h), a non-specific inhibitor of plasminogen and serine protease, may reduce blood loss by inhibiting fibrinolysis and excessive activation of coagulation (neuhaus et al. 1989; cottam et al. 1991 ). however, clinical use of aprotinin declined even before the drug was withdrawn by the manufacturer: clinical reports did not show a significant reduction in blood loss (ickx et al. 1993; groh et al. 1993) , and fibrinolysis can be treated with eaca or amca more efficiently with negligible side effects boylan et al. 1996) . recombinant factor viia (rfviia) has been suggested to improve coagulation and reduces bleeding by actively enhancing coagulation and stimulating fibrin formation in the presence of tissue factor. its beneficial effects have been shown in patients with fulminant hepatic failure, "critical bleeding," and a ruptured liver (meadows et al. 2011; merchant et al. 2004; yamaguchi et al. 2015) . however, results of clinical trials are controversial (planinsic et al. 2005; gasperi and baudo 2006; niemann et al. 2006 ) and a european consensus concluded that a paucity of data from clinical trials with rfviia limits both the strength and the scope of clinical recommendations (vincent et al. 2006) . recently, the use of prothrombin complex concentrate has been assessed in a limited number of centers. prothrombin complex concentrate is prepared from ffp and contains clotting factors ii, vii, ix, and x, protein c, and protein s, and its use may improve coagulation without increasing preload (arshad et al. 2013 ). however, it has limited components of coagulation and its clinical advantage requires further investigation. desmopressin acetate (ddavp), a synthetic analog of 8-arginine vasopressin, increases the endothelial release of factor viii, von willebrand factor, and plasminogen. its beneficial effects have been demonstrated in vitro and in patients with liver disease, and it may be used to improve coagulation (0.3 μg/kg) . conjugated estro-gen has been reported to improve coagulation and reduce blood loss (frenette et al. 1998) , although its use has not been accepted widely. calcium metabolism patients with hepatic dysfunction invariably develop ionic hypocalcemia during massive blood transfusion, which is caused by chelation of serum calcium with citrate in the banked blood. ionic hypocalcemia begins to appear during the dissection stage (marquez et al. 1986 ) and becomes severe during the anhepatic stage. the serumionized calcium level is inversely related to the serum citrate level, as the absence of hepatic metabolism of citrate increases the serum citrate level close to that in the banked blood (fig. 8) . significant hypocalcemia (ca 2+ <0.55 mmol/l) is associated with a prolonged q-t interval and decreases in the cardiac index, stroke-work index, and blood citrate level ca ++ level mg ++ level fig. 8 intraoperative changes in serum calcium, magnesium, and citrate level (results of two studies (marquez et al. 1986 and scott et al. 1996) are superimposed, with the permission of the publisher) pressure. therefore, the ionized calcium concentration is monitored hourly or more frequently, and cac1 2 (15 mg/kg) or calcium gluconate (30 mg/kg) is administered to maintain a normal level (martin et al. 1990 ). ionic hypocalcemia improves gradually as the engrafted liver begins to metabolize citrate, unless the speed of the transfusion exceeds the metabolic function of the liver. hypokalemia is not uncommon in patients with liver disease due to poor dietary intake of potassium and its loss from chronic diuretic therapy and diarrhea. severe hypokalemia (<2.5 mmol/l) is treated with potassium chloride to increase its level to 3.0-4.0 mmol/l. moderate hypokalemia (<3.5 mmol/l) is not treated because it is welltolerated by patients and self-corrected by blood transfusion. hyperkalemia is a serious concern because it interferes with myocardial conduction and contractility, particularly in the presence of acidosis and hypocalcemia. progressive hyperkalemia (up to 6-7 mmol/l) may occur in patients with renal dysfunction or those requiring massive blood transfusion. mild hyperkalemia (up to 5.5 mmol/l) is treated with insulin (10 units) and glucose (12.5 g). it has been shown that glucose and insulin therapy is effective in lowering the serum potassium level even in the absence of hepatic function (dewolf et al. 1993a) . for moderate-to-severe hyperkalemia (>5.5 mmol/l), in addition to insulin therapy, prbc or phlebotomized blood can be washed to remove potassium using an autotransfusion system before transfusion (ellis et al. 1987) . reperfusion hyperkalemia is caused by potassium influx from the preservation solution and hepatocytes, and its systemic effects and treatment have been described previously. acute hyperkalemia returns to a normal range within 5-10 min as a result of redistribution. the potassium level gradually returns to the baseline value as the rbcs and the engrafted liver take up excess potassium. hypokalemia (<3.5 mmol/l), which occurs toward the end of procedure, is treated using a kcl infusion (20 mmol increments). hyponatremia (<130 mmol/l) is a common occurrence in patients with liver disease, particularly those with fluid retention, ascites, diuretic therapy, and restricted sodium diet. the serum sodium level gradually increases towards normal during surgery via administration of blood products and a balanced salt solution. a rapid rise in the serum sodium level (>10 mmol/l) is a clinical concern because it may contribute to the development of central pontine myelinolysis, a serious neurological injury caused by the destruction of the myelin sheath in the pons (videira et al. 1991) . therefore, the preoperative serum sodium level should be raised to >130 mmol/l, if possible, and a rapid increase in sodium should be prevented by administration of low sodium-containing crystalloids during surgery. in addition, tromethamine (tham) is the preferred drug for treatment of metabolic acidosis as it does not contain sodium. hypernatremia may be seen in some patients who receive a large dose of nahco 3 preoperatively. this hypernatremia is gradually normalized by administration of blood products and a balanced electrolyte solution. a clinical investigation showed that the serum ionized magnesium level, similar to the ionized calcium level, has an inverse relationship with the serum citrate level as magnesium ion chelates with citrate in banked blood (scott et al. 1996) . although the clinical significance of ionic hypomagnesemia during liver transplantation is unclear, mgso 4 (1-4 g) can be administered to minimize potential cardiac irritability and myocardial depression. metabolic acidosis begins to appear during the dissection and anhepatic stages because of impaired hepatic metabolism of the acid load from the banked blood and the peripheral tissues. the base deficit and lactate level increase further (approximately 5 mmol/l) on reperfusion due to the acid load from the graft and congested viscera and lower extremities. it gradually improves as hepatic function is restored and tissue perfusion improves during the neohepatic stage. persistent lactic acidosis (>15 mmol/l) appears to be associated with graft dysfunction (begliomini et al. 1989) . metabolic acidosis is aggressively corrected by administration of nahco 3 to maintain base deficit levels <5 mmol/l because acidosis is frequently progressive and leads to myocardial depression, inadequate cellular respiration, and decreased sensitivity to catecholamines. as described earlier, tham is preferred in hypo-or hypernatremic conditions to minimize fluctuation of the serum sodium level: 150 ml of 0.3 m tham is equivalent to 50 mmol of nahco 3 . alternatively, dichloroacetate (40 mg/kg every 4 h) appears to reduce lactate production by stimulating pyruvate oxidation (shangraw and robinson 1997) . metabolic alkalosis may develop during the neohepatic stage, and this was believed to be associated with nahco 3 -administered and citrate metabolism-generating bicarbonate. however, it has been shown that the degree of metabolic alkalosis is unrelated to the citrate and nahco 3 load (fortunato et al. 1987) and may be associated with residual hyperaldosteronism. body temperature may gradually decrease to 34 c during the dissection stage as a result of the exposure of the abdominal contents to the cold environment, vasodilatation, and lack of shivering. hypothermia continues during the anhepatic stage as energy production decreases further. an abrupt decrease in core temperature (2-3 c) occurs on reperfusion as cold preservation solution enters systemic circulation. the temperature increases during the neohepatic stage, and the surgery ends with a body temperature of approximately 35-36 c. hypothermia is difficult to avoid, although raising the room temperature, application of forced warm air devices, use of a warming blanket, and a heat exchanger in the venovenous bypass system may be beneficial. the blood glucose level is relatively wellmaintained (100-200 mg/dl) with blood transfusion, as the banked blood contains glucose (approximately 200 mg/dl). a gradual decrease in glycogenolysis reduces the blood glucose level during the dissection and anhepatic stages. in patients with fulminant hepatic failure or severe hepatocellular disease, the blood glucose level may decrease precipitously, making glucose supplementation necessary. hyperglycemia (up to 300 mg/dl) occurs on reperfusion as glucose is released from the engrafted liver (dewolf et al. 1987 ). insulin does not appear to be effective in treating reperfusion hyperglycemia because glucose reuptake requires restoration of hepatic function. the insulin level is relatively steady during surgery, and the glucagon level increases after reperfusion. the blood glucose level usually returns to normal within 12-24 h. persistent hyperglycemia caused by impaired hepatic glucose reuptake and hormonal imbalance is an early sign of poor graft function (mallett et al. 1989 ). urine output is well-preserved in most patients once the intravascular volume is optimized. oliguria or anuria, however, may persist in patients with the hepatorenal syndrome or underlying renal disease. the presence of oliguria and hematuria during the anhepatic stage of the simple cross-clamping technique has been described earlier. urine output increases during the neohepatic stage as a result of the restoration of renal function and circulation. various agents have been tried to protect or improve renal function: the role of dopamine is controversial, dopexamine appears to be beneficial, and triple-drug therapy (dopamine [2-3 μg/kg/min], mannitol [250 mg/kg], and furosemide) improves urine output but not renal function (gray et al. 1991; planinsic et al. 1997 ). when fluid overload or severe electrolyte imbalance is a concern, intraoperative venovenous ultrafiltration or hemodialysis may be utilized. the restoration of hepatic function is evident about 2 h after reperfusion: levels of citrate and lactate decrease, the glucose level returns toward normal, coagulopathy improves, and bile production begins. persistent citrate intoxication, acidosis, hyperglycemia, coagulopathy, and pale-colored bile are poor prognostic signs. recently, tracheal extubation in the operating room has been successful in several centers when the patient meets the liver transplantationspecific extubation criteria, including the severity of pre-existing liver disease, blood loss, and hemodynamic stability (mandell et al. 2002; biancofiore et al. 2005; glanemann et al. 2007 ). however, most patients are still transported to the intensive care unit (icu) while receiving invasive monitoring and ventilatory support. upon arrival to the icu, the ventilator setting is reported to the respiratory therapist, the lungs are auscultated, and vital signs are displayed on the icu monitor. detailed intraoperative information is reported to the icu physician and nursing staff. primary non-function primary non-function is defined as graft failure occurring within 90 days after liver transplantation in the absence of either rejection or technical factors such as hepatic arterial thrombosis (bzeizi et al. 1997) . this complication occurs in up to 10 % of patients and is frequently caused by hepatic dysfunction of the donor liver or prolonged cold ischemia (>18 h). the patient develops progressive multi-organ failure including encephalopathy, coagulopathy, minimal bile production, and oliguria. supportive therapy may be helpful until the liver resumes its function, although urgent retransplantation is the only solution in many patients. worsening liver function without technical complications in the second week after liver transplantation suggests acute cellular rejection. biopsy findings are inflammation of the intrahepatic endothelium and bile duct and a mononuclear cell infiltration with eosinophilia (wiesner 1996) . hepatic arterial stenosis occurs in approximately 5 % of patients, and is four times more common in children. common clinical signs are biliary tract breakdown, recurrent bacteremia, hepatic abscess, and occasionally massive hepatic necrosis (tzakis et al. 1985) . hepatic arterial stenosis is suspected when an ultrasound examination reveals increased focal arterial flow velocities and is confirmed by angiography. in the immediate postoperative period, direct repair or reconstruction using an infrarenal arterial conduit is usually successful. stenosis occurring several weeks after transplantation is treated with percutaneous hepatic arterial angioplasty, which has a success rate of more than 90 % in achieving long-term patency. vena caval stenosis and thrombosis occur in 1-2 % of patients. in traditional liver transplantation, outflow obstruction is managed by balloon angioplasty with or without a metallic stent placement (simo et al. 1993) . in the piggyback technique, it is treated with end-to-side anastomoses between the donor infrahepatic ivc and the recipient retrohepatic ivc (stieber et al. 1997) . portal venous stenosis and thrombosis are relatively uncommon in the adult population and present with graft dysfunction, massive ascites formation, and hemodynamic instability. this complication is corrected by an urgent reconstruction of the portal vein or construction of a superior mesenteric venous graft to the liver, together with a ligation of large collaterals that may reduce the portal flow. biliary complications are more common in children and have an overall incidence of 8-15 %. early recognition is difficult, leading to high morbidity and mortality. bile leaks usually occur at the anastomotic site, although they may be found at the t-tube site or aberrant ducts. most biliary complications occur within the first 3 months and are diagnosed by liver function tests (serum bilirubin, γ-glutamyltransferase, and alkaline phosphatase) and imaging techniques. these complications are treated by percutaneous or endoscopic drainage of bile collections. in cases of roux-en-y choledochojejunostomy, surgical reconstruction is required. intra-abdominal bleeding occurs in about 7-15 % of patients and requires exploration in about half of these cases (ozaki et al. 1994) . gastrointestinal bleeding may develop from ulcers, viral enteritis, varices, and an afferent roux-en-y loop. variceal bleeding is usually associated with portal vein thrombosis and requires an urgent ultrasound or angiographic evaluation. bleeding from the roux-en-y limb occurs 1 week after surgery and is usually self-limited. additionally, bleeding can be caused by persistent thrombocytopenia associated with splenic sequestration, drug toxicity, heparin-induced thrombocytopenia, and immunologic reactions. intestinal perforation is caused by serosal injury to the intestines and usually occurs in patients who have had a technically difficult hepatectomy, prolonged portal venous clamping, or a massive blood transfusion. intestinal perforation or leakage is treated by urgent surgery and antifungal therapy. cardiac complications any type of cardiac complication can develop in the postoperative period. hypotension can occur due to hypovolemia, either from underresuscitation or ongoing bleeding, decrease in contractility secondary to the pre-existing myocardial disease, or new onset of dilated or ischemic cardiomyopathy. other potential causes are acidosis, hypocalcemia, or vasodilation from sepsis or graft failure. management of cardiac complications is based on the underlying cause. hypertension occurs in patients with pre-existing hypertension, inadequate pain control, hypoglycemia, and cerebral edema. restoration of normal liver function may increase systemic vascular resistance, and calcineurin inhibition can increase systemic blood pressure. calcium channel blockers (i.e., diltiazem and verapamil) are avoided because they can increase the levels of the calcineurin inhibitors. myocardial infarction is relatively rare due to thorough preoperative evaluation being undertaken to detect cad. however, when it does develop, a cardiologist should be consulted for possible emergent cardiac catheterization during surgery and revascularization. pulmonary edema is commonly seen postoperatively and may be caused by significant transfusion requirements, increased capillary permeability, prolonged intubation, and reversible dilated cardiomyopathy. reversible dilated cardiomyopathy with pulmonary edema may develop in the first 5 days after transplantation. sampathkumar et al. reported that 1 % of patients who did not have ventricular dysfunction developed dilated cardiomyopathy postoperatively, most of whom recovered completely without any long-term complications (sampathkumar et al. 1998) . the cause of this condition is unknown, although it may be a form of stress-induced cardiomyopathy. atrial fibrillation, ventricular tachycardia, and other arrhythmias may develop as a result of electrolyte abnormalities (i.e., hypomagnesemia, hyperkalemia, and hypocalcemia), cardiac ischemia, or irritation from cvp or pa catheters. in a study by xia et al., atrial fibrillation was observed in 7.4 % of patients and was associated with increased mortality, graft failure, and acute kidney injury (xia et al. 2015) . all arrhythmias are treated following the standard guidelines. thromboembolism is a common cause of sudden postoperative death. deep vein thrombosis should be prevented by early extubation and mobilization, use of compressive stockings, and administration of heparin (subcutaneous or low molecular weight). most patients require mechanical ventilation for only a few hours or days after transplantation. however, prolonged ventilatory support is required in some patients with atelectasis, pleural effusions, and central nervous system (cns) depression. intraoperative cross-clamping of the ivc occasionally results in right phrenic nerve crush injury and diaphragmatic paralysis in the immediate postoperative period (mcalister et al. 1993 ). ards may develop in patients with intra-abdominal infection, pancreatitis, hepatic necrosis, acute cellular rejection, and occasionally with muromonab-cd3 (okt3) treatment. bronchoalveolar lavage and bacterial culture are frequently performed to rule out pulmonary infection from any other pulmonary pathology. pre-existing pulmonary hypertension may persist postoperatively and is controlled by epoprostenol or nitroglycerin. neurological complications occur in 12-20 % of patients, mostly in the first week of transplantation (singh et al. 1994 ). these are more common in adults and present as mental status changes ranging from dysphasia to frank coma. dysfunction of the cns is commonly caused by medications, such as cyclosporine, tacrolimus, histamine h 2 -blockers, acyclovir, and antibiotics such as imipenem. non-convulsive seizures may occur, and an eeg is performed for patients with unexplained mentation changes. intracranial hemorrhage and watershed infarcts are ruled out using ct scans. hyponatremia and hypomagnesemia can also delay awakening. central pontine myelinolysis may develop several days after transplantation, and recovery is often slow and incomplete (winnock et al. 1993) . hepatic encephalopathy may be present for several days after transplantation in patients with persistent portosystemic shunting. meningitis should be ruled out when the mental status change is accompanied by fever. disseminated aspergillosis is a devastating complication in a patient with multiple brain infarcts and fever. peripheral neuropathy presenting as weakness is usually myopathic in nature and is more common in patients with preoperative severe liver disease, poor graft function, high steroid doses, and uremia, and are confirmed by electromyography and muscle biopsy. in patients with fulminant hepatic failure, cerebral hyperemia and hypertension usually decrease gradually, and the patient regains consciousness as the liver begins to function. renal dysfunction is usually transient and is commonly associated with intraoperative hypovolemia and hypotension, allograft dysfunction, and nephrotoxicity of cyclosporine and tacrolimus. oliguria is an early sign of renal dysfunction and is managed by restoring intravascular volume and renal perfusion. the hepatorenal syndrome may persist after transplantation, and its recovery depends on its preoperative severity and allograft function. in some patients, addition of vasoconstrictive immunosuppressants (cyclosporine and tacrolimus) may lead to acute tubular necrosis. in general, renal function returns to the normal range in most patients, and approximately 10 % of patients require temporary dialysis (mccaulley et al. 1990 ). long-term prognosis is fair, although hypertension, diabetes, and chronic nephropathy induced by steroids and the calcineurin inhibitors may result in chronic renal failure. more than half of the postoperative infections following liver transplantation are bacterial in origin. these infections typically occur in the first 2 weeks, when blood levels of immunosuppressants are high. the most common sites of infection are the liver, biliary tract, peritoneal cavity, and pulmonary system. common organisms in the abdomen are aerobic gram-positive organisms (streptococci and staphylococci) and gram-negative bacilli (escherichia coli, enterobacter species, and pseudomonas), while pseudomonas infection is most common in the lungs. approximately 20 % of infections are caused by fungus, with candida species accounting for more than 80 % of all fungal infections. the risk factors are a high steroid dosage, usage of broad-spectrum antibiotics, and prolonged surgical time. candida infection is treated with amphotericin or fluconazole. aspergillus infection accounts for 15 % of all fungal infections and is associated with a very high mortality; high-dose liposomal amphotericin b followed by prolonged itraconazole is the treatment of choice. viral infections are seen 2-3 months after transplantation, with cytomegalovirus and herpes simplex accounting for the bulk of these infections. epstein-barr virus is not usually seen until approximately 6 months after transplantation, but is an important cause of lymphoproliferative disease. pneumocystis pneumonia, an opportunistic infection, responds to trimethoprim-sulfamethoxazole. late metabolic complications following liver transplantation include diabetes, hyperlipidemia, weight gain, and hypertension. diabetes is induced by steroids, cyclosporine, and tacrolimus and may respond to oral hypoglycemic agents or insulin. hyperlipidemia is associated with diabetes, obesity, steroids, and immunosuppressive drugs and is treated by diet and exercise. hypertension is seen in as many as 85 % of patients after transplantation; the use of steroids or tacrolimus is the most likely cause. hypomagnesemia has been implicated as the cause of the hypertension in some cases. approximately 15 % of all patients require retransplantation of the liver. early retransplantation is performed within several days after the primary transplantation to rescue patients from primary non-function (graft factor), acute rejection, and technical failure (vascular thrombosis), or secondary non-function (host factor) associated with poor hepatic perfusion. hepatic necrosis is the common pathway of graft non-function and results in progressive, severe encephalopathy, ards, lactic acidosis, coagulopathy, hypoglycemia, and significant circulatory instability. although infrequent, hepatectomy with a portacaval shunt may be performed to protect the patient from the ill effects of the necrotizing liver on extrahepatic organ functions. in such a case, retransplantation should be performed as soon as the donor organ is available. the surgical procedure itself is relatively simple because surgical dissection has already been made and adhesions have not yet formed. anesthetic management of these patients is similar to that of patients undergoing primary transplantation. late retransplantation is performed in patients with chronic rejection, vascular complications, and recurrence of the original disease. the physical condition of the patient may have improved, but complications of immunosuppression (i.e., hypertension, renal insufficiency) may be present. adhesions and the steroid-induced fragile tissues frequently complicate late retransplantation. anesthetic management is similar to that of primary liver transplantation, but a large amount of blood loss is anticipated. in pediatric liver transplantation, rapid-sequence iv induction is preferred, although mask induction is chosen in patients in whom there is difficulty obtaining iv access (borland et al. 1985) . large-bore iv catheters are placed in the upper extremities after induction of anesthesia. a central venous catheter with cvp monitoring is the usual procedure, and pulmonary arterial catheterization is rarely indicated. blood pressure is monitored using a femoral intra-arterial catheter. it appears that children tolerate cross-clamping of the ivc and portal vein reasonably well without significant hemodynamic changes, possibly by compliant vasomotor tone. therefore, venovenous bypass is rarely used in children under 20 kg. coagulation changes that occur during liver transplantation are not as severe as those of adults, and this may be associated with more prevalent cholestatic diseases in children ). blood loss in children with biliary atresia can be large due to the technical difficulty associated with previous biliary surgery (i.e., ksai procedure). maintenance of body temperature is difficult, as the large surface area promotes heat loss. live-donor hepatectomy is usually a challenging procedure. the young and healthy donors (asa physical status [ps] 1 or 2) undergo a complete evaluation by hepatologists, surgeons, anesthesiologists, and psychologists. the anesthetic goals are minimizing surgical blood loss and allogeneic blood transfusion, maintaining liver blood flow, facilitating early extubation, preventing deep venous thrombosis and infection, and providing adequate postoperative pain control. preoperatively, donors may be given erythropoietin to boost rbc production and they can donate 2 units of autologous whole blood 2-3 weeks before surgery. on the day of surgery, donors may be given heparin (5000 units, subcutaneous injection) to prevent deep venous thrombosis, and 6 units of typed and cross-matched prbcs are prepared. in the holding area, a peripheral iv catheter is secured, anxiolytics are administered, and donors may elect to receive thoracic epidural anesthesia for postoperative analgesia. the need for epidural local anesthetics with or without narcotics is determined by the attending anesthesiologist and pain service. in the operating room, unasyn ® (3 g iv) or vancomycin (if allergic to penicillin) is administered to prevent infection, and the patient is positioned with minimal stress to the brachial plexus to avoid neurologic injury (dulitz et al. 2005) . induction and maintenance of anesthesia follows the standard guidelines of any major surgical procedure. ultra-short-acting narcotics such as remifentanil may be beneficial for early extubation after surgery as it is rapidly metabolized by plasma esterase and does not have a prolonged effect in the presence of hepatic and renal dysfunction. intraoperative monitoring is similar to that of patients undergoing major surgery, and a radial arterial catheter and cvp are placed for hemodynamic monitoring. additional ivaccess is secured to prepare for the potential need for rapid infusion of fluids using a rapid-infusion system. immediately after induction of anesthesia, isovolemic hemodilution may be performed: 2 units of the patient's whole blood is collected in cpda (citrate phosphate dextrose adenine) blood collection bags, agitated to prevent clot formation, stored at room temperature, and returned to the patient within 8 h. intraoperatively, physiologic condition should be maintained at all times to ensure adequate perfusion of all tissues including the liver by monitoring the cardiopulmonary system and stat laboratory. metabolic acidosis should be avoided, and use of a balanced salt solution (lactated ringer's solution or plasmalyte-a ® ) is the preferred choice in order to avoid the acid load from normal saline (waters et al. 2001 ). blood loss is not excessive and pre-and intraoperatively donated autologous blood and intraoperative autotransfusion are sufficient in most patients. the relationship between the cvp level and surgical blood loss is controversial: chhibber et al. reported that intraoperative blood loss did not correlate with cvp (<5 mmhg) in their study (chhibber et al. 2007 ), while jones et al. demonstrated a significant reduction in blood loss with low cvp (jones et al. 1998 ). the authors recommend euvolemia to maintain hepatic blood flow during dissection of the liver. however, fluid overloading should be avoided after hepatectomy because relatively high portal venous flow to the reduced liver mass may lead to liver congestion and small-for-size syndrome (dahm et al. 2005) . at the conclusion of surgery, the patient can be extubated safely in the operating room and transported to the icu. all types of surgical procedures may be necessary in the early postoperative period. within the first 2 months after transplantation, surgical procedures are performed to treat complications of transplantation, such as exploratory laparotomy for abdominal bleeding or reconstruction of the biliary system. some degree of hepatic dysfunction may still be present, and ventilatory and circulatory support and invasive monitoring may be required. regional anesthesia is not recommended because of potential bleeding and infectious complications. anesthesia care of these patients is similar to that of other urgent abdominal procedures. patients may return to the operating room at any time for biliary reconstruction, replacement of a hip joint, or almost any other procedure. liver function and drug metabolism are usually within the normal range, and anesthetic management differs little from that of other patients. side effects of immunosuppressants (hypertension and renal insufficiency) and drug interactions should be considered. the main goal of organ procurement is the maintenance of optimal conditions for all organ systems to promote as normal as possible an environment for the organs prior to harvesting. specifically, integrity of organs should be maintained by optimizing organ perfusion and preventing further damage associated with pre-existing illness or trauma. therefore, donor care during procurement is a continuum of the intensive care provided before brain death. the donor is reviewed and examined by the anesthesia team to evaluate their medical history and vital organ function. the equipment and medications necessary for multiple organ procurement are shown in table 5 . a multiple-channel vital-sign monitor is an essential piece of equipment because of the unavoidable hemodynamic changes associated with the absence of brain stem function, surgical manipulation, and fluid shift. a volume ventilator may be required for donors requiring high levels of peep or airway pressure. a large volume of crystalloids and colloid solutions is prepared, and 5 units of prbcs are frequently required. the transit from the icu to the operating room is a crucial period; the anesthesia care team directs the transportation while the donor is continuously monitored, ventilated, and treated. intraoperatively, blood pressure is monitored by an indwelling radial or brachial arterial catheter as abrupt changes in blood pressure are anticipated. cvp monitoring is essential, and a pa catheter may be used in unstable donors. general anesthesia is provided as donors respond to surgical stimulation by dramatic hemodynamic changes such as tachycardia, hypertension, perspiration, and involuntary movement (wetzel et al. 1985) . this so-called mass reflex is caused by the neurogenic vasoconstriction and stimulation of adrenal medulla by reflex spinal arc. isoflurane is the most commonly used agent, because its myocardial depression is relatively benign, and short-acting narcotics (i.e., fentanyl, up to 5 μg/kg/min) may be used in unstable donors. rocuronium bromide or vecuronium bromide is administered for muscle relaxation. the specific goals of ventilatory care are to maintain normal pao 2 (70-100 mmhg), arterial hemoglobin oxygen saturation (>95 %), and paco 2 (35-45 mmhg) as well as to avoid pulmonary complications. this goal is frequently achieved by ventilating with a tidal volume of 10-15 ml/kg, fio 2 of 30-40 %, respiratory rate of <20/min, and a low level of peep (<5 cmh 2 o). however, in donors with pulmonary complications, adjustments are made in tidal volume (up to 20 ml/kg), respiratory rate (up to 20/min), and peep (up to 10 cmh 2 o). aggressive circulatory care is essential because hemodynamic instability may impair organ perfusion. specifically, hypotension (systolic blood pressure <80 mmhg or mean arterial pressure <40 mmhg) is associated with a high incidence of acute tubular necrosis, non-function of the graft kidneys, and poor hepatic function. it is generally agreed that systolic blood pressure should be within the normal range (100-120 mmhg) and cvp should be <10 cmh 2 o with minimal vasopressor support. maintaining circulatory homeostasis, however, can be challenging. preload is frequently decreased because of blood loss, vasomotor paralysis, diuretic therapy, and diabetes inspidus, although fluid resuscitation may result in overload. the heart rate may vary depending on the degree of brain injury, ranging from tachycardia to bradycardia. arrhythmia is not uncommon, and myocardial contractility is frequently impaired by myocytolysis, myocardial necrosis, coronary spasm, and reduction of myocardial energy storage (novitzky et al. 1988 ). afterload may be high, from excessive sympathetic tone, or low, from vasomotor paralysis. volume deficit is usually corrected with lactated ringer's or colloid solution, and transfusion of prbcs (1-3 units) may be necessary to maintain hematocrit between 25 and 35 % (hardesty and griffith 1986) . once the fluid deficit is corrected, a glucose-containing hypotonic solution (5 % dextrose in 0.45 % nacl 1 ml/kg/h) is administered to replace urine output and insensible loss, guided by cvp and urine output. excessive urine output (>200-250 ml/h) is replaced using a hypotonic electrolyte solution with supplementation of kcl (20 meq/l). tachycardia with hypertension should be avoided as it may cause pulmonary edema, decrease organ perfusion, and increase myocardial oxygen consumption. a β-antagonist (i.e., labetalol hydrochloride or esmolol hydrochloride) or a calcium channel blocker (verapamil hydrochloride) is used to treat tachycardia and arrhythmia (novitzky et al. 1984) . for bradycardia, isoproterenol or epinephrine is used for positive chronotropic effects because donors are unresponsive to centrally acting chronotropic drugs (i.e., atropine). supraventricular or ventricular arrythmia is treated using antiarrhythmic drugs. low afterload is compensated for by increasing preload because α-vasopressors increase the myocardial work load and decrease splanchnic and coronary blood flow. in severely hypertensive donors, an α-blocker (hydralazine or sodium nitroprusside) may be given to reduce the afterload. when cardiac output and organ perfusion are impaired, inotropes (dopamine hydrochloride, dobutamine hydrochloride, and isoproterenol hydrochloride) are recommended to improve cardiac contractility. in brain-dead animal models, serum levels of triiodothyronine, insulin, and cortisol have been found to be low, and the administration of triiodothyronine may improve hemodynamic stability by maintaining myocardial high-energy stores and glycogen (james et al. 2010) . circulatory arrest, which occurs in 10 % of potential donors (emery et al. 1986) , is managed in the standard fashion, except atropine is not effective. adequate diuresis (>0.5 ml/kg/h, preferably 1-1.5 ml/kg/h) is recommended as urine output (>100 ml/h) is the most significant factor that determines the outcome of the kidney and liver graft. oliguria is generally caused by hypovolemia and hypotension and frequently responds to fluid administration. diabetes insipidus leads to polyuria, hypovolemia, and electrolyte imbalance. in addition to the fluid replacement, ddavp (0.5-1 units/h) may be administered (richardson and robinson 1985) , although an excessive dose of ddavp may increase the risk of acute tubular necrosis and reduce hepatic blood flow (burggraaf et al. 1994) . donors are poikilothermic, and hypothermia plays a major role in hemodynamic instability. body temperature should be kept above 35 c by raising the operating room temperature, infusing all fluids through a blood warmer, and using heating lamps, a warming blanket, and a heated humidifier in the ventilation circuit. metabolic acidosis, caused by inadequate tissue perfusion, is corrected by administration of nahco 3 or tham. commonly seen electrolyte imbalances are hypernatremia, hypokalemia, hypocalcemia, hypophosphatemia, and hypomagnesemia, and they are treated in a standard fashion. glucose metabolism is relatively well-maintained, and any abnormality in glucose metabolism is corrected by administration of insulin or glucose on the basis of the serum glucose level. dilutional coagulopathy is common, and consumption coagulopathy may develop secondary to the release of tissue thromboplastin from injured tissues and the ischemic organs (kaufman et al. 1984) . fibrinolysis is not uncommon in donors, possibly as a result of the release of tpa from the necrotic brain. replacement of coagulation factors and platelets or any pharmacologic therapy is rarely indicated as donors are fully heparinized when the aorta is cannulated. once cardiac arrest is induced by cardioplegia, no further supportive care is necessary. liver transplantation is one of the most stressful procedures for patients with multiple organ dysfunction and it is a challenge for anesthesiologists. it is remarkable that anesthesiologists have played a major role in the progress of liver transplantation and its successful outcome. it cannot be overemphasized, however, that a 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orthotopic human liver transplantation pulmonary aspects of chronic liver disease and liver transplantation hepatopulmonary syndrome: a prospective study of relationships between severity of liver disease, pao (2) response to 100% oxygen, and brain uptake after (99m)tc maa lung scanning hepatic enzymic zonation: a reevaluation of the concept of the liver acinus relationship between hepatic blood flow and overall metabolism: the hepatic arterial buffer response regulatory processes interacting to maintain hepatic blood flow constancy: vascular compliance, hepatic arterial buffer response, hepatorenal reflex, liver regeneration, escape from vasoconstriction adenosine as a putative regulator of hepatic arterial flow (the buffer response) pulmonary hypertension complicating portal hypertension cardiac abnormalities in liver cirrhosis desensitization of myocardial beta adrenergic receptors in cirrhotic rats agreement between radial and femoral arterial blood pressure measurements during orthotopic liver transplantation cardiac disease evaluation and management among transplantation candidates: a scientific statement from the american heart association and the american college of cardiology foundation liver transplantation: intraoperative changes in coagulation factors in 100 first transplants antithrombin iii during liver transplantation intraoperative blood salvage during liver transplantation in patients with hepatocellular carcinoma: efficiency of leukocyte depletion filters in the removal of tumor cells intracranial pressure monitoring in liver transplantation for fulminant hepatic failure biochemical factors in alcoholic liver disease cardiopulmonary dysfunction in cirrhosis cirrhotic cardiomyopathy role of altered beta adrenoceptor signal transduction in the pathogenesis of cirrhotic cardiomyopathy in rats prognostic significance of reperfusion hyperglycemia during liver transplantation reduced use of intensive care after liver transplantation: patient attributes that determine early transfer to surgical wards portal axis thrombosis with spontaneous portocaval shunt and resulting cor pulmonale cardiovascular depression secondary to citrate intoxication during hepatic transplantation in man hepatic transplantation, anesthetic and perioperative management ionization and hemodynamic effects of calcium chloride and calcium gluconate in the absence of hepatic function effects of phlebotomy and phenylephrine infusion on portal venous pressure and systemic hemodynamics during liver transplantation adult respiratory distress syndrome associated with acute liver allograft rejection: resolution following hepatic transplantation right phrenic nerve injury in orthotopic liver transplantation acute and chronic renal failure after liver transplantation primary pulmonary hypertension and cirrhosis: are they related? clinical usefulness of recombinant factor vii in patients with liver failure undergoing invasive procedures negative impact of systemic catecholamine administration on hepatic blood perfusion after porcine liver transplantation perioperative outcomes of major hepatic resections under low central venous pressure anesthesia: blood loss, blood transfusion, and the risk of postoperative renal dysfunction recombinant factor viia in management of spontaneous subcapsular liver hematoma associated with pregnancy liver regeneration: molecular mechanisms of growth control long term terlipressin administration improves renal function in patients with cirrhosis with type 1 hepatorenal syndrome: a pilot study effect of aprotinin on intraoperative bleeding and fibrinolysis in liver transplantation recombinant factor viia reduces transfusion requirements in liver transplant patients with high meld scores electrocardiographic, hemodynamic and endocrine changes occurring during experimental brain death in the chacma baboon injury of myocardial conduction tissue and coronary artery smooth muscle following brain death in the baboon american college of cardiology/american heart association expert consensus document on electronbeam computed tomography for the diagnosis and prognosis of coronary artery disease surgical complications of liver transplantation hemodynamic changes in clinical orthotopic liver transplantation portal and hepatic arterial blood flow measurements of human transplanted liver by implanted doppler probes: interest for early complications and nutrition glucose metabolism in cirrhosis: a review with some perspectives for the future albumin gene expression is down regulated by albumin or macromolecule infusion in the rat molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis dopamine, furosemide and mannitol infusions and changes in serum creatinines after liver transplantation safety and efficacy of a single bolus administration of recombinant factor viia in liver transplantation due to chronic liver disease dobutamine stress echocardiography for preoperative cardiac risk stratification in patients undergoing orthotopic liver transplantation pathologic aspects of cirrhosis systemic effects of tissue plasminogen activator-associated fibrinolysis and its relation to thrombin generation in orthotopic liver transplantation cardiovascular risk assessment of the liver transplant candidate effect of hypothermia on the coagulation cascade platelet function in chronic liver disease; relationship to disease severity myocardial ischemia after orthotopic liver transplantation perioperative risk predictors of cardiac outcomes in patients undergoing liver transplantation surgery complications associated with percutaneous placement of venous return cannula for venovenous bypass in adult orthotopic liver transplantation postliver transplantation myocardial dysfunction intraoperative fluid management during orthotopic liver transplantation ionized hypomagnesemia in patients undergoing orthotopic liver transplantation: a complication of citrate intoxication pericardial effusion and left ventricular dysfunction associated with ascites secondary to hepatic cirrhosis oxygen metabolism during liver transplantation: the effect of dichloroacetate safety of cardiac catheterization in patients with end-stage liver disease awaiting liver transplantation venous bypass in clinical liver transplantation prognostic value of cardiac risk factors and coronary artery calcium screening for all-cause mortality treatment of refractory hepatic hydrothorax with transjugular intrahepatic portosystemic shunt: long term results in 40 patients stenosis of the inferior vena cava after liver transplantation: treatment with gianturco expandable metallic stents central nervous system lesions in adult liver transplant recipients: clinical review with implications for management hemodynamic interaction between portal vein and hepatic artery flow in small-for-size split liver transplantation increased concurrence of cirrhosis and bacterial endocarditis role of the molecular adsorbent recycling system (mars) in the treatment of patients with acute exacerbation of chronic liver failure experience in hepatic transplantation. saunders, philadelphia starzl te, marchioro tl, von kaulla kn et al (1963) homotransplantation of the liver in humans fk 506 for liver, kidney, and pancreas transplantation a simple solution to a technical complication in piggy back liver transplantation intraoperative transesophageal echocardiography during liver transplantation non alcoholic cirrhosis associated with neuropsychological dysfunction in the absence of overt evidence of hepatic encephalopathy cardiac hemodynamic and coronary angiographic characteristics of patients being evaluated for liver transplantation clinical presentation of hepatic artery thrombosis after liver transplantation in the cyclosporine era orthotopic liver transplantation with preservation of the inferior vena cava low levels of thrombin activatable fibrinolysis inhibitor (tafi) in patients with chronic liver disease complications and use of intracranial pressure monitoring in patients with acute liver failure and severe encephalopathy a rapid increase in sodium is associated with cpm after liver transplantation recommendations on the use of recombinant activated factor vii as an adjunctive treatment for massive bleeding-a european perspective alterations in plasminogen activator and plasminogen activator inhibitor levels during liver transplantation vasopressin decreases portal vein pressure and flow in the native liver during liver transplantation hepatic and portal vein thrombosis in cirrhosis: possible role in development of parenchymal extinction and portal hypertension intraoperative pulmonary embolism and intracardiac thrombosis complicating liver transplantation: a systematic review identification of osmosensitive and ammonia-regulated genes in rat astrocytes by northern blotting and differential display reverse transcriptase-polymerase chain reaction normal saline versus lactated ringer's solution for intraoperative fluid management in patients undergoing abdominal aortic aneurysm repair: an outcome study hemodynamic responses in brain dead organ donor patients is hepatic histology the true gold standard in diagnosing acute hepatic allograft rejection pontine myelinolysis following liver transplantation: a report of two cases postoperative atrial fibrillation in liver transplantation successful anesthetic management of a patient with critical bleeding during hepatectomy using recombinant activated factor vii and intraoperative blood salvage endotoxemia and human liver transplantation key: cord-022483-hdmwv540 authors: nan title: gastrointestinal disease date: 2009-06-05 journal: equine neonatal medicine doi: 10.1016/b978-1-4160-2353-1.50016-8 sha: doc_id: 22483 cord_uid: hdmwv540 nan straining to defecate. '03 rockn'roll at the time of presentation to the veterinary teaching hospital at approximately 26 hours of age. the foal is exhibiting signs of straining to defecate: raised tail head, slightly arched back, hindlimbs slightly under the trunk. note the meconium staining on the tail and perineum. the white tape on the foal's back was used as a landmark to serially measure the circumference of the foal's abdomen. compare this foal's stance to the stance in united states in 1998 and 1999, the incidence of colic in foals less than six months of age was approximately 18 times less than the incidence in mature horses. 1 however, when considering aliments affecting foals, gastrointestinal tract problems and infection were most commonly reported. 2, 3 because some etiologies of colic are unique in the neonatal period, special consideration must be given to the evaluation of abdominal pain in this age group. in some respects, the diagnostic approach to colic in neonatal foals is similar to that used in mature horses: rarely will any single fact be useful in determining the exact etiology. however, careful and simultaneous inspection of multiple historical, physical, and diagnostic findings may be formative in determining the anatomical location of the lesion (stomach, small intestine, large intestine, peritoneal cavity), the etiologic category (congenital, nonstrangulating obstruction, strangulating obstruction, inflammatory, or other), or even possibly the specific diagnosis (table 11 -1). in obtaining a history, particular attention should be given to the farm history, use of medications (especially analgesics), risk factors for septicemia or failure of passive transfer, and problems with other foals on the farm. as some differentials are strictly age-dependent (see table 11 -1), knowing the age of onset of clinical signs is important. in the neonatal period, commonly reported causes of abdominal pain are meconium impaction, small-intestinal volvulus, enteritis or colitis, uroperitoneum, intussusception, gastric ulcers, and ileus secondary to prematurity, septicemia, or neonatal encephalopathy. [4] [5] [6] [7] [8] clinical signs of lethal white syndrome, meconium impaction, and uroperitoneum most commonly manifest in the first 12 to 24 hours, 12 to 96 hours, and 48 to 96 hours of life, respectively. however, if uroperitoneum is the result of urachal or urinary bladder infection, clinical signs may be delayed until 7 to 14 days of age. although the potential spaces created by congenital or traumatic umbilical, inguinal, and diaphragmatic hernias may be present since birth, incarceration of bowel into these spaces may occur at any age, if at all. enteritis, colitis, intussusception of small intestine, small-intestinal volvulus, and clinically significant gastric ulcers may develop at any age. the breed and sex of the foal may provide supportive evidence for certain differential diagnoses. lethal white syndrome is most commonly reported in all-white to almost-all-white offspring of overo cross overo paint horses. in one study of 168 horses with congenital umbilical hernias, the incidence was two times greater in fillies compared to colts, and thoroughbreds were twice as likely to have an umbilical hernia compared to standardbreds. 9 in this later study, incarceration of bowel into the umbilical hernia was not reported. scrotal hernias most commonly occur in standardbred and tennessee walking horse colts, 8 and fecaliths are frequently reported in american miniature foals. 6, 10 in one study, meconium impactions occurred twice as often in colts compared to fillies. 11 although the incidence of uroperitoneum is often quoted to occur more commonly in colts, in one recent study, the incidence was approximately equal among the sexes. 12 additional historic information that should be carefully scrutinized is the general health of the dam, the foal's gestational age, the foaling history and general perinatal health, such as ingestion of colostrum, age at passage of meconium, nursing frequency (see chapter 1 for review of normal perinatal health), and farm history of potentially infectious causes of enteritis or colitis (salmonella, rotavirus, clostridium). most foals will have passed meconium within 9 to 12 hours of life; however, the gastrocolonic reflex stimulated by ingestion of colostrum frequently initiates earlier passage of meconium. evacuation of meconium may be delayed (meconium retention) as the result of ileus secondary to another primary nongastrointestinal disease, such as septicemia or neonatal encephalopathy. in these cases, although passage of meconium may be slower than expected, the delay in passage may not be accompanied by clinical signs of abdominal pain. passage of "milk feces" or yellow pasty feces does not necessarily indicate that all meconium has been removed from the colon. clinical signs of abdominal pain in foals can be highly variable, and the intensity of the signs is not necessarily indicative of the etiology. it is important to note that foals with inflammatory lesions of the intestinal tract or those suffering from general functional ileus secondary to systemic disease can act as violently painful as foals with obstructive or strangulating lesions of bowel. however, in general, foals suffering from uroperitoneum and gastric ulcers have less intense abdominal pain than do foals with inflammatory or obstructive lesions. repeatedly thrashing or rolling from side to side is generally accepted as a highly representative clinical sign of abdominal pain. however, early signs of abdominal pain in neonatal foals may only manifest as reduced frequency of nursing and prolonged recumbency. other subtle but significant signs of abdominal pain in foals include general restlessness, especially in recumbency, and/or frequent adjustment of recumbent positions (figure 11 -2). often recumbent foals with abdominal pain will stretch their limbs, twist their head or neck, roll into dorsal recumbency ( figure 11-3) , and make frequent attempts or strain to either defecate and/or urinate. in the standing foal, signs that are classically associated with straining to defecate include frequent tail swishing, a "water spout" tail, and a "camped under" leg stance with a dorsiflexed back (figure 11-1) . in contrast, a flat or ventroflexed back with the hindlimbs stretched backward and the tail held up is associated with urination ( figure 11-4) . other signs of abdominal pain include lip curling, flank biting or watching, pawing at the ground, and kicking at the abdomen. it is important to recognize that often critically ill foals that are stuporus will not overtly demonstrate classic signs of the colicky neonatal foal should receive a thorough general physical examination with careful attention given to identification of clinical signs or physical evidence of sepsis (see chapter 5) . sepsis may be directly associated with the primary etiology of the abdominal pain or it may develop secondary to bacterial translocation if the integrity of the gastrointestinal mucosa is compromised. the presence of fever, depression, petechiae, injection, synovitis, uveitis, or diarrhea may be cardinal clues that the abdominal pain has an inflammatory etiology. although the presence of diarrhea may be an important sign of enterocolitis, intense abdominal pain frequently precedes the onset of diarrhea. tachycardia and/or tachypnea are expected findings despite etiology. persistent tachycardia (heart rate >120 beats per minute) has been suggested to be more commonly associated with surgical disorders of the abdomen, compared to medical etiologies. 13 gross abdominal distension will develop with gas or fluid accumulation in the bowel or abdomen. severe abdominal distension with intense pain that elicits high-pitched "pings" with percussion is consistent with gas-distended large intestine or cecum. repeated measurement of the abdominal circumference may be helpful in more objectively determining the course of progression of abdominal distension. typically, the absence of intestinal sounds is not helpful in determining etiology; however, increased borborygmi may be associated with enteritis or colitis. if diarrhea is present with signs of abdominal pain in a foal, additional fecal diagnostics may be indicated (see . unlike mature horses, transabdominal wall palpation can be performed in the neonate and may identify gastric distension, hepatomegaly, thickened or distended bowel, meconium, and the urinary bladder. the ventral abdomen and inguinal rings should be carefully examined for evidence of herniation. ballottement or succussion of the abdomen may verify a fluid wave compatible with excess fluid in the peritoneal cavity. with adequate restraint or sedation, a careful digital rectal examination may identify meconium or ingesta in the pelvic inlet. a nasogastric tube should be passed in all foals with clinical signs of abdominal pain. compared to mature horses, successful retrieval of gastrointestinal reflux can be frustrating in the neonate, even in the presence of proximal intestinal disease. frequently, nasogastric tubes that are designed for neonatal enteral feeding are of insufficient size for gastric decompression. with this in mind, the largest-bore tube that can be comfortably passed should be used, and persistent attempts should be made to obtain gastric reflux, either by suction or priming the tube with water. a complete blood count and serum biochemical profile were obtained from '03 rockn'roll. the pcv was normal at 32%. the total white blood cell count was 9,600/ml, characterized by a normal neutrophil count (6, (figure 11-5 changes in the leukogram or serum biochemical analysis may provide indirect etiologic evidence. neutropenia, neutrophilia, a significant left shift, toxic changes within the neutrophils, and hyperfibrinogenemia are indicative of an inflammatory or infectious etiology and if present in the equine neonate, aseptic collection of additional blood for culture is recommended. hypoglobulinemia is indicative of failure of passive transfer and should be verified by specific igg testing (see case 3-1). panhypoproteinemia is indicative of loss and may accompany severe inflammatory lesions of bowel. azotemia, hyponatremia, hypochloremia, and hyperkalemia may occur with enteritis, colitis, or uroperitoneum. marked acidosis most commonly occurs in foals with inflammatory lesions of the bowel. many clinicians are hesitant to perform an abdominocentesis on a neonatal foal as the risk for complications such as inadvertent enterocentesis or omental prolapse occur more commonly in foals undergoing an abdominocentesis, as compared to adult horses. many clinicians perform a transabdominal ultrasound examination first to assess potential risks of performing an abdominocentesis versus the likelihood of successfully obtaining peritoneal fluid. if an ultrasound examination reveals widespread and/or grossly distended loops of intestine, and/or free peritoneal fluid is difficult to identify, the chances of an uncomplicated and successful abdominocentesis are reduced. an abdominocentesis may be completed on the standing foal or with the foal in lateral recumbency. in either position, proper restraint is imperative and is best facilitated by light sedation (table 11 -2). the procedure for performing an abdominocentesis in a foal is similar to the adult horse, using either a 20-to 18-gauge 1.5-inch needle or a teat cannula to collect the peritoneal fluid. first, the hair from the ventral midline of the rostral abdomen should be clipped and the skin sterilely prepared. the subcutaneous tissue of the abdominocentesis site in the rostral midline of the abdomen should be infiltrated with 0.5 ml of 2% lidocaine. when a teat cannula is used, entry through the abdominal wall must be preceded by a stab incision made with a number 15 blade through the skin and external portion of the linea alba. although use of an 18-gauge needle is expeditious, there may be a greater risk of inadvertent enterocentesis or bowel laceration; however, enterocentesis may also occur with a teat cannula if the intestine is grossly distended. localized or generalized peritonitis may develop subsequent to an enterocentesis, and prophylactic use of parenteral antimicrobials is recommended. fig. 11 -5 ultrasonographic image obtained on '03rockn'roll at 26 hours of age with acute abdominal pain. this image was obtained using a 7-4 mhz curvilinear probe set to a depth of 6 cm. note the four "balls" of meconium of mixed echogenicity in small colon (1, 2, 3, 4) . this image was obtained from the left caudoventral abdomen. more commonly, use of a teat cannula results in inadvertent prolapse of omentum through the abdominocentesis site upon collection of peritoneal fluid or removal of the cannula. if this happens, the omentum should be sharply transected with a sterile blade at the abdominal wall and tucked back into the abdominal cavity with another sterile teat cannula. normal peritoneal fluid should be light yellow in color and clear. the normal mean nucleated cell count in the peritoneal fluid of healthy foals was reported to be 450 cells/μl (range 60 to 1,400 cells/μl) for 17 foals that were 13 to 134 days of age 14 and 1,400 cell/μl (±1077) for 32 thoroughbred foals that were 14 to 75 days of age. 15 both of these reference ranges for nucleated cell counts are lower than means reported for healthy adult horses. the mean protein concentration by biochemical biuret determination and refractive index in foals was reported to be 1.2 g/dl and 1.6 g/dl, respectively. 14 the interpretation of peritoneal fluid nucleated cell count and protein concentration is similar to adult horses with abdominal pain and does not necessarily definitively distinguish etiology or medical from surgical causes of colic. sanguinous fluid can be present with either severe enterocolitis or strangulating lesions of bowel. special attention should be given to the cytologic examination. the presence of degenerative neutrophils or bacteria is worrisome (figure 11 -6) and is indicative of loss of the mucosal integrity, bowel rupture, or primary sepsis in the peritoneal cavity. in one study, 78% of colicky foals with peritoneal fluid protein concentrations >2.5 g/dl that subsequently underwent an exploratory laparotomy were euthanized. 8 when uroperitoneum is suspected, the creatinine concentration of both peritoneal fluid and serum should be simultaneously assessed. a peritoneal fluid creatinine to serum creatinine ratio >2 is supportive evidence of uroperitoneum. when determining peritoneal fluid creatinine concentration on an automated chemistry analyzer, it is important to use plasma or serum methodology on the abdominal fluid. if urine methodology is used to determine the creatinine concentration on a peritoneal fluid sample, erroneously increased creatinine concentration may lead to an incorrect diagnosis of uroperitoneum. unlike mature horses, abdominal radiography can provide useful information in a colicky neonatal foal. grid, rare-earth screens and sufficient mas (5 to 28) and kvp (80 to 120) should be used. 16 radiographs may be taken with the foal either standing or in lateral recumbency; however, keep in mind that fluid-gas interfaces are easier to detect in the standing foal, when the radiographic beam is horizontal or perpendicular to the dorsoventral plane of the gas/fluid interface in the abdomen. both right and left lateral views should be obtained as some structures will be more obvious, depending upon which side of the abdomen is closer to the film. ventrodorsal views may only be possible in small foals or those that are stuporus, sedated, or anesthetized. this view will optimize identification of the pylorus, the descending duodenum, the base of the cecum, the left colon, and the transverse colon. 17 some degree of gas is normally visible in the stomach, small intestine, cecum, and small colon. in general, plain film abdominal radiology is more likely to provide information on the anatomic location of the problem than information directly leading to the exact etiology. 8, 16, 17 for example, gas distension of the small intestine is a nonspecific finding that may occur with enteritis, functional obstruction (ileus), or mechanical obstruction (figure 11-7) . however, if small-intestinal distension is accompanied by "hairpin" or "u-shaped" turns of the bowel ( figure 11 -8) or multiple, uneven intraluminal gas-fluid interfaces ( figure 11-9 ), it is more likely associated with mechanical obstruction, though these findings can also occur with enteritis or functional ileus. thickened walls of small intestine may appear uneven or "corrugated" when caused by enteritis (figure 11 -10). severe generalized gas distension of large intestine is more commonly associated with mechanical obstruction than with inflammatory lesions of large colon ( figure 11 -11) . meconium frequently appears as granular contents in the ascending or descending colon ( figure 11 -12) . pneumoperitoneum suggestive of bowel this neonatal foal had radiographic signs of smallintestinal obstruction, as evidenced by isolated loops of gasdistended small intestine that form a u-turn or "hairpin" turn (arrows). an exploratory celiotomy confirmed a jejunal intussusception. fig. 11-9 radiographic appearance of small-intestinal disease. the presence of multiple gas-fluid interfaces at different levels is often considered radiographic evidence of smallintestinal obstruction; however, it can also be seen in foals with intense ileus from enteritis, as was the situation for the foal depicted in this figure. rupture should be suspected if there is a gas cap in the dorsal aspect of the abdominal cavity, if the serosal surfaces of bowel are enhanced, or if visualization of the renal silhouettes is improved. iatrogenic pneumoperitoneum should be considered if an abdominocentesis was performed prior to abdominal radiography. finally, abdominal radiography obtained on foals with necrotizing enterocolitis may demonstrate the pathognomonic sign of pneumatosis intestinalis, or intramural air. radiographic signs of pneumatosis intestinalis are spectacular and include localized cystic collections that appear as radiolucent "bubbles" in the bowel wall, or diffuse linear strips or flat oval-shaped areas of radiolucency in the bowel wall flanked by the radiopaque serosa and mucosa, when the bowel wall is viewed end-on. 18 contrast radiography also has some specific indications in neonatal foals. an upper gastrointestinal contrast study can be used to document delayed gastric emptying of older foals with suspected pyloric outflow obstruction. 16 foals less than two weeks of age ideally should be fasted for four hours, and foals consuming solid feeds should be fasted for 12 hours prior to contrast radiography. 17 barium is administered by gravity flow via a nasogastric tube (5 ml/kg as a 30% weight/volume solution), and abdominal radiographs are obtained every 30 minutes. if barium remains in the stomach for longer than two hours, delayed gastric emptying should be suspected but does not necessarily distinguish between mechanical or functional obstruction. barium should normally reach the cecum and transverse colon by two hours and three hours, respectively, in 10-to 12-day-old foals. 16, 17 barium transit time to the transverse colon is five to eight hours in foals one to two months of age. in addition to documenting transit time, stenosis or abnormal bowel wall may be highlighted by the contrast (figure 11-13 ). lower-intestinal contrast studies (i.e., barium enema) have been reported to have 100% sensitivity and 100% specificity for identifying mechanical obstruction (meconium impaction, atresia coli) of the transverse colon or small colon in foals less than 30 days of age ( figure 11-14) . 19 the foal should be restrained or lightly foals with enteritis may have numerous nonspecific radiographic findings, such as gas-distended small intestine and multiple gas-fluid interfaces. in the radiograph depicted here, the finding of individual loops of mildly to moderately gas-distended small intestine with irregular or "corrugated" walls is highly indicative of enteritis. fig. 11-11 radiographic appearance of a large-or smallcolon obstruction. unlike small-intestinal gas distension, generalized gas distension of large colon is considered to be fairly specific for large-or small-colon obstruction. this foal had a distal meconium impaction. sedated and placed in lateral recumbency. a 24-french foley catheter is placed into the rectum and the bulb gradually inflated. by gravity flow, administer up to 20 ml/kg of 30% weight/volume barium. two-dimensional transabdominal ultrasonography is a valuable diagnostic tool in the colicky neonatal foal. scanning techniques for the foal abdomen are extensively reviewed elsewhere, 20 though a brief review follows here. standard linear array 4 to 7 mhz transducers are sufficient for visualization of most intra-abdominal structures in the neonatal foal; however a curvilinear transducer will optimize image quality. the foal may be scanned in either lateral recumbency or standing; however keep in mind that fluid-filled, thickened, or enlarged structures may descend to the dependent portion of the abdomen and could be overlooked if only the nondependent side of a recumbent foal is scanned. furthermore, gas in nondependent bowel, especially gas in large colon, may preclude examination of deeper structures in laterally recumbent foals. visualization of intra-abdominal structures is facilitated by clipping the abdominal hair; however thorough wetting of the hair with water or alcohol, in addition to acoustic coupling gel, may be sufficient. in the colicky neonate, transabdominal ultrasonography can be used to identify fluiddistended structures (i.e. stomach, small intestine, large intestine, urinary bladder), gastric or intestinal wall thickness, abnormal intestinal contents (i.e. meconium, fecaliths, phytobezoars, or trichophytobezoars), peritoneal fluid, and to determine intestinal motility. normal gastric wall thickness in foals should be less than 7 mm, whereas intestinal wall thickness is 3 to 4 mm. foals with gastritis or gastric ulcers may have a thickened or irregular gastric wall. likewise, foals with enteritis or colitis will frequently have diffusely increased small and/or large intestine wall thickness ( figure 11-15 ). this feature alone does not definitively identify enteritis, as edematous bowel that develops as the result of strangulation (volvulus or intussusception) will also appear regionally thickened ( figure 11 -16), with distended fluid-filled intestine proximally. the presence of gas echoes in the intestinal wall, a thickened hypoechoic wall that appears "wavy," or the presence of sloughed mucosa in the lumen are more consistent with a diagnosis of enteritis and should be administration of barium as an enema (see text) reportedly has 100% sensitivity and 100% specificity for identification of small or large-colon obstructions. the abrupt end of contrast in the small colon in this foal was highly indicative of atresa coli (thin arrow), which was later confirmed at necropsy. also note the stenosis of the rectum above the pelvis (thick arrow). 200 considered as distinguishing features from strangulating lesions (figure 11 -17). foals with enteritis or colitis may demonstrate either hypermotile or hypomotile bowel, whereas functional ileus and mechanically obstructed intestine more often will appear hypomotile. neonatal foals with mechanical obstruction of small intestine may have hairpin or u-shaped turns of the small intestine when viewed in long axis ( figure 11 -18). the unique regional presence of a double intestinal wall or what appears to look like a "bull's-eye" of multiple concentric rings in a short axis view of small intestine is consistent with an intussusception (figure 11-19) , which most commonly is seen in the dependent portion of the abdomen. meconium may appear hyperechoic, hypoechoic, or as a mixture of echogenicities in hypomotile intestine. fluid-or gas-distended intestine may be present proximal to the obstruction ( . when present in the small colon, retained or impacted meconium often appears as a row of "balls" and is most easily identified in the dependent portion of the left caudal abdomen in the standing foal. it may be traceable dorsal to the urinary bladder and often is surrounded by a thin layer of hyperechoic gas in sacculated intestine (small colon). meconium in large colon typically is more amorphous. extensive gas in the large colon frequently hinders visualization of other intra-abdominal structures and can be associated with mechanical obstruction, though it may also occur with acute colitis. the sonographic appearance of abdominal abscesses is variable, but in the neonatal foal, intra-abdominal abscesses are often associated with umbilical remnants, previously devitalized bowel, or mesenteric lymph nodes. finally, the presence of excessive anechoic fluid may be indicative of uroperitoneum (see chapter 12), though anechoic transudate may also develop as a result of enteritis, colitis, or strangulation. hyperechoic densities within peritoneal fluid may represent fibrin strands, leukocytes, or adhesions ( figure 11 -22) . the presence of gas echoes free within the peritoneal fluid is indicative of a ruptured viscus. gastroscopy may be accomplished with a 1-meter endoscope in a neonatal foal and is covered elsewhere in more detail later in this chapter (see . for most neonatal foals, suckling can be allowed prior to gastroscopy, but solid-feed consumption should be withheld for 6 to 10 hours. 21 caution should be exercised in the interpretation of the clinical significance of gastric ulcers in foals, as up to 50% of young asymptomatic foals have gastric ulcers along the margo plicatus of the greater curvature of the stomach, and gastric ulcers may develop secondary to another primary gastrointestinal disorder. 22, 23 however, multiple deeper ulcers, bleeding ulcers, ulcers along the lesser curvature or in the glandular mucosa, or those accompanied by clinical signs that are consistent with gastric ulceration should be considered clinically significant. most information on colic in the neonate is obtained from referral institutions or, more often, from foals undergoing exploratory celiotomy for acute abdominal pain; therefore the data may not precisely reflect the true incidence of each reported etiology. the only published study on 20 foals less than two weeks of age with acute abdominal pain reported that an exploratory celiotomy revealed functional ileus (45%), meconium impaction (25%), large-colon displacement (15%), small intestine displaced around the base of the cecum (10%), ruptured gastric ulcer, and small colon obstructed by the ovarian ligament. 7 other retrospective studies of abdominal surgery in the foal include foals three to six months of age and thus it was difficult to ascertain the etiology of abdominal pain exclusive to the neonatal period. in 53 foals that underwent an exploratory celiotomy from birth to three months of age, meconium impaction, uroperitoneum, enteritis, small-intestinal strangulation (herniation with incarceration, small-intestinal volvulus, and intussusception), and enteritis accounted for 80% of the total cases. 4 in a study that reviewed 83 foals up to six months of age, the most commonly reported diseases identified at surgery were small-intestinal volvulus, meconium impaction, and intussusception. 8 these reports underscore the difficulty in definitively identifying the cause of abdominal pain prior to exploratory celiotomy in neonatal foals, as clearly some of these cases, such as enteritis and functional ileus, would not be considered to be predominantly surgical diseases. furthermore, it is noteworthy that if the etiology of the abdominal pain cannot be ascertained in the violently painful patient, an exploratory celiotomy may be indicated solely as a diagnostic tool. the purpose of this section is to provide a brief review of the conditions most commonly reported in the neonatal foal. meconium is the sticky caramelized feces of the newborn foal that comprises intestinal secretions, swallowed amniotic fluid, and cellular debris. in one study of 30 newborn foals, it was reported that the total weight of meconium is equal to 1% of the foal's body weight. 24 most foals will start to evacuate meconium shortly after the first ingestion of colostrum, which acts both as a laxative and stimulator of the gastrocolonic reflex. the majority of the meconium is evacuated within the first 12 hours of birth and is replaced by "milk" feces, which are pasty and yellow in appearance. however, concurrent disease such as neonatal asphyxia, prematurity, septicemia, and encephalopathy may delay passage. meconium impaction implies failure to evacuate sufficient quantities of meconium with subsequent development of signs of colonic obstruction: pain, straining to defecate, and abdominal distension secondary to accumulation of gas in bowel proximal to the impacted meconium. it has been suggested that meconium impaction is more likely to occur in colts 11 and in foals greater than 340 days of gestational age. 25 to the author's knowledge, prophylactic use of an enema at birth has not been shown to prevent meconium retention or impaction. foals with meconium impaction are disinterested in nursing, strain to defecate, and typically stand with a slightly arched back and frequently "swish" their tail ( figure 11 -1). digital examination may detect meconium within the rectum. colic, abdominal distension, tachypnea, general restlessness, and tachycardia are frequently reported clinical signs. 11 intestinal borborygmi are usually present 11 and are not a reliable sign of obstruction. meconium can usually be identified by either plain or contrast abdominal radiography or ultrasonography (see "diagnostic approach to colic in neonatal foals" section above). in more severe obstructions, excessive amounts of gas may accumulate in the large colon, proximal to the obstruction. rupture of the urinary bladder may occur in foals that strain excessively from meconium impaction. 11 furthermore, extensive mural damage lends to bacterial translocation and secondary septicemia. other causes of acute abdominal pain that closely mimic meconium impaction and primarily manifest during the first 24 to 48 hours of life are the rarely reported cases of ileocolonic aganglionosis, atresia coli, and atresia recti. ileocolonic aganglionosis, or "lethal white" syndrome, is a fatal autosomal recessive disorder principally of overo cross overo paint horses that is caused by a point mutation in amino acid 118 in endothelin receptor b. 26 the endothelin receptor is critical for the proper development and migration of cells from the neural crest that ultimately form melanocytes in the skin as well as neurons in the intestinal tract. thus, foals that are homozygous for the mutation are essentially all white (though some small areas of pigmentation can occur) and develop signs of functional ileus in the first few hours of life ( figure 11 -23). color patterns in the dam and sire that have the highest incidence of heterozygote carriers of the mutation are frame overo, highly white calico overo, and frame blend overo horses. 26 however, the heterozygote mutation has been occasionally rarely detected in other white-patterned paints and "nonpaint" whitepatterned bloodlines. it has not been detected in solidcolored breeding-stock paint horses without white, but heterozygote adult solid-colored miniature horses of paint lineage and white-patterned horses of breeds other than the paint horse have been rarely identified. 26 lethal white foals typically are born "normal" in appearance and behavior, with the exception of the mostly white coat. clinical signs of abdominal pain usually develop in the first few hours of life, after ingestion of colostrum, and progressively develop gross abdominal distension. evidence of passage of meconium is often missing, though some meconium may be passed. the only way to definitively identify a lethal white foal is by dna testing for the mutation or histopathologic demonstration of insufficient intestinal ganglia. unfortunately, it can take weeks to obtain the results of dna testing and hours to days to obtain the results of intestinal biopsies. diagnosis is often initially presumptive, based on signalment, heterozygote parents, lack of response to symptomatic treatment, and rule out of other causes by additional diagnostics or surgical exploratory. affected foals and heterozygote adults can be identified by submitting plucked hair with intact root bulbs from the mane (preferred sample) to the veterinary genetics laboratory at the university of california, davis, available at www.vgl.ucdavis.edu/service/horse/coatcolor.html. it should be noted that not all white foals of paint lineage are lethal whites and when tested, these unaffected, rare, all-white paint horses are not homozygous for the mutation. atresia coli, recti, or ani are rarely reported causes of colic in the newborn foal that may mimic meconium impaction, based on age of onset and signs of obstructive disease. 27 it may be possible to identify atresia recti on visual or digital examination of the rectum or by protoscopy. retrograde contrast barium enema usually identifies an abrupt obstruction (see , but definitive identification may require an exploratory celiotomy. successful surgical repair has been described; however, other congenital abnormalities have been simultaneously reported and should be ruled out prior to consideration of surgery. the heritability of these defects is not fully known. 27 fecaliths are hard concretions of ingesta ( figure 11 -24) that may also contain undigested material, such as hair. they most commonly occur in the american miniature breed and have been reported in foals as young as 19 days of age. 6, 10 affected foals typically present with progressive unresponsive abdominal pain that is accompanied by gross abdominal distension. as fecaliths obstruct the small colon or rectal lumen, abdominal radiographs typically demonstrate gas distension of the large colon. successful treatment almost inevitably involves a celiotomy with a small colon enterotomy. strangulating lesions of the small intestine that require surgery occur more frequently in neonatal foals, as compared to strangulating lesions of the large intestine. volvulus of the small intestine is the most commonly reported strangulating lesion in foals less than three months of age. 8 factors leading to development of 204 these lesions are undetermined, but alterations in motility may contribute. intussusceptions are most commonly reported in three-to five-week-old foals 2 and may be acute or chronic and intermittent. smallintestinal intussusceptions are most frequent, but ileocecal and cecocolic intussusceptions have been reported in young foals. 28 most inguinal and umbilical hernias in foals resolve spontaneously without incarceration of intestine. 8, [29] [30] [31] [32] in a large retrospective study of 147 foals with umbilical hernias, only 13 required surgical repair. 29 only 4 of the 13 foals that needed surgery had incarcerated bowel in the umbilical hernia. although the hernias were present since birth, and strangulation may rarely occur shortly after birth, the majority of foals with strangulating umbilical hernias were not neonates, but most often, affected foals were presented in the first six months of life. 31 the most common presenting complaint in foals with an umbilical hernia with incarcerated bowel was an acute firm enlargement of the hernia that was sensitive upon palpation. only 30% of foals with strangulated umbilical hernias presented with signs of abdominal pain. in separate retrospective reports, inguinal hernias in neonatal foals that resulted in incarceration of small intestine that required surgical correction were exclusively found in newborn foals as a result of rents in the vaginal tunic (direct inguinal hernia). 30, 32 onset of clinical signs was typically within 48 hours of birth. diagnosis should be suspected by the presence of an irreducible mass in the scrotal sac, edema in the scrotum and prepuce, and colic. diagnosis of either an umbilical or scrotal hernia with incarceration can usually be confirmed by palpation and ultrasonography. it has been suggested that elective herniorrhagy should be considered if an umbilical hernia has been present in foals greater than six months of age or if the defect is larger than 10 cm. 31 diaphragmatic hernias are rare in foals and can result from failure of fusion of its embryonic components or from traumatic rupture of the diaphragm in utero, at birth, or after birth. 33 affected foals can remain asymptomatic for prolonged periods of time, but the presenting complaint typically is acute abdominal pain. the intensity and onset of clinical signs is related to the amount of intestine that has herniated into the thoracic cavity and whether the herniated bowel is simply displaced or strangulated. 33 in addition to abdominal pain, tachypnea and/or dyspnea may be present. the diagnosis can be confirmed by either radiographic or ultrasonographic evidence of an incongruous diaphragmatic line, the presence of gas-fluid interfaces indicative of intestine in the thoracic cavity, and/or pleural effusion. enterocolitis, gastric ulceration, and uroperitoneum are commonly reported causes of abdominal pain in the neonate and are described separately later in this chapter and in chapter 12. functional ileus secondary to prematurity, sepsis, neonatal asphyxia, neonatal encephalopathy, electrolyte abnormalities, concurrent gastrointestinal disease, hypoxic or ischemia bowel, overfeeding, use of milk replacer, botulism, and many other concurrent diseases may induce significant abdominal pain in the neonatal foal. if these underlying conditions are present, careful consideration to additional diagnostics should be used to rule out more serious gastrointestinal disorders (see "diagnostic approach to colic in neonatal foals" section above). simple solutions, such as reducing or eliminating enteral feeding, changing the source of milk, or correction of electrolyte derangements and other underlying disorders should be tried first. however, it may be necessary to consider an exploratory celiotomy for definitive diagnosis and for therapeutic decompression of bowel before initiation of prokinetic therapy. cecal impaction, large-colon volvulus, large-colon displacement, intestinal infarction, and ileal impaction are considered to be rare disorders of the neonatal foal. medical therapy for meconium impaction includes judicious use of analgesics, intravenous polyionic isotonic fluids, oral laxative therapy, and enemas. foals with meconium impactions are expected to exhibit some degree of pain. judicious use of analgesics (table 11 -2) is required to balance the necessity to provide relief from pain and the ability to appropriately assess the patient's progress. mineral oil (4 to 8 ounces administered via a nasogastric tube) is used for its lubricating effect. milk of magnesia (1 to 2 ounces) provides an osmotic laxative effect, but should be used sparingly as it may be dehydrating. the detergent dioctyl sodium sulfosuccinate can be quite irritating, and it should be avoided in both oral and rectal therapy. 8 castor oil therapy has been described, 34 but can provoke violent abdominal pain in the foal. enemas are a mainstay of treatment for small-colon meconium impactions. warm-water liquid detergent (i.e., palmolive ® ) enemas ( 1 / 2 teaspoon liquid detergent to 500 ml water) are purportedly gentle to the rectal mucosa and effective. commercial phosphate enemas (i.e., fleet ® ) can also be used, but repeated administration may increase the risk of phosphate toxicity. recently, acetylcysteine retention enemas have been reported to be a highly successful treatment for meconium impactions in foals. 11 it is hypothesized that the acetylcysteine cleaves disulphide bonds in the mucoprotein molecules in meconium, decreasing its overall tenacity. a 4% acetylcysteine solution, ph 7.6, is made by adding 20 g of baking soda and 8 g of acetylcysteine to 200 ml of water. a 30-french foley catheter with a 30 ml bulb is inserted approximately 2.5 to 5 cm into the rectum and, the bulb is slowly inflated to occlude the rectum. one hundred to 200 ml of the 4% acetylcysteine solution is administered by gravity flow and retained for 30 to 45 minutes. the acetylcysteine retention enema was effective in eliminating 78% of meconium impactions within 12 hours. 11 if needed, the acetylcysteine therapy can be repeated in 12 hours, and was repeated up to three times in some cases before resolution of the impaction. occasionally, repeated use of an enema generates significant rectal and small colon mucosal irritation that sustains signs of straining despite effective removal of the impaction. this continued straining may confound determination of successful treatment. surgical intervention should be considered if medical therapy is unsuccessful. 35, 36 although there often is no single criterion that distinguishes the course of treatment in any particular case, an exploratory celiotomy should be considered in a foal with abdominal pain (of any etiology) if there is: the prognosis for foals with a meconium impaction is generally considered good to excellent with short-term survival reported to be 100% 11 and long-term survival, following either medical or surgical treatment, reported to be 80% to 94%. 35, 36 most meconium impactions will resolve with medical intervention. at the university of california, davis, from 1987 to 2002, 41 out of 44 foals (93%) with meconium impactions were successfully treated medically with acetylcysteine enemas. 11 about 40% of these former cases required more than one acetylcysteine enema, and in about 20% of cases it took more than 12 hours for the impaction to resolve. once hospitalized, '03 rockn'roll received one acetylcysteine enema and oral laxative and intravenous fluid therapy over 12 hours. prior to surgery, the foal exhibited only mild to moderate signs of intermittent pain (straining to defecate with recumbency and rare dorsal recumbency) and his abdominal size did not significantly increase. the discovery of pneumoperitoneum before celiotomy was worrisome. however, because an abdominocentesis had been performed several hours prior to taking the abdominal radiograph, it was speculated that the pneumoperitoneum was iatrogenic from introduction of room air through the abdominocentesis needle into the peritoneal cavity. the subsequent detection of a ruptured transverse colon during surgery was an unexpected complication. spontaneous rupture of the colon as a sequela to meconium impaction must indeed be rare, as there are no reports of its occurrence in the american literature. it remains unclear as to when the colon rupture occurred. at presentation, the foal did not exhibit clinical signs of sepsis, though a small but significant left shift was present on the leukogram. this may have been a result of early sepsis from an unrelated cause or may have been the result of bacterial translocation across compromised mucosa along the colonic impaction. the foal did not exhibit intense signs of abdominal pain, in fact, he only showed signs of straining to defecate during the first 10 hours of hospitalization. although he received an analgesic dose of flunixin meglumine prior to referral, he only received one other shortacting analgesic prior to surgery: 1 mg of butorphanol. the abdominocentesis was performed at approximately 10 hours of hospitalization, after the foal was observed to roll into dorsal recumbency. although the nucleated cell count and protein concentration were both increased, there were no signs of sepsis cytologically. either rupture had not yet occurred or it had already occurred and the peritoneal fluid sample obtained by abdominocentesis was not representative. in retrospect, the pneumoperitoneum seen radiographically at 12 hours of hospitalization most likely was indicative of a localized rupture. the decision to perform an exploratory celiotomy was based upon the lack of passage of a significant amount of meconium, the presence of extensive amounts of meconium in the colon (as seen ultrasonographically and radiographically), the presence of free air in the peritoneal cavity on abdominal radiographs, and the development of persistent tachycardia, with a sustained heart rate greater than 120 beats/minute. although the majority of foals with meconium impactions are treated medically, surgery is indicated in some cases. postoperative intra-abdominal adhesion formation is the most common anticipated complication, thus surgery is often delayed for fear of poor long-term survival. two short-term survival (i.e., survival until discharge) studies on foals undergoing exploratory celiotomy for meconium impactions had a combined survival rate of 90% (9 out of 10 foals). 4, 6 long-term survival to maturity was 67% (4 out of 6) for foals undergoing surgery with either enterotomy or manual reduction. 36 in a 2002 retrospective study in germany on 42 foals undergoing an enterotomy for a meconium impaction, long-term survival to six months was 60%. 35 in general, for foals undergoing an exploratory celiotomy, the survival rate is better for foals with lesions of the large colon, compared to the small intestine. 8 although studies before 1994 indicate that survival rate for foals undergoing surgery for colic is lowest for neonates, data from the early 1990s showed significantly higher survival rates, as compared to the 1980s. [4] [5] [6] with advancements in diagnostics and critical care, assuredly the long-term survival has continued to improve, though specific data is unavailable for recent years. (figure 11-27) . over the next several hours, the foal could rise with assistance and would attempt to nurse, but would stop nursing shortly after beginning. dorsal recumbency became increasingly frequent. gastric ulceration has been diagnosed in foals as young as 24 hours of age and can been seen throughout the neonatal period and beyond. 1 the incidence of gastric ulceration in foals has been cited as high as 50% in normal foals less than 50 days of age. 2, 3 because of this, it is difficult to know what role it plays in the health of the asymptomatic foal. 4 stress has been implicated as a cause for gastric ulceration in the sick foal. in the neonate, ulcers are often present with concurrent diseases, such as enteritis or sepsis. the history should be utilized to determine if the foal had any prior illnesses, including diarrhea, colic, lethargy, or anorexia. furr found that foals stressed by disease were more likely to have abnormal cortisol, t3, t4, and reverse t3 levels than age-matched normal controls foals. these stressed foals had a higher incidence of gastric ulceration than the normal foals. 4 the clinical findings associated with ulcers can range from asymptomatic to death secondary to severe peritonitis from gastric rupture. a study in japan of 40 foals with gastric ulceration found that the most prevalent clinical signs were depression and intermittent nursing (82.5% of affected foals) followed by diarrhea (65%), colic (37.5%), bruxism (10%), and ptyalism (7.5%). 5 affected foals will often lay in dorsal recumbency trying to be more comfortable. spontaneous gastric reflux from the nostrils can be seen on rare occasion. foals with suspected or confirmed ulcers should be evaluated closely for sepsis or signs of peripartum asphyxia, as ulcers may develop secondary to these conditions. some clinicians associate the stress of orthopedic disease in foals to be a risk factor for the development of gastric ulcerations. because of the role that other diseases may play in the development of gastric ulcers, the diagnostics surrounding the neonate with gastric and/or duodenal ulceration should include a thorough investigation of the systemic status of the foal, including complete blood count, serum biochemistries, and urinalysis. blood cultures should be taken to evaluate for concurrent sepsis. because of the potential role for hypoperfusion in the development of ulcer disease, perfusion should be monitored through serial blood pressure measurements (direct or indirect) and blood or plasma lactate concentrations. specific diagnostics for ulcer disease include gastroscopy or gastroduodenoscopy (including esophagoscopy for reflux esophagitis) as the most sensitive and specific means of diagnosing ulcer disease in the newborn. usually this can be accomplished with a 1-meter endoscopy. the foal should be fasted for approximately two hours before scoping so that the gastric mucosa can be viewed better. the examination should be systematic being sure to view the distal esophagus and the squamous and glandular portions of the stomach. lesions may vary from a mild hyperemia or gastritis to deep bleeding ulcerations ( figure 11-28, a and b) . another finding of gastroscopy in young foals is a desquamation of the squamous epithelia. this occurs in 80% of foals less than 35 days of age. sheets of squamous epithelia shed during this time period. this can occur with or without ulceration (figure 11-29 ). 6 radiographs of the abdomen may be useful in ruling out other causes of colic in the foal. the presence of free gas in the abdomen may be suggestive of a gastric perforation. contrast radiography should be performed in foals with suspected gastric outflow obstruction (emptying disorders) such as those with a b fig. 11-28 a, endoscopic view of ulceration of the squamous mucosa of a foal's stomach. b, ulcerative esophagitis from gastric reflux. note the "cobblestone" appearance of the mucosa. pyloric or duodenal ulceration (see . delayed gastric emptying should be suspected if the barium is still in the stomach after two hours. foals with ulcer disease should also have abdominal ultrasonography performed to evaluate for bowel wall thickness, ileus, and the possibility of peritoneal effusion. ascites due to peritonitis in response to ulcers is a negative prognostic sign, and can mean perforation is imminent or has occurred. fecal or gastric occult blood may be suggestive of bleeding ulcers, but these tests are neither sensitive nor specific. neonatal ulcer disease occurs in a number of different syndromes, including silent ulcers, clinical or active ulcers, perforating ulcers, and duodenal ulcers. silent ulcers are subclinical, but on occasion they have gone on to perforate. they may also be found incidentally on gastroscopy or postmortem examination. clinical ulcers are consistent with the foal of this case; clinical signs included depression, partial anorexia, bruxism, ptyalism, dorsal recumbency, and colic. ulcers can develop both in the glandular and nonglandular regions of the stomach. duodenal ulcers, most common in the older (two-to five-month-old) foal, can result in pyloric strictures and gastric outflow obstruction and appear to represent a different syndrome than neonatal ulcers. these foals may develop reflux esophagitis as a result of the obstructive nature of this problem. healthy neonatal foals have an acidic baseline gastric ph, with an average ph of approximately 3.2 to 3.7. 7 they intermittently develop a gastric ph level below 1. prolonged recumbency, greater than 20 minutes, results in a more acid gastric ph, often below 2.5 for prolonged periods. 7 suckling milk raises the gastric ph to above 4.0, and sustains this high ph for several minutes. 7 this emphasizes the importance of frequent feedings in minimizing the potential for development of ulcers in the critically ill foal that is unable or not allowed to nurse. the acid secretory profile of the critically ill foal is highly variable and differs widely from foal to foal. in one study, 43% of 23 hospitalized foals demonstrated alkaline profiles continuously, while another 43% had profiles typical of healthy foals. 8 the etiology of alkaline ph is unknown, but may represent parietal cell dysfunction as a consequence of hypoperfusion, or may alternatively reflect ileus with enterogastric reflux from the duodenum. approximately 13% of foals have atypical profiles with periods of marked acidity. the pathophysiology of gastric ulcer disease is multifactorial. in human infants, concurrent illness is strongly associated with gastric ulcers, with risk factors in some studies including mechanical ventilation (one of the most consistent risk factors), abnormal mode of delivery, delayed delivery, and hypotension. 9 luminal factors that may predispose to ulcer formation include hydrochloric acid (hcl), pepsin, bile acids, and volatile fatty acids. 10 additional pathophysiologic mechanisms for ulcer development include impairment of mucosal perfusion, reduction in mucus or bicarbonate secretion, neutrophil-and inflammation-mediated injury, and inhibition of nitric oxide. normal defenses of the mucosa vary anatomically. the squamous regions, including the esophagus, cardia, and fundus, are protected from acids by intercellular tight junctions. the glandular epithelium is protected by a mucous and bicarbonate barrier, prostaglandins, a rich mucosal blood supply, and rapid cellular restitution upon injury. mucosal blood flow is highly dependent upon prostaglandins e1 and e2 as well as nitric oxide. normal gastric and duodenal motility and duodenal sphincter tone are other protective mechanisms that minimize duodenal reflux of bile acids and other cytotoxins. ulceration in the squamous mucosa, particularly the region adjacent to the margo plicatus, must be interpreted with caution in terms of clinical significance because of the finding that up to 50% of clinically normal foals may have ulcers in this area. 2,3 only 3% of these healthy foals had ulcers in the glandular mucosa. however, up to 40% of critically ill foals had glandular ulcers. 4 it appears that concurrent illness (sepsis, diarrhea, pneumonia) increases the incidence of glandular ulcers in the neonatal foal. while luminal factors such as hcl may be the primary mechanisms of nonglandular mucosal injury, damage to the glandular mucosa may be multifactorial. hypoperfusion and reduced oxygen delivery, as may occur with septic shock or hypovolemia, are believed to be important in the pathogenesis. hyposecretion of sodium bicarbonate or mucus is also important, and these often occur secondary to hypoperfusion. inflammatory mediators also play a role in mucosal injury. the use of nonsteroidal anti-inflammatory drugs, such as phenylbutazone, may lead to gastric ulceration as a consequence of prostaglandin e inhibition, leading to a reduction in mucosal blood flow, and bicarbonate or mucus secretion. 11, 12 candidiasis has been associated with gastric ulcers in neonatal foals, however in the author's opinion it is not clear whether they were primary or secondary invaders. 13 '04 belly dancer was treated with a combination of supportive intensive care and gastric protectants. supportive care consisted of crystalloid administration and parenteral dextrose administration, with a conservative approach to enteral feeding. blood pressure was monitored and maintained with crystalloid therapy. urine output was also monitored closely as a marker of renal perfusion. treatment treatment of the neonatal foal with gastric ulcer syndrome consists of supportive care and the use of antiulcer medications. supportive care consists of maintenance of fluid volume, pressure support, and supplementation of oxygenation through insufflation as necessary for hypoxemia. acid-base and electrolyte disorders should be corrected. antimicrobials should be administered to treat and/or prevent sepsis in the neonate. nutritional support is also critical to the management of the neonate with gastric ulcers. small, frequent meals of milk should be provided in order to buffer gastric acids and to maintain higher intraluminal ph. 7 continuous rate infusions of milk can also be provided, but caution must be taken that the foal's stomach does not become overdistended. 14 the nasogastric tube should be intermittently checked for gastric residual accumulation. specific therapy of gastric ulcers includes mucosal adherents, histamine type 2 receptor antagonists, and proton pump inhibitors. sucralfate, a hydroxy aluminum salt of sucrose called sucrose octasulphate, is a mucosal adherent. at acid ph (<2), sucralfate forms a viscous gel that binds ulcers. sucralfate also inhibits pepsin, buffers acid, and stimulates the production of prostaglandin e. this latter action stimulates bicarbonate and mucus secretion. 10 sucralfate also binds epidermal growth factor and thus may play a role in epithelial restitution. suggested doses for sucralfate include 10 to 20 mg/kg po q 6 to 8 h (table 11-3) . histamine-2 antagonists include cimetidine, ranitidine, and famotidine. these drugs decrease acid secretion by competitively binding to the histamine receptor, thereby reducing stimulation of gastric acid secretion by histamine (see table 11 -3). suggested dosages include the following: cimetidine 20 mg/kg po q 6 h or 6.6 mg/kg iv q 6 h; ranitidine 6.6 mg/kg po q 8 h or 1.5 mg/kg iv q 8 h; famotidine 2.8 mg/kg po q 12 h or 0.3 mg/kg iv q 12 h. 15 because cimetidine is less potent, has variable absorption in horses, and is associated with inhibition of hepatic microsomal enzymes, it may be the least preferred drug compared to the other h-2 antagonists. intravenous or oral administration of ranitidine significantly increased intragastric ph for four and eight hours, respectively, in healthy experimental neonatal foals. 7 however, in critically ill foals, ranitidine may have a blunted duration in terms of alkalinizing response. 8 in addition, these drugs will only be beneficial when gastric ph is acidic, which may not be the case in all foals. 8 ranitidine and sucralfate have been shown to provide partial protection against clinical, clinicopathologic, and pathologic manifestations of phenylbutazone in foals. 16 proton pump inhibitors have been more recently added to the treatment options for gastric ulcers in horses. the most commonly used agent is omeprazole, which decreases acid production through irreversible binding to the hydrogen-potassium atp pump of the parietal cell. omeprazole has been studied in healthy neonatal foals. 17, 18 an oral dose of 4 mg/kg resulted in an increase in gastric ph within two hours of administration (see table 11 -3). for foals with delayed gastric emptying, prokinetic therapy may be in order. metaclopramide is a dopaminergic receptor antagonist that can be given as a constant rate infusion (0.02-0.04 mg/kg/hour). its actions are to tighten the esophageal sphincter, increase gastric motility, and relax the pyloric sphincter. caution should be used, as overdosage can result in neurologic excitement (see table 11 -3). while the therapy of ulcers certainly includes pharmaceuticals to raise gastric ph, prevention of ulcers through their use is controversial. 10, 19 some clinicians feel that alkalinizing the foal's stomach without evidence of ulceration may lead to increased survival of ingested bacteria and a risk of translocation of bacteria through the gastrointestinal mucosa. also as stated above, the acid-secreting profile in the stomach of the critically ill foal may already be alkaline. excellent supportive care along with intensive management of hemodynamics for the at-risk foal is at least as important to prevention of ulcers. until research studies are available for defining the risks and benefits of the prophylactic use of antiulcer medications, definitive recommendations cannot be given. the outcome of foals with gastric ulcers is highly variable. in a retrospective study by taharaguchi, mortality from gastric ulceration ranged from 7.1% to 16.2% of deaths in foals between 1997 and 1999. 5 foals with silent ulcers due to hemodynamic perturbations that go on to perforate have a grave prognosis. foals with ulcers that do not progress to the point of perforation have a better prognosis, particularly those whose primary problem responds to therapy. foals with duodenal ulcers have a guarded prognosis in general, because of the potential for duodenal strictures, fibrosis, and gastric outflow obstruction. 20 (figure 11-30) . the peripartum history and history of the days prior to the development of diarrhea are important in differentiating the causes of diarrhea in foals. the potential for peripartum asphyxia, immature gestational age, and sepsis can be assessed through history. foals with perinatal hypoxic-ischemic injury are at significant risk for gastrointestinal dysfunction, including diarrhea. premature foals are predisposed to necro-tizing enterocolitis. additional information that is pertinent to the workup and support of the diarrheic foal include nursing ability and appetite, mentation and activity level, housing environment, and character and frequency of the diarrhea. assessment of passive transfer of colostral antibodies is also important, as foals with failure of passive immunity are at increased risk of developing sepsis and possibly infection with pathogenic enteric organisms. on the other hand, foals with high intakes of colostrum and milk may be at increased risk for clostridial enteritis, as it has been hypothesized that colostral trypsin inhibitors may protect clostridial toxins from acid degradation in the stomach. ingestion of large quantities of milk from heavy lactating mares may provide for additional enteral substrate for proliferation of clostridial organisms. additional historic information centers on diet. foals provided milk replacer or a nonequine source milk rather than mare's milk are at increased risk for development of osmotic diarrhea. foals from poor milkers may be ingesting water or foreign material such as dirt or bedding and are at risk for diarrhea associated with pica. some level of epidemiologic data is also very useful in determining the etiology of diarrhea in foals. information as to whether the foal is part of an outbreak situation (suggesting an infectious, nutritional, or toxic cause) or the sole foal with diarrhea on a farm is useful in directing the diagnostic approach. hygiene and husbandry practices should be assessed for aiding in education toward minimizing the incidence of diarrhea on farms. stall versus pasture housing and number of foals per paddock or pasture should also be considered. neonatal foals can present with diarrhea at any age between hours and several days of age. early diarrhea is consistent with hypoxic-ischemic damage or sepsis. diarrhea in the slightly older foal is indicative of infectious or dietary causes. the neonate with diarrhea should be evaluated for the following: 1) circulatory volume and hydration status, 2) clinical signs consistent with systemic inflammation (e.g. sirs, sepsis) such as mucosal hemorrhages, congested or hyperemic mucous membranes, injection of vessels, tachycardia, tachypnea, or fever, 3) nutritional status through assessment of body condition or weight, and 4) indicators of concurrent disease such as bacteremia, signs of asphyxial injury, or congenital defects. circulatory status should be assessed in the physical examination by close attention to mentation status, capillary refill time, jugular vein fill time, extremity temperature, mucous membrane color, heart rate, and pulse quality. heart rate can be misleading in the neonatal foal, however, as those with hypoxemia, hypothermia, or hypoglycemia can be bradycardic despite significant hypovolemia. hydration status, reflecting interstitial and intracellular volumes, is indicated by skin turgor, mucous membrane texture, sunken eyes, and corneal quality. the abdomen should be evaluated for distention. ballottement can indicate ascites or gas distention. the perineum should be assessed for scalding and dermatitis associated with fecal adherence to hair and skin. a cautious digital examination can reveal sand or dirt, rectal impactions, and rectal edema. body condition scoring can be performed in the neonatal foal, and a subjective evaluation should be performed as a minimum because many diarrheic foals lose substantial amounts of weight (see chapter 4). 1 because foals with enterocolitis are at risk for bacterial translocation and subsequent sepsis, they should be evaluated for signs of localized sepsis such as effusive joints or enlarged external umbilical stumps. the pathophysiology of diarrhea is multifactorial. factors resulting in diarrhea from enterocolitis include hypersecretion, osmotic draw, altered motility, altered starling's forces (as with increased vascular permeability or hydrostatic pressure, or decreased colloid osmotic pressure), maldigestion/malabsorption, and inflammation. though one mechanism may predominate in some forms of diarrhea, most of them contribute. foals with enterocolitis often exhibit signs consistent with abdominal pain. differential diagnoses that should be considered in foals with acute abdominal disease include strangulating and nonstrangulating obstructions, uroabdomen, and other nongastrointestinal causes of colic, such as liver disease. foals with impending enterocolitis can be very difficult to differentiate from those with obstructive diseases, because they often exhibit signs of abdominal pain and distention prior to the onset of diarrhea. the presence of fever and leukopenia are suggestive of enterocolitis, but not specific. abdominal ultrasonography and radiography can help in differentiation of these conditions. causes of diarrhea in neonatal foals include both infectious and noninfectious causes. noninfectious causes include "foal heat diarrhea," pica, dietary intolerance, gastric ulcer disease, hypoxic-ischemic injury, and necrotizing enterocolitis. infectious causes include bacterial, viral, parasitic, and protozoal causes (table 11-4) . foals with foal heat diarrhea are otherwise bright and nursing well. they do not experience a systemic inflammatory response, and therefore have normal hematologic and vital sign findings. 2, 3 the name foal heat diarrhea is a misnomer because the diarrhea does not appear to be a result of hormonal changes in the mare. 3 rather, it is a result of intestinal maturational changes. it usually occurs between seven and 10 days of life and is self-limiting. pica results from ingestion of sand, dirt, bedding, or hair. 4 inflammatory or motility alterations secondary to the presence of foreign material can result in obstructions or impactions as well as diarrhea. another dietary cause of diarrhea in the foal is dietary intolerance. it occurs when either a foal is lactoseintolerant (secondary to infectious diarrheas such as clostridiosis or rotaviral infection) or is an orphan foal with diarrhea resulting from feeding excessive amounts of milk replacer or milk replacers mixed at higher than 11% total solids. 5 milk replacers with sucrose or maltose are particularly offensive. 5 gastric ulcer disease is associated with diarrhea in some neonatal foals. clinical signs can be inapparent or include bruxism, ptyalism, dorsal recumbency, diarrhea, or colic (see . hypoxic-ischemic insult as occurs with peripartum asphyxia can result in gastrointestinal injury, leading to diarrhea. hypoxic and subsequent reperfusion injury may both contribute to mucosal compromise. smooth muscle and serosal dysfunction may also result. energy depletion as occurs secondary to hypoxic-ischemic injury leads to cell membrane pump failure. subsequent reperfusion injury leads to oxidant injury as well as inflammatory cell activation and "no-reflow" phenomenon. necrotizing enterocolitis (nec) is a state of gastrointestinal necrosis that occurs from mucosal injury, as might occur with asphyxial injury, the presence of enteral nutrition, and bacterial invasion of the gi wall. ileus, gastric reflux, abdominal distention, intestinal perforations, or diarrhea can result from nec. 6 prematurity is a risk factor for development of necrotizing enterocolitis. infectious causes of diarrhea often occur in large groups of foals that are housed together; sporadic cases can also occur, particularly with clostridial agents. viral etiologies include rotavirus and coronavirus. rotavirus infects and denudes small intestinal microvilli, resulting in maldigestion and malabsorption. 7 it thus interferes with lactase function and sodium-glucose cotransport. because of relative sparing of crypt epithelium, the virus also results in increased net secretion. clinical signs vary from mild to severe diarrhea, requiring minimal to intensive therapeutic intervention, respectively. experimentally, the incubation period is as short as two days. 7 most affected foals are five to 35 days of age, although younger and older foals can be affected. older foals (up to 60 days of age) usually have mild diarrhea, and asymptomatic animals can shed the virus. 7, 8 vaccination of gravid mares may reduce the incidence of disease on farms, as well as the severity of clinical signs. 9,10 phenolic compounds are the optimal disinfectant type used, as rotavirus is resistant to many others. coronavirus has been associated with diarrhea in foals. 11, 12 it infects the small intestine during the first few days of life, with persistence of viral antigen in the crypt cells for three to four days after onset of diarrhea. 11 complications of coronaviral infections can include coronitis and limb edema associated with inadequate perfusion of the extremities. 11 other potential viral etiologies of diarrhea include adenovirus and parvovirus, although their exact role in foal diarrhea has not been clearly defined. 13 the most common bacterial causes of diarrhea include clostridial agents, especially clostridium difficile and c. perfringens, and salmonella. the clostridial infections can result in outbreaks or sporadic cases of diarrhea. c. difficile is a primary pathogen in neonatal foals and does not require prior antimicrobial administration as a risk factor for development of diarrhea as is usually the case with adult horses. 14, 15 the primary virulence factors for pathogenicity include two large exotoxins, toxins a (enterotoxin) and b (cytotoxin). c. difficile-associated disease ranges from mild diarrhea to highly fatal, hemorrhagic, or necrotizing diarrhea. 16 it has also been associated with lactose intolerance in foals. 14 it should be noted that toxigenic clostridium difficile can be cultured from the feces of clinically normal foals, just as it can from healthy infants, and it is unknown what circum-stances or agent-host interactions lead to clinical disease. 17, 18 clostridium perfringens types a and c have been reported to cause enterocolitis and diarrhea in neonatal foals. as with c. difficile, the pathogenicity of c. perfringens a and c is dependent upon virulence factors, especially the production of enterotoxins. 19 in a recent epidemiological study, c. perfringens was found in 90% of normal three-day-old foals and 64% of normal foals at eight to 12 hours of age. the most common genotype was type a (85%), and c. perfringens type c was found in less than 1% of the foals. these results suggest that type a is likely part of the normal microflora of the neonatal foal (and thus the clinical relevance of positive cultures without presence of enterotoxin are questionable), whereas type c is rarely found in the normal neonatal and has been associated with the clinical signs of watery to hemorrhagic diarrhea, but also includes abdominal distention, colic, and ileus. 20 therefore, typing of isolates is critical to pursuing positive cultures, because the type of c. perfringens varies between clinically normal and affected foals. foals with c. perfringens, type c infection can develop rapidly progressive obtundation and colic and may die before the onset of diarrhea. c. perfringens-associated diarrhea is associated with a high mortality rate, 54% in one study, and is highly associated with fatal diarrhea in another study. 8, 19 adult horses may serve as reservoirs for spores in the environment. salmonellosis is another bacterial form of enterocolitis that can affect the neonatal foal as it can adult horses. concurrent sepsis is a concern with salmonellae, due to invasiveness and the potential for translocation across the compromised gut barrier. studies have found an association between isolation of salmonellae from foals with diarrhea and fatality. 8 the severity of the disease, in part, relates to the serotype involved, with group b salmonellae, such as s. typhimurium, being among the most pathogenic. salmonella of other groups can also cause disease, for example, a group c1 salmonella ohio has been reported to cause an outbreak of neonatal salmonellosis. 21 mares of infected foals may be asymptomatic carriers of salmonella and the source of the foal's infection. other bacteria that can cause diarrhea sporadically in the neonate include actinobacillus sp. (in association with bacteremia), enterotoxigenic e. coli, bacteroides fragilis, clostridium sordelli, aeromonas hydrophila, and streptococcus durans. [22] [23] [24] [25] [26] [27] the significance and pathogenesis of these agents in neonatal foal diarrhea requires further study. the primary parasitic agent that has been associated with diarrhea in the young foal is strongyloides westeri. 8 it appears that heavy infestations with more than 2000 eggs per gram of feces are required for development of diarrhea, and it is generally not a cause of severe 216 diarrhea. 8 strongyloides westeri is transmitted to foals through suckling milk. the primary protozoal enteric pathogen of foals is cryptosporidium parvum. it was initially believed that cryptosporidium infections were limited to immunocompromised foals such as arabians with severe combined immunodeficiency syndrome. 28 more recently, it has been identified as a pathogen of immunocompetent foals as well. 29 the organism invades the microvillus, occupying an intracellular but extracytosolic space. it thus causes villus blunting and maldigestion with a secondary osmotic diarrhea. most affected foals are five days of age and older, but younger foals can develop disease with high-level exposure. diarrhea usually lasts between five and 14 days, with recovering animals and asymptomatic older foals and adult horses shedding oocysts. one study identified cryptosporidium parvum causing an outbreak of neonatal diarrhea to be genotypically identical to that associated with bovine diarrhea. 30 however, exposure to cattle was not found to be an important source of infection for foals in another epidemiologic study. 29 sources of cryptosporidial infection in foals have not been definitively elucidated. 29 mares, cattle, and municipal water sources have all been suspected as the source of infection in foals, but these remain speculative. 29 because of the zoonotic risk associated with cryptosporidium, affected foals should be handled carefully with particular attention to good hygiene. the role of other protozoal agents, including eimeria leukarti, trichomonas equi, and giardia sp., is poorly understood and requires further study. daily fecal cultures for salmonella sp. were negative. immunoassays for clostridium difficile toxin a were negative, as was fecal culture for c. difficile. the clinical pathology of foals with diarrhea can vary depending upon whether there is a concurrent systemic inflammatory response syndrome. the hematologic (cbc) findings can vary from unremarkable to those of hemoconcentration, leukopenia, and hypoproteinemia. a left shift with immature (band, metamyelocytes) neutrophils, with variable toxic cytologic changes, may also be present, reflecting a profound inflammatory response such as sepsis. hypoproteinemia often reflects protein-losing enteropathy, and hyperfibrinogenemia may be present in the face of inflammatory mediators induced by endotoxemia. thrombocytopenia occurs when coagulopathies such as disseminated intravascular coagulation are triggered. as for hematology, serum biochemistry analyses are normal when diarrhea occurs without gastrointestinal inflammation. enteritis or enterocolitis with subsequent activation of sirs can lead to organ dysfunction as might occur with severe sepsis. increases in creatinine and/or bun and liver enzymes or bilirubin concentrations reflect renal or hepatic dysfunction, respectively. hypoalbuminemia usually reflects gastrointestinal protein loss. similarly, hypoglobulinemia may be present, although it may be due to failure of passive transfer of colostral antibodies as well as gi loss. a variety of electrolyte derangements may reflect enteric losses, renal dysfunction, or acid-base derangements and often include hyponatremia, hypochloremia or hyperchloremia, hypokalemia or hyperkalemia, and an increased anion gap. decreases in total co 2 concentrations reflect metabolic acidemia. anion gap may also be increased with hyperlactatemia. metabolic acidemia results from both organic and inorganic acidosis. lactate is the primary organic anion contributing to acidemia. hyperlactatemia results from a combination of hypoperfusion associated with hypovolemia and hypotension, hypermetabolism, increased glycolysis from inhibition of pyruvate dehydrogenase by inflammatory mediators, enhanced activity of na-k atpase due to circulating catecholamines, and reduced hepatic or renal clearance of lactate. inorganic causes of acidemia in diarrhea cases include hyponatremia or hyperchloremia resulting in a decrease in strong ion difference. the acid-base status is reflected on blood gas analyses as reduced ph and increased base deficit. as part of intensive management of foals with severe enterocolitis, additional diagnostic monitoring should include indirect blood pressure, urine output measurements, and urinalysis with fractional excretion of electrolytes. because foals with diarrhea and sirs are at risk for coagulopathies, monitoring should also include clotting times (pt, aptt) and measurement of fibrin degradation product or d-dimer concentrations. antithrombin concentrations may be low because of induction of coagulopathies or loss of this small protein through the compromised gi mucosa. imaging should be a part of the diagnostic workup of these foals with diarrhea. abdominal ultrasonography and radiography can aid in differentiating causes of acute abdominal disease. ultrasound evaluation should include assessment of bowel wall thickness, luminal diameter, and small-intestinal motility (figures 11-16 and 11-31) . ascites or effusion can also be detected. radiography can be utilized to assess the degree of gastrointestinal distention (figures 11-9 , 11-12, and 11-32) . it can also be utilized to detect pneumatosis intestinalis, or linear gas shadows within the wall of the gi tract, a hallmark of necrotizing enterocolitis. sand and radiopaque foreign material will also be apparent on abdominal radiographs. specific diagnostic evaluation of the neonatal foal with diarrhea includes a number of tests to rule out pathogenic agents (table 11 -5) . fecal samples can be screened for viral particles with electron microscopy. fecal elisa and immunoassays are available for rotavirus and coronavirus. fecal cultures can be performed for salmonella sp. and clostridial organisms. to increase the sensitivity of identifying salmonella shedders, repeated daily cultures should be performed (up to five consecutive samples). alternatively, pcr techniques are available for salmonellae, although the findings must be interpreted in light of the high-level sensitivity of this test. positive cultures for c. difficile and c. perfringens must be interpreted with caution as nontoxigenic and incidental colonization is possible. the diagnosis of c. difficile-associated disease should be coupled to toxin assays. commercial immunoassays test for toxin a or both toxin a and b. these kits are easy and rapid, however they have variable sensitivities. stool cytotoxin assays for toxin b are also available and are considered the gold standard for toxin testing, but are time-consuming. pcr techniques for c. difficile toxin gene sequences are currently moving from the experimental arena to the clinical setting. diagnosis of c. perfringens enterocolitis is difficult to make. unfortunately, immunoassays are not available for most toxins, except for enterotoxin. therefore, cultured isolates must be typed using pcr techniques, such as the multiplex pcr. the diagnosis is made from positive cultures of toxigenic and pathogenic isolates of c. perfringens (e.g., type a or c), as well as exclusion of other pathogenic agents. gram staining of feces can serve as a rapid screen for clostridial overgrowth, however this technique is neither sensitive nor specific (figure 11-33) . there are a number of diagnostic tools available for diagnosis of cryptosporidium parvum infection. immunofluorescence assay (ifa), acid-fast (af) staining, and flow cytometry have been developed for identification of oocysts. 29 in one study, af and flow cytometry were determined to be more sensitive than ifa. 29 af staining was less specific, however. because of their small size, oocysts are very difficult to visualize microscopically after fecal flotation. fecal flotation is useful for identifying strongyloides westeri ova, with the mcmaster method used for quantification. if one is concerned that lactose intolerance is playing a role in the continuation of a foal's diarrhea, then the use of a lactose tolerance test may be indicated. after a four-hour fast, the foal is administered lactose (1 g/kg) through a nasogastric tube. blood samples collected in fluoride oxalate tubes are taken for glucose levels at preadministration, immediately postadministration, and every 30 minutes for three hours. a normal absorption curve would show a doubling of glucose in 60 to 90 minutes. an absence of increase in glucose or a delay in the increase would indicate a problem with lactose digestion and absorption. 31 tiofur (10 mg/kg, iv, q 12 h) and amikacin (21 mg/kg, iv, q 24 h) were administered to provide broad-spectrum antimicrobial coverage with potent gram-negative efficacy. kaolin pectate and bismuth subsalicylate were utilized intermittently as gastrointestinal protectants. the treatment of foals with diarrhea is highly variable, depending upon the severity of clinical signs and physiologic derangements. in general, the management of enterocolitis includes a number of goals. these include hemodynamic, metabolic, and nutritional support; gastrointestinal protectants; antibiotic coverage; and infectious disease control. if a specific agent is identified, then specific therapy for that agent can be instituted. hemodynamic disturbances are common among foals with enterocolitis, particularly those with sirs or sepsis syndrome. hypoperfusion results from hypovolemia, altered myocardial contractility, inappropriate vasomotor responses, and hypooncotic states associated with hypoalbuminemia. hemodynamic support occurs primarily in the form of intravenous fluid therapy, consisting of both crystalloids and colloids. a number of crystalloids are appropriate for foals with diarrhea, but because of the tendency for metabolic acidemia, fluids with higher strong ion differences such as normosol r, plasma-lyte 148, or lactated ringers solution are more ideal than 0.9% saline. 32 volume replacement in the neonatal foal should be done with great attention; overzealous administration of crystalloids may potentiate gastrointestinal edema. monitoring of central venous pressure, urine output, and serial lactate concentration measurements can aid in guiding volume replacement. colloid therapy is indicated in the hypooncotic and hypovolemic foal. plasma or synthetic colloid solutions, such as hetastarch, can be used. plasma has the advantages of providing not only albumin, but also immunoglobulin, antithrombin, clotting factors, and other proteins. if hetastarch is used, platelet counts and clotting times should be monitored, as coagulopathies associated with reduced concentrations of von willebrand's factor and factor viii may develop. once volume replacement has occurred and the central venous pressure (cvp) is at maximum (10 cm h 2 o), hypotension should be addressed with inotropes, such as dobutamine (2 to 20 μg/kg/minute, iv cri). if hypotension and clinical parameters of hypoperfusion persist after initiation of inotropy, then vasopressors should be considered (norepinephrine or vasopressin cri-see chapter 7). metabolic support occurs in the form of managing acid-base and electrolyte balance. metabolic acidemia is treated depending upon the cause of acidosis; organic acidoses (lactic acidosis) should be treated with optimizing peripheral oxygen delivery. inorganic acidoses, as occur with hyponatremia or hyperchloremia associated with electrolyte loss into the gi tract, can be treated by correcting the underlying electrolyte disorder. sodium bicarbonate is indicated in these situations. potassium, calcium, and magnesium may need to be supplemented in foals with severe diarrhea. glucose concentrations should be monitored frequently in the neonatal foal, particularly if the foal is anorexic or receiving parenteral dextrose. both hypoglycemia and hyperglycemia should be avoided. recently, tight glucose regulation with insulin has received a great deal of study in human critical care and is associated with improved survival in septic patients (see chapter 7) . 33 in cases with severe diarrhea, ileus and abdominal distention, abdominal discomfort, or suspected osmotic diarrhea, partial or complete withholding of milk should be considered. parenteral nutritional support is required in these foals (see chapter 4) . antimicrobial therapy should be provided in order to minimize the risk for bacterial translocation across the compromised gut barrier. broad-spectrum coverage, as with a beta lactam and aminoglycoside combination or second-or third-generation cephalosporins, is indicated (see chapter 5). gastrointestinal protectants include di-tri-octahedral smectite and activated charcoal, which act as adsorbents. smectite has been shown to bind clostridial toxins in vitro. 34 however, to the author's knowledge, this product has not been evaluated in neonatal foals and should be used with caution. other protectants include kaolin/pectin combinations and bismuth subsalicylate. nonspecific therapy for diarrhea may include antiulcer medication and probiotics. sucralfate is also protective, stimulating mucosal blood flow and mucus production because of increased local prostaglandin synthesis. treatment of gastric ulcers with proton pump inhibitors and/or histamine type 2 receptor antagonists is indicated when ulcers are suspected from clinical signs (bruxism, ptyalism, dorsal recumbency) or documented via gastroscopy (see . probiotics are often utilized to modulate colonic flora. however, evidence documenting their efficacy in horses is lacking, and their use in neonatal foals prone to bacteremia warrants further study. if a specific offending agent is identified, then treatment may be more directed. metronidazole, administered orally or intravenously (10 to 15 mg/kg, q 8 to 12 h), is indicated in clostridiosis cases. the early use of metronidazole in even suspect cases of c. perfringens is important because foals can die before the onset of diarrhea. metronidazole is also the first-line therapeutic for treating c. difficile infection in foals, however, cases of metronidazole resistance have been documented. 35, 36 in these cases, vancomycin can be used, although it should be restricted to foals in isolation and foals that have severe clinical scores. unfortunately, bacitracin is uniformly ineffective against equine isolates of c. difficile. 36, 37 ivermectin is effective in treating strongyloides westeri infestations. 38 paromomycin has been suggested as a treatment for cryptosporidium infections, although its efficacy and safety are unproven in foals. 39 exogenous lactase enzyme (3000 to 6000 u/50 kg foal, po, q 3 to 6 h) should be provided to foals with rotaviral, cryptosporidial, and streptococcal infections and those with osmotic diarrheas to aid in digestion and prevention of chronic osmotic diarrhea. nursing care is very important for the diarrheic neonate. cleanliness and hygiene are important not only for infectious disease control, but also for preventing secondary complications such as thrombophlebitis in the patient. the foal with diarrhea should be isolated so as to prevent spread of infectious agents. consideration must be given to efficacy of particular disinfectants for specific agents. though bleach is effective against most pathogens, rotavirus is best killed with phenolic compounds. cryptosporidium is resistant to most disinfectants. high heat and ultraviolet light may be helpful in eliminating this organism. owners should be warned about the potential zoonotic nature of salmonella and cryptosporidium. frequent handwashing and use of separate clothing when working with sick foals are prerequisites of curtailing the spread of the infection. cryptosporidium can also cause disease through any mucous membrane contact. protective eyewear and the use of surgical masks may be an important part of disease prevention in caretakers. physical examination revealed the foal to be severely depressed and responsive only to noxious stimuli. the foal exhibited a systemic inflammatory response syndrome, with tachycardia, tachypnea, and pyrexia. clinical signs compatible with sepsis were found on ophthalmologic exam, including yellow-discolored irides, hyphema, hypopyon, and aqueous flare. the foal was markedly icteric (mucous membranes and sclera) and was in hypovolemic shock as exhibited by prolonged capillary refill time, tachycardia, poor pulse quality, and cold extremities. important historical information for the foal with icterus and altered mentation includes farm epidemiologic data such as the number and percentage of affected foals. the spectrum of clinical signs and course of the disease encountered in outbreak situations are important, as is the age range. administered supplements or medications should be evaluated for potential hepatotoxicity. for isolated cases, the history leading up to the disease should be investigated. age of onset is important; for example tyzzer's disease is limited to foals a few days of age to six weeks of age. 1,2 management practices, including housing, diet, peripartum history, and additional concurrent disease in the herd, should be considered. treatment prior to veterinary examination or therapies provided by the referring veterinarian, in the case of specialty practices, should be evaluated. a detailed physical examination of the foal with liver dysfunction is important not only for elucidating the etiology, but also for management and therapy. with acute hepatic disease, foals may exhibit a systemic inflammatory response syndrome with tachycardia, tachypnea, and/or pyrexia or hypothermia. hypovolemia and dehydration are common due to lack of nursing, third space losses (diarrhea or ascites), or vascular permeability alterations. icterus or jaundice is a key finding with liver disease, and mucous membranes, including gingiva, vulvar, and ocular mucous membranes, as well as sclera should be evaluated closely for yellow discoloration (figure 11-35) . fecal character should be evaluated, as some forms of liver disease can cause diarrhea or changes in fecal color. for example, foals with biliary atresia may have gray feces while those with tyzzer's disease can exhibit diarrhea. [3] [4] [5] abdominal distention may be present, and abdominal ballottement can be used to reveal ascites or hepatomegaly. foals with subacute or chronic liver disease may exhibit weight loss or poor growth, which may be apparent as poor body condition. those with acute hepatopathy, such as tyzzer's disease, have a normal body weight and appear normal until acute onset of signs. 2, 6 the perinatal foal with hepatopathy should be closely evaluated for concurrent signs of hypoxicischemic injury, including encephalopathy, enteropathy, and nephropathy. hepatic dysfunction may occur in these foals as well. similarly, foals with sepsis can develop hepatitis. hepatoencephalopathy is present in many foals with acute liver disease or end-stage liver failure. signs range from subtle to severe and fulminate. subtle signs include mild depression and yawning. more obvious signs include central blindness, circling, head pressing, seizures, behavioral changes, and obtundation to coma. fundic examinations should be performed in icteric foals because herpes-positive cases often demonstrate dark and red-discolored optic discs and irregularly dilated vessels. 7 the pathophysiology of liver disease in the neonatal foal is multifactorial and complex. because of a large number of etiologies, insult or abnormalities may occur in the form of congenital, genetic, toxic, infectious, and hemodynamic causes. the differential diagnoses for foals with icterus include fasting hyperbilirubinemia, increased erythrocyte destruction, hepatocellular disease, and cholestatic disorders. causes of hemolysis include neonatal isoerythrolysis, drug-induced lysis, and increased red blood cell turnover from sepsis or peripartum asphyxial injury. sepsis may impair bilirubin metabolism due to the presence of endotoxemia contributing to hyperbilirubinemia. liver disease occurs from a number of causes. infectious causes include bacterial hepatitis secondary to sepsis, and infection with actinobacillus equuli or clostridium piliformis. bacteremia and sepsis in general can cause hepatic dysfunction by means of inducing hypoperfusion and tissue hypoxia. inflammatory mediators stimulated by the systemic inflammatory response syndrome (sirs) will contribute to hepatic damage. cholangiohepatitis from enteric microbes can result from ileus, enteritis, or ulcerative duodenitis/ stricture syndrome. bacterial hepatopathy may also occur as extension of umbilical vein phlebitis or abscessation. hepatoencephalopathy was associated with rhodococcus equi infection in one foal. although not cultured, a liver biopsy suggested a bacterial etiology and the authors speculated that it may have been due to r. equi bacteremia or ascending biliary infection. 8 tyzzer's disease, caused by clostridium piliformis, formerly known as bacillus piliformis, is an acute and multifocal hepatic necrosis associated with a high mortality rate. most commonly associated with sporadic cases on farms, outbreaks of disease can also occur. foals present with a rapidly progressive clinical course including recumbency, hyperthermia or hypothermia, and stupor or coma, icterus, and seizures with marked hypoglycemia and metabolic acidosis. 9 some foals may be found dead without preceding clinical signs. viral hepatitis from equine herpes virus 1 (ehv-1) neonatal infection can occur in association with respiratory distress, neurologic signs, and bone marrow necrosis resulting in leukopenia. foals are infected in-utero from repeated cell-associated viremia in the dam, 9 so signs are seen immediately at birth. icterus is a common finding in neonatal herpes infections as a result of multifocal and acute hepatic necrosis. despite profound necrosis, liver enzymes were not increased in one retrospective report. 10 in the report, herpes-positive foals were more likely to have total white blood cell counts less than 3,000/μl and to be icteric (and hyperbilirubinemic) compared to septic or premature foals. foals with neonatal herpes infections have a poor prognosis; however, those with milder infections may survive. treatment with acyclovir can be attempted. 11 foals with hypoxic-ischemic injury from peripartum asphyxia most commonly exhibit clinical signs consistent with encephalopathy, nephropathy, or gastroenteropathy, however hepatopathies may also result. such foals exhibit icterus from biliary stasis and hepatic dysfunction, and have increased concentrations of liver enzymes. foals with neonatal isoerythrolysis sporadically develop hepatopathies with increases in direct bilirubin concentration in addition to indirect hyperbilirubinemia and increased liver enzymes. 12 liver disease is suspected to occur as a result of tissue hypoxia, iron overload from hemolysis, or bile stasis caused by the increased amounts of conjugated bilirubin. 12 another form of liver disease occurs secondary to gastrointestinal disease, primarily strangulating or obstructive lesions and ileus. endotoxin or bacteria can enter the portal circulation from translocation. congenital causes of hepatopathies include portacaval shunts and biliary atresia. 3, 13 portosystemic vascular anomalies include both intrahepatic and extrahepatic shunts. [13] [14] [15] persistent vitelline arteries have been reported in conjunction with intrahepatic portosystemic shunts. 16 foals with portosystemic shunts have recurrent episodes of blindness, seizures, or altered mentation associated with hepatoencephalopathy. the shunts may not be clinically apparent until affected foals are eating grain or hay. congenital hepatic fibrosis and cystic bile duct formation is a syndrome suspected to be an autosomal recessive genetic trait of swiss freiberger horses. 17 this report included 30 foals that demonstrated jaundice, neurologic signs, colic, and unthriftiness. hyperammonemia of morgan foals and equine glycogen storage disease iv are syndromes suspected to have a genetic basis with some degree of hepatopathy. [18] [19] [20] persistent hyperammonemia in morgan horses is suspected to be similar to a syndrome of hyperornithinemia, hyperammonemia, and homocitrullinuria (hhh) in humans. 18 these foals exhibit neurologic signs including seizure activity, aimless wandering, yawning, and circling. supportive care, lactulose, and a low-protein diet resulted in temporary clinical improvement in one of two foals that was treated, however the foal relapsed and was euthanized. 18 glycogen-branching enzyme deficiency has been recently attributed to a mutation in the glycogen branching enzyme 1 (gbe 1) gene in quarter horses. 20 the syndrome has recently been termed glycogen storage disease iv and is believed to be inherited as an autosomal recessive trait, with clinically affected foals being homozygous for the mutation. 19, 20 affected foals are sometimes stillborn, and otherwise exhibit flexural deformities, seizure activity, respiratory or cardiac failure, or recumbency. leukopenia, hypoglycemia, and high serum ck, ast, and ggt concentrations were common, and all foals died by seven weeks of age. 19 the livers of affected foals contained periodic acid schiff's (pas)-positive intracellular inclusions and no gbe activity. an additional consideration for congenital hepatic disease is hepatoblastoma. 21 polycythemia, hyperbilirubinemia, and increased liver enzymes occur with this liver neoplasm. 21 these foals may survive to weaning or adulthood, but hepatoblastoma has been reported in an aborted fetus and may thus be congenital. 22 toxic hepatopathies have been reported in neonatal foals following administration of oral pastes contain-ing ferrous sulfate in the first one to two days of life. 23 experimental administration of iron to foals confirmed that the hepatotoxicity from the oral paste was indeed due to the contained iron. 23, 24 pathologic changes included liver atrophy, bile duct hyperplasia, lobular necrosis, cholestasis, and periportal fibrosis. druginduced hepatopathies can also occur during this time period because of relatively immature hepatic function and increased absorption of macromolecules during colostral igg absorption. 12 oral medications should be used with caution in foals less than 24 hours of age because of the potential for increased bioavailability with subsequent hepatotoxicity. other potential hepatotoxins reported in horses include aflatoxins, pyrrolizidine alkaloids, leukoencephalomalacia, and chlorinated hydrocarbons, although these are considered unlikely in the young foal because of lack of ingestion of feedstuffs by neonates. steroid hepatopathy has been reported in three-and 10-year-old horses, secondary to intramuscular administration of high doses of triamcinolone acetonide in both cases. 25, 26 (figure 11-36) . clinical pathologic findings in foals with liver disease include increases in concentrations of both hepatocel-224 lular and biliary enzymes. aspartate aminotransferase (ast) and sorbitol dehydrogenase (sdh) are examples of hepatocellular enzymes. ast is not liver-specific, however, having significant components originating from both skeletal and cardiac muscle. sdh is considered to be liver-specific and has a short (two-to three-hour) half-life. biliary-derived enzymes include γ-glutamyl transferase (ggt) and serum alkaline phosphatase (sap). ggt is nearly biliary-specific, however pancreatic origins must be considered when concentrations are increased. sap has isoenzymes from multiple sources, including fast-growing bone in the neonatal foal. it should be noted that serum biochemical indicators of liver status in neonatal foals have some distinct features as compared to adult horses. 27 serum alkaline phosphatase (152 to 2835 iu/liter), ggt (13 to 39 iu/liter), and sdh (0.2 to 4.8 iu/liter) activities are increased during the first two weeks of life. 27 serum cholesterol, triglycerides, and total and unconjugated bilirubin concentrations peak during this same neonatal period. foals with cholangiohepatitis and infectious hepatopathies usually have increases of both hepatocellular and biliary enzymes. similarly, those with toxic insults and hypoxic-ischemic disease also have increased liver enzymes. glycogen-branching enzyme deficiency results in increased ggt concentrations. those with portacaval shunts often have normal liver enzymes, but liver function tests are abnormal. foals with herpes hepatopathies also usually have normal enzymes, but increased bilirubin concentrations. both indirect-reacting (unconjugated) and directreacting (conjugated) bilirubin concentrations increase with liver disease. cholestatic disease results in increases in both components of bilirubin as well, while the percentage attributed to direct bilirubin is usually higher than with primary hepatocellular disease, often exceeding 20% to 25% of the total bilirubin concentration. on the other hand, fasting hyperbilirubinemia and that resulting from hemolysis are due exclusively to indirect bilirubin. serum bile acid concentrations increase with liver dysfunction, as occurs with portacaval shunts. they represent a good screening test of liver function. bile acids, such as choleic and chenodeoxycholic acids, are synthesized by the liver from cholesterol, and are excreted into bile after conjugation with amino acids. these conjugated forms form micelles with fat in the intestine and are reabsorbed and recirculated via enterohepatic circulation. serum concentrations of bile acids increase in the face of hepatocyte damage, biliary stasis, and shunting of portal blood to the systemic circulation as occurs with portosystemic shunts. hyperammonemia is often present, also reflecting liver dysfunction. the liver is the primary site of ammonia removal by converting it to urea for renal excretion. foals with portacaval shunts commonly have high resting ammonia concentrations. morgan foals with hhh syndrome have very high concentrations of blood ammonia (>200 μmol/liter), while liver enzymes are only mildly to moderately increased and bilirubin is normal to mildly elevated. proper handling procedures are critical to accurate measurement and interpretation of blood ammonia. 28 heparinized blood should be used, and the sample should be collected gently (to avoid hemolysis) and kept on ice. the sample should be run within one hour of collection or otherwise spun in order to freeze the plasma at −20°c for subsequent measurement within 48 hours. blood from aged-matched control foals should be run concurrently. 28 additional biochemical indicators of liver function include serum glucose, bun, albumin, and cholesterol concentrations. glucose concentrations are variable, and reflect the degree of hepatic necrosis and milk intake. foals with severe hepatitis or necrosis, such as those with tyzzer's disease, have profound hypoglycemia. those with portacaval shunts also commonly exhibit hypoglycemia. as bun, albumin, and cholesterol are synthesized by the liver, dysfunction is reflected by reduced concentrations of these analytes. however, hypoalbuminemia is uncommon with liver disease, as only 18% and 6% of horses with chronic and acute liver disease had albumin concentrations below the reference value in one report. 29 it has been estimated that greater than 80% of liver mass must be lost for longer than three weeks before hypoalbuminemia develops. 30 fractionation of blood amino acids and determination of the branch chain amino acid (bcaa) to aromatic amino acid (aaa) ratio can aid in determining hepatic function. decreases in the ratio indicate insufficiency, with normal ratios falling between 3.5 and 4.5. 30 because of liver dysfunction and reduced conversion of blood ammonia to bun, a decrease in bun concentration is often observed. it should be noted, however, that bun concentrations in the normal neonatal foal are lower than those in the adult horse and low concentrations are therefore difficult to interpret in the neonatal liver patient. bromsulphalein (bsp) and indocyanine green are dyes that are excreted primarily by hepatic clearance. clearance and half-life reflect hepatic excretory function and are prolonged in animals with biliary obstruction or hepatocellular failure. it should be noted that alterations in hepatic blood flow alter interpretation of bsp clearance; significant decreases in blood flow as occurs with portosystemic shunts will reduce the delivery of bsp and hence cause an increase in halflife. high bilirubin and low albumin concentrations will result in increased and decreased bsp half-life, respectively. quantification of serum bile acids has largely replaced the use of bsp clearance as an indicator of hepatic function in horses. 30 the concentration of triglycerides may increase in foals with liver disease because of reduced hepatic clearance and increased mobilization from adipose tissue associated with a catabolic state. cholesterol and vldl concentrations may decrease, however, because of reduced hepatic synthesis. it should again be noted that triglyceride and cholesterol concentrations are higher in healthy neonatal foals compared to adults, and vary with age. 27 in one report, foals had concentrations of 24 to 88 and 30 to 193 mg/dl for triglycerides in <12-hour and in one-day-old foals, respectively. cholesterol concentrations were 111 to 432 and 110 to 562 in those same age groups. 27 cbc findings are dependent upon the cause of liver disease. leukocytosis may be present with cholangiohepatitis. when liver disease is secondary to sepsis or hypoxic-ischemic disease, however, leukopenia with band neutrophils is more common. hyperfibrinogenemia is usually present with infectious or inflammatory hepatopathies, however fibrinogen may be decreased when coagulopathies such as disseminated intravascular coagulation exist. blood cultures should be performed in the neonatal foal with liver disease because of the possibility of sepsis causing hepatitis. clotting times may be prolonged with liver failure. prolongation of prothrombin time (pt) and partial thromboplastin (ptt) occur due to a lack or reduction of synthesis of several clotting factors, including factors ii, v, vii, ix, and x. this is an important clinical finding because liver biopsies may have to be delayed in hypocoagulable animals. for similar reasons, plasma antithrombin concentrations may be reduced, leading to coagulopathies such as disseminated intravascular coagulation. in one review, almost half of horses with liver disease had a prolonged pt or ptt. 31 coagulopathies are common in horses with liver dysfunction due to concurrent systemic inflammatory response syndrome, and fibrin degradation products may be increased and thrombocytopenia may be present. the acid-base status of foals with liver disease should be evaluated closely through blood gas analysis. with liver dysfunction, acidemia may occur as a result of increased formation and reduced clearance of organic acids such as lactate, pyruvate, and amino acids. impaired urea synthesis may reduce renal buffering through urine acidification. serial monitoring of lactate should be included as part of the diagnostic monitoring of the foal with liver disease because lactate clearance may be reduced. ultrasonography is a critical component of the diagnostic workup of foals with liver disease. it can be utilized to determine the size of the liver, changes in parenchyma, and dilated bile ducts. with infectious etiologies, such as cholangiohepatitis, hepatitis secondary to sepsis, and tyzzer's disease, the liver appears subjectively enlarged. biliary distention is evident in some cases. evidence of fibrosis (hyperechogenicity) may be present in subacute to chronic cases. hepatic scintigraphy will confirm the diagnosis of portacaval shunts. injection of technetium 99m-labeled sulfur colloid demonstrates alterations in blood flow or the presence of masses. biliary scans can also be performed looking for biliary obstruction or atresia. portograms are necessary in cases of portosystemic shunts where surgical correction is contemplated. these are done intraoperatively using radiopaque agents that are injected into a mesenteric vein with subsequent radiography. simultaneous filling of the portal and azygous veins and caudal vena cava, and lack of intrahepatic filling indicate portacaval shunting. 30 histopathology provides the most specific information as to the etiology of liver disease and can aid in diagnostic, prognostic, and therapeutic directions. the safest means of performing liver biopsies is under ultrasonographic guidance and using an automated needle biopsy instrument. it should be pointed out that focal lesions such as neoplasia are easily missed by liver biopsy. special stains (silver or warthin-starry stains) may be helpful in specific diagnoses such as tyzzer disease. fine-needle aspirates may also be helpful in the diagnosis of a specific etiology ( figure 11-37) . culture of liver biopsies is indicated when infectious etiologies are suspected. cholangiohepatitis is usually associated with gram-negative enteric aerobes and gram-negative or gram-positive anaerobes. foals with tyzzer's disease develop a peracute to acute multifocal necrotizing hepatitis. grossly, hepatomegaly is evident. these foals generally have marked increases in ast and sdh concentrations. other liver enzymes, both hepatocellular and biliary (ldh, sap, ggt), are also increased, as are both components of bilirubin. striking hypoglycemia should be a clue to the presence of infection with clostridium piliformis. these foals often have a metabolic acidosis, with hyperlactatemia due to increased production (hypoperfusion, inflammation, catecholamine surges) and reduced hepatic clearance. complete blood counts often reveal leukopenia with a left shift and cytologic evidence of toxicity, although leukocytosis is present in some cases. 32 hemoconcentration reflects hypovolemia and dehydration. hyperfibrinogenemia is common. physical examination is consistent with septic shock, with petechial hemorrhages and injection being common. hypovolemic shock is also evident. mentation is usually depressed, ranging from lethargy to comatose, and icterus is a prominent finding. clinical signs of hepatoencephalopathy may include depression, weakness, and seizures. diarrhea is an inconsistent finding. fever is usually present, although hypothermia from shock may be predominating when initially examined. 32 affected foals are often found dead. serologic testing for recovered or suspected cases can be helpful. pathology reveals hepatomegaly with rounded margins (figure 11-38 ). histopathologic findings with tyzzer's disease include multifocal and random necrosis with neutrophilic inflammation (figure 11-39 ). long bacilli are often but not always seen, particularly in hepatocytes at the periphery of necrotic areas. they are best delineated with warthin-starry stains (figure 11-36 ). an intranuclear location of the microbe has been found using electron microscopy. 33 myocardial necrosis with warthin-starry stained bacilli has also been reported. 32, 34 intestinal necrosis may also be present. and organisms consistent with c. piliformis may occasionally be observed in sections of intestine, particularly the large intestine. 36 clostridium piliformis is an obligate intracellular spore-forming anaerobe. it is very difficult or nearly impossible to culture in vitro using routine microbiologic methods. inoculation of embryonated eggs has been one means of trying to assess antimicrobial susceptibility. penicillin, tetracycline, erythromycin, and streptomycin appear to be effective in vitro, while sulfonamides and chloramphenicol do not. 37 treatment with dextrose, volume replacement, and sodium bicarbonate can result in temporary improvement of mentation, however most foals are reported to die. the exact pathophysiology of infection is unknown but is believed to be oral with subsequent distribution to the liver. ingestion of spores from feces shed by subclinical carriers may play a role in development of disease, and the disease has been experimentally reproduced. 35 it has also been suspected that rabbit and rodent populations could serve as reservoirs of infection. 33 most reported cases have been sporadic, however a number of cases have been noted on particular premises. 38 preventive measures are unknown, however efforts should be directed at environmental hygiene on affected farms, including the use of 0.3% sodium hypochlorite to eliminate spores from barns housing affected foals. 39 risk factors for clostridium piliforme infection in foals include foaling season, farm residency and age of mare. in one study, foals born between march 13 and april 13 were 7.2 times as likely to develop infection as those born at any other time of the foaling season. 38 foals of nonresident visiting mares were 3.4 times as likely to develop the disease as were foals born to mares that were permanent residents of the farm. foals born to young mares were 2.9 times as likely to develop tyzzer's disease as those born to mares ≥6 years of age. 38 (22 mg /kg iv, q 8 h) and gentamicin (6.6 mg/kg iv, q 24 h) . seizure activity was controlled with phenobarbital. (0.1 ml/kg po, q 8 treatment treatment of liver disease is largely supportive, with specific therapies available for certain diseases. foals with acute hepatopathies often present in hypovolemic crises. a combination of crystalloids and colloids is indicated in volume replacement. ideal crystalloids for liver disease include those containing acetate and/or gluconate instead of lactate, such as plasma-lyte 148 or normosol-r (abbott laboratories, abbott park, il). the liver is the primary site of lactate clearance, whereas muscle and renal metabolize acetate and gluconate is utilized by most tissues. though accumulation of lactate from lrs does not lead to lactic acidosis (the cation associated with lactate in lrs is sodium as opposed to hydrogen ion), lactate is depressive to the myocardium; therefore iatrogenic increases in lactate concentration should be avoided. foals with septic shock, such as those with tyzzer's disease, should be treated with aggressive fluid therapy. boluses of 10 to 20 ml/kg of isotonic replacement crystalloids or 3 to 5 ml/kg of colloids should be administered over 20 to 30 minutes until the hypovolemic crisis is improved or fails to improve with further volume. if hypotension persists despite volume resuscitation, inotropes and pressors are indicated. if systemic blood pressure does not normalize in the face of a maximum or high cvp (>10 cm h 2 o), dobutamine should be administered as a β-agonist inotrope (2 to 20 μg/kg/ minute). lastly, vasopressors should be administered. norepinephrine (0.1 to 3 μg/kg/minute) or vasopressin (0.25 to 1 mu/kg/minute) can be used in an attempt to normalize blood pressure and perfusion (see chapter 7). oxygen insufflation (5 to 10 liters/ minute) should be provided to foals with central depression, hypoventilation, or concurrent respiratory tract disease. because hepatic biotransformation and elimination of drugs is reduced, medications metabolized by the liver should be used judiciously and dosage alterations should be considered. antimicrobial therapy is indicated in cases of bacterial cholangiohepatitis or hepatitis. broad-spectrum antimicrobials should be administered to foals with cholangiohepatitis. a combination of beta lactam, such as penicillin or ampicillin, and aminoglycoside, such as amikacin or gentamicin, is a reasonable initial choice. ceftiofur is also a good choice, and potentiated sulfonamides can be used for long-term therapy. foals with tyzzer's disease may be treated with any of a number of antimicrobials as the agent appears very susceptible to penicillin, gentamicin, tetracycline, and metronidazole. plasma transfusions (20 to 40 ml/kg) can be beneficial to foals with liver disease. plasma provides additional colloid support, albumin and immunoglobulin replacement, and provision of antithrombin or clotting factors for foals with coagulopathies. dextrose supplementation in polyionic nonlactated fluids should be provided. potassium chloride should be supplemented even if potassium concentrations are normal because it aids in renal excretion of ammonia. neomycin (4 to 8 mg/kg po, once) or metronidazole (low doses because of reduced hepatic metabolism (10 mg/kg po q 12 h) may be administered to reduce enteric bacterial production of ammonia. alternatively, lactulose can be used and is preferred by the author (0.1 to 0.25 ml/kg po q 6 to 8 h). 28 lactulose is a synthetic disaccharide that bypasses the small intestine. fermentation in the colon results in increased nitrogen incorporation into the enteric flora, with less available for absorption. lactulose also reduces the ph of ingesta, resulting in inhibition of ammonia generation and increased ionization of ammonia with ion trapping within the gi lumen. for severe neurologic signs, mannitol (0.25 to 1.0 g/kg) may be administered if increased intracranial pressure is suspected. sodium bicarbonate should not be administered unless a significant inorganic acidosis is present; rapid correction of acidemia can increase the concentration of ionized ammonia and exacerbate clinical signs. benzodiazepines, such as diazepam, should be avoided in foals with hepatic insufficiency as they can potentiate clinical signs of hepatic encephalopathy by enhancing the effects of gaba. seizures should be controlled with phenobarbital or pentobarbital. flumazenil (0.5 to 1.0 mg/kg slowly iv), a benzodiazepine antagonist, can be tried in foals with clinical signs of hepatic encephalopathy that are unresponsive to other therapeutics. nutritional support of foals with hepatic disease is vital to a successful outcome. parenteral nutrition should be administered to foals with fulminate hepatitis or liver dysfunction, including those with tyzzer's disease. formulations for patients with hepatic failure should be utilized. protein requirements should be met but not exceeded in order to avoid contributing to hyperammonemia. formulations with increased branch chain amino acids (valine, isoleucine, leucine) and decreased aromatic amino acids (phenylalanine, tryptophan, tyrosine, methionine) are being evaluated for use in human liver patients. vitamin k 1 should be administered in foals with long-term liver disease. since the liver is the primary source of vitamin c synthesis and storage of vitamins a, d, and riboflavin, a multivitamin should be provided to foals with chronic liver disease. b vitamins should be administered diluted in fluids. vitamin e can be supplemented as an antioxidant. foals with portacaval shunts should be stabilized for hepatic encephalopathy, and then surgical correction can be attempted after diagnostic venography to localize the shunt. 13 gastric ulcer prophylaxis is indicated in select cases, particularly those with hypovolemic or hypotensive shock. sucralfate is advantageous in that it does not undergo hepatic biotransformation. for treating ulcers, famotidine or ranitidine should be utilized, because unlike cimetidine, they are excreted primarily in the kidneys and do not inhibit hepatic metabolism of other drugs. tyzzer's disease, which is uniformly fatal. the outcome in tyzzer's disease is generally grave. there are currently no reports of survival in documented (definitively diagnosed) cases. some cases will improve temporarily with volume replacement and administration of dextrose and sodium bicarbonate. however, there are three reported cases and a few anecdotal reports of survival in presumptive cases of clostridium piliforme infection. 38, 40 early therapy with antimicrobials and aggressive intensive care including parenteral nutrition appear to be key to a successful outcome. 40 gastric ulceration in an equine neonate endoscopic appearance of gastric lesions in foals: 94 cases pathophysiology of peptic disorders in foals and horses: a review the effects of stress on gastric ulceration, t3, t4, reverse t3 and cortisol in neonatal foals gastric ulceration in foals in the hidaka district gastroduodenal ulceration effect of ranitidine on intragastric ph in clinically normal neonatal foals intragastric ph in critically ill neonatal foals and the effect of ranitidine stress-induced gastric findings in critically ill newborn infants: frequency and risk factors gastric and duodenal ulceration in the critical neonate. proc international veterinary emergency and critical care society clinical and pathological effects of flunixin meglumine administration to neonatal foals phenylbutazone toxicosis in the foal gastroesophageal ulceration and candidiasis in foals nutritional support: enteral and parenteral treatment and prevention of equine gastric ulcer syndrome the protective effects of sucralfate and ranitidine in foals experimentally intoxicated with phenylbutazone effect of omeprazole paste on intragastric ph in clinically normal neonatal foals safety, acceptability, and endoscopic findings in foals and yearling horses treated with a paste formulation of omeprazole for twenty-eight days is prophylaxis for gastric ulcers necessary in critically ill equine neonates: a retrospective study of necropsy cases 1995-1999. proc 18 th american college veterinary internal medicine scientific forum ulcerative duodenitis in foals nutritional support: enteral and parenteral faecal composition in foal heat diarrhea mares' milk composition as related to "foal heat" scours sand-induced diarrhea in a foal effect of protein source in liquid formula diets on food intake, physiologic values, and growth of equine neonates necrotizing enterocolitis in two equine neonates rotavirus infection in foals foal diarrhea between 1991 and 1994 in the united kingdom associated with clostridium perfringens, rotavirus, strongyloides westeri, and cryptosporidium spp field study of the safety, immunogenicity, and efficacy of an inactivated equine rotavirus vaccine prevention of rotavirus diarrhoea in foals by parenteral vaccination of the mares: field trial neonatal enterocolitis associated with coronavirus infection in a foal: a case report characterization of a coronavirus isolated from a diarrheic foal adenovirus in the intestinal epithelium of a foal with prolonged diarrhea association of clostridium difficile with enterocolitis and lactose intolerance in a foal isolation of clostridium difficile and detection of cytotoxin in the feces of diarrheic foals in the absence of antimicrobial treatment hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals asymptomatic neonatal colonization by clostridium difficile clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility enterocolitis associated with clostridium perfringens infection in neonatal foals: 54 cases populationbased study of fecal shedding of clostridium perfringens in broodmares and foals an outbreak of equine neonatal salmonellosis actinobacillus sp. bacteremia in foals: clinical signs and prognosis experimental inoculation of foals and pigs with an enterotoxigenic e. coli isolated from a foal isolation of enterotoxigenic escherichia coli from a foal with diarrhea streptococcus durans: an unexpected enteropathogen of foals clostridium sordelli isolated from foals diarrhea associated with enterotoxigenic bacteroides fragilis in foals cryptosporidium in immunodeficient arabian foals prevalence of and risk factors for fecal shedding of cryptosporidium parvum oocysts in horses identification of cryptosporidium parvum "cattle" genotype from a severe outbreak of neonatal foal diarrhea foal diarrhea; pathogenesis, etiology and therapy volume replacement in the neonatal icu: crystalloids and colloids intensive insulin therapy in the critically ill patient evaluation of the ability of di-tri-octahedral smectite to adhere to clostridium difficile toxins and clostridium perfringens enterotoxin in vitro. proc 48 th annual convention of the american association of equine practitioners characterization of clostridium difficile isolates from foals with diarrhea: 28 cases antimicrobial susceptibilities of equine isolates of clostridium difficile and molecular references 1. brown cm, ainsworth dm, personett la et al: serum biochemical and haematological findings in two foals with focal bacterial hepatitis (tyzzer's disease) bacillus piliformis infection (tyzzer's disease) in foals in northwestern united states: a retrospective study of 21 cases the pathology of a case of biliary atresia in a foal tyzzer's disease in an 11-day-old foal tyzzer's disease in a foal nutritional support: enteral and parenteral a comparison of the clinical, clinicopathological, and bone marrow characteristics of foals with equine herpes and neonatal septicemia pulmonary abscessation, hepatoencephalopathy and igm deficiency associated with rhodococcus equi in a foal four possible causes of hepatic failure and/or icterus in the newborn foal clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus-1 infection compared with septic and premature foals neonatal equine herpesvirus type 1 infection on a thoroughbred breeding farm urinary and hepatic disorders in neonatal foals the diagnosis and surgical correction of congenital portosystemic vascular anomalies in two calves and two foals clinical signs and radiographic diagnosis of a portosystemic shunt in a foal clinical and diagnostic features of portosystemic shunt in a foal persistent vitelline arteries in a foal congenital hepatic fibrosis and cystic bile duct formation in swiss freiberger horses persistent hyperammonemia in two related morgan weanlings glycogen branching enzyme deficiency in quarter horse foals glycogen branching enzyme (gbe1) mutation causing equine glycogen storage disease iv hepatoblastoma in a foal sm: hepatoblastoma in an equine fetus iron toxicity in neonatal foals hepatotoxicosis in neonatal foals (letter) steroid hepatopathy in a horse with glucocorticoid-induced hyperadrenocorticism glucocorticoid-induced laminitis with hepatopathy in a thoroughbred filly serum biochemical indicators of liver function in neonatal foals liver failure and hemolytic anemia serum protein concentrations in horses with severe liver disease: a retrospective study and review of the literature disorders of the liver clinicopathological features of equine primary hepatic disease: a review of 50 cases clinical and clinicopathologic findings in two foals infected with bacillus piliformis tyzzer's disease in a foal tyzzer's disease in foals multifocal hepatic necrosis and hepatitis in foals caused by bacillus piliformis naturally-occurring tyzzer's disease (bacillus piliformis infection) in horse foals tyzzer's disease risk factors for clostridium piliforme infection in foals tyzzer's disease in an 11-day-old foal neonatal hepatic failure in a thoroughbred foal: successful treatment of a case of presumptive tyzzer's disease key: cord-353633-a4pu6rlu authors: perakakis, nikolaos; stefanakis, konstantinos; mantzoros, christos s. title: the role of omics in the pathophysiology, diagnosis and treatment of non-alcoholic fatty liver disease date: 2020-07-23 journal: metabolism doi: 10.1016/j.metabol.2020.154320 sha: doc_id: 353633 cord_uid: a4pu6rlu non-alcoholic fatty liver disease (nafld) is a multifaceted metabolic disorder, whose spectrum covers clinical, histological and pathophysiological developments ranging from simple steatosis to non-alcoholic steatohepatitis (nash) and liver fibrosis, potentially evolving into cirrhosis, hepatocellular carcinoma and liver failure. liver biopsy remains the gold standard for diagnosing nafld, while there are no specific treatments. an ever-increasing number of high-throughput omics investigations on the molecular pathobiology of nafld at the cellular, tissue and system levels produce comprehensive biochemical patient snapshots. in the clinical setting, these applications are considerably enhancing our efforts toward obtaining a holistic insight on nafld pathophysiology. omics are also generating non-invasive diagnostic modalities for the distinct stages of nafld, that remain though to be validated in multiple, large, heterogenous and independent cohorts, both cross-sectionally as well as prospectively. finally, they aid in developing novel therapies. by tracing the flow of information from genomics to epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and glycomics, the chief contributions of these techniques in understanding, diagnosing and treating nafld are summarized herein. nafld is recognized as the most common liver disease in developed countries with its incidence continuously rising in parallel to the increasing incidences of obesity and type 2 diabetes (t2dm) [1] . significant efforts are currently underway to delve deeper into the pathophysiology of the disease, to create non-invasive tools for its diagnosis and staging, as well as to develop highly effective and specific treatments. the disease is characterized initially by hepatic lipid accumulation (nonalcoholic fatty liver; nafl), that can often progress to non-alcoholic steatohepatitis (nash), liver fibrosis or cirrhosis, as outlined in detail elsewhere in this special issue [1] . several triggers and series of events stimulating necroinflammatory processes in the liver have been recognized and described as "the multiple hit" pathogenetic mechanism leading to advanced nash [2] . additionally, multiple factors (such as lack of physical activity, unhealthy nutrition, concomitant alcohol consumption and presence of other metabolic diseases) have been associated with increased risk of disease progression [3] . still, we are not able to predict with great accuracy which patient and when will develop hepatic inflammation or fibrosis. j o u r n a l p r e -p r o o f 13 assessed [43] , and histologically-proven gwas [44] of more than 10000 total patients each, re-affirmed the robust clinical and histological genome-wide associations of pnpla3, tm6sf2, gckr, and hsd17b13. novel fibroinflammatory loci in slc30a10 and slc39a8 were also studied [43] . however, a bench-to-bedside conversion of current results through tangible diagnostic and therapeutic applications has yet to take place. pnpla3 rs738409 constitutes the chief nafld predictor, adjusted for known risk factors (or=3.12) [21] . yet its addition in an early predictive model of nafld, using routine variables, contributes trivially to score accuracy (area under the receiver operator characteristics (auroc) = 0.866 and 0.872 respectively) [45] . pnpla3 is included in the nash score (aspartate transaminase (ast, insulin, pnpla3 genotype) and the clinlipmet score (nash clin score, metabolomic and lipidomic variables) with aurocs of 0.778 and 0.866 respectively, yet its contribution to nash prediction is not underlined [46] . in a more recent phenome-genome wide study of 27744 patients, clinical data (age, sex, bmi, blood counts) comprise a fitter nafld predictor than rs738409 genotyping (auroc = 0.785 and 0.574 respectively) and have 34% higher sensitivity compared to genotyping alone. even though rs738409 carriage was associated with 3.1 years earlier nafld detection, its addition does not markedly amplify the forecasting model (auroc = 0.788) [17] . cumulative genetic risk scores (grs) are frequently discussed (table 2) . a grs of previously mentioned snps in pnpla3, tm6sf2, gckr and mboat7 indicates a 3-fold increase in nafld risk starting from its second tertile [21] . when coupled with a multivariate insulin resistance score, a grs j o u r n a l p r e -p r o o f 14 of pnpla3 and tm6sf2 snps detects nas ≥ 3 (auroc=0.74) and fibrosis ≥ 3 when age included (auroc = 0.82), yet grs alone have aurocs of ≤ 0.65 [41] . pnpla3 and tm6sf2 polymorphisms are also included in a novel nash score alongside diabetes, insulin resistance, ast and c-reactive protein levels, which predicts nash more effectively compared to the older nash score (aurocs of 0.787 and 0.729 respectively) and has enhanced accuracy in diabetic cohorts (auroc = 0.835) [47] . lastly, a recent investigation of 445452 individuals formulates a grs comprising pnpla3, tm6sf and hsd17b13 snps. a maximum risk score of 6 designates a 26% increase in alanine aminotransferase (alt) levels and a pronounced risk of cirrhosis and hcc (or=12 and 29 respectively) [48] . genetic testing in nafld is currently not recommended by the american association for the study of liver diseases [49] . thus far, genomics have not identified a consistent marker that reflects the accurate histologic features of nafld, partially owing to limited population sizes, inconsistent nafld validation methods and scarcity of biopsy-confirmed models. most importantly, genomics are not dynamic processes, thus they cannot reflect the impact of environmental factors (high caloric intake, presence of obesity, t2dm etc.) in the pathogenesis and progression of nafld. multi-gene models are promising tools of patient risk stratification but require additional validation. pnpla3 has also been investigated through early genetic interventions. preand post-translational silencing of pnpla3 in p.i148m knock-in mice j o u r n a l p r e -p r o o f 15 challenged with a sucrose-rich diet drastically improves intrahepatic fat profiles [50, 51] . additionally, pnpla3 silencing ameliorates steatosis and nas independent of genotype, and improves inflammation, fibrosis and levels of acute phase, chemo-attractant and pro-fibrotic proteins, in p.i148m knockin mice fed a nash-inducing diet [51] . however, pnpla3 protects from hypercholesterolemia, gallstones, gout and acne in humans [52] , therefore future pilot studies must be carefully considered. tm6sf2 is another possible target, yet its substantial cardioprotective effects [53] indicate the complexity of such an approach, since the chief cause of death in nafld patients is cardiovascular disease [49] . patient genotyping prior to therapeutic scheme initiation implies compelling curative applications. for instance, pnpla3 p.i148m carriers would not respond to statins and inhibitors of de novo lipogenesis, in contrast to tm6sf2 and gckr carriers [54] . moreover, hsd17b13 has already been implied as a therapeutic target [42] , while it was recently established that carriage of hsd17b13 rs6834314 diminishes the fibrotic effects of pnpla3 rs738409, thus indicating a novel objective for pathway-targeting therapies [55] . further research could uncover powerful hierarchical and therapeutic tools in nafld genomics, jumpstarting a new era of clinicogenomics. j o u r n a l p r e -p r o o f 16 progression of nafld. given the continuous evolvement of personalized medicine, we expect the information from genomics to be used in the near future for identifying the patients at higher risk for presenting a more aggressive course of disease and/or for potentially predicting the response to specific treatments. these may justify more intensive follow-ups or guide treatment decisions in high-risk populations. sfince genomics are not dynamic processes and do not reflect the effects of environmental factors on nafld, we see less potentials for them as diagnostic markers and we expect for them to have only a small, if any, contribution in non-invasive diagnostic tests. finally, due to the technical and clinical challenges related to geneengineering as well as due to the complex pathophysiology of the disease, we recognize at the moment limited perspectives in the development of gene therapies for nafld. epigenomics investigates the epigenetic modifications on cellular genetical material. several studies have assessed the impact of epigenetic modifications in the development and progress of nafld ( figure 2 ) as well as in the association of nafld with other metabolic diseases by focusing on dna methylation, histone modifications and mirna expression profiles that can significantly affect transcriptional activity. significant alterations in dna methylation are observed when moving from nafl to nash and liver fibrosis [56] [57] [58] . these changes include alterations j o u r n a l p r e -p r o o f in dna methylation that affect the expression of genes involved in glucose, lipid or acetyl-coenzyme a (coa) metabolism (pc, plcg1, acly) [57] , insulin-like signaling (e.g. igf-1, igfbp2) and mitochondrial function (nadh dehydrogenase 6) [59] . additionally, given that a specific dna methylation signature develops with increasing age in humans, nash seems to accelerate epigenetic age by promoting changes in methylation that are associated with hepatic collagen content [60] . an untargeted evaluation of dna methylation in liver tissues of patients with nafld identified almost 70,000 cpg sites that were differentially methylated in patients with advanced liver fibrosis (f3-f4) compared to those with no or mild fibrosis (f0-f1). 76% of these sites were hypomethylated and 24% were hypermethylated in advanced liver fibrosis in nafld, whereas 7% of the reported methylations correlated with gene expression levels. the hypomethylation was associated with higher expression of genes related to tissue repair, liver inflammation, fibrosis and carcinogenesis (e.g. fgfr2, col1a1, casp1, ccr7, ccl5) whereas the hypermethylation with lower expression of genes involved in lipid and aminoacid metabolism [56, 61] . these findings were also replicated in other studies [62] . dna methylation seems to be particularly involved in the activation of hepatic stellate cells and their differentiation to myofibroblast that are crucial procedures for hepatic fibrogenesis [63, 64] . changes in methylation of specific genes have been linked with these processes. genes promoting fibrogenesis such as tgfβ1 and pdgfα are hypomethylated and thus highly expressed whereas genes inhibiting hepatic stellate cells activation such as the pparα, pparδ and pparγ are hypermethylated and j o u r n a l p r e -p r o o f 18 thus lower expressed in the liver of patients with advanced fibrosis compared to the ones with mild disease [65, 66] . apart from their involvement in nafld development and progress, hepatic alterations in dna methylation may be associated with systemic metabolic outcomes. specifically, a case-control study in nafld patients has shown that increased methylation in specific sites of the promoter of the ppargc1a, which encodes the major regulator of mitochondrial biogenesis, is associated with lower mrna expression of ppargc1a in the liver, lower ratio of mitochondrial to nuclear dna and increased insulin resistance, thus linking hepatic mitochondrial dysfunction of nafld with peripheral insulin resistance [67] . in agreement with the above finding, a study following untargeted procedures has identified 30 methylations that are affecting mrna expression of hepatic genes involved in insulin signaling that are also highly correlated with fasting insulin independently of the presence of t2dm, thus providing further evidence for the relation of hepatic dna methylation with insulin sensitivity [58] . finally, a recent study focused on the identification of differentially methylated regions that form networks which are associated with the progression of nafld. they identified two important networks, one that included genes that affect cytoskeleton organization, transcriptional activity and cell proliferation and another that was associated with metabolic pathways. the cpg methylation levels in both networks were affected by age and fasting plasma glucose levels and for the second network the changes in methylation levels were partially corrected by controlling weight and blood glucose levels [68] . j o u r n a l p r e -p r o o f 19 histone modifications are also important epigenetic changes that affect transcriptional activity and refer to several posttranslational procedures such as acetylation, phosphorylation, methylation and ubiquitination. among them, acetylation status has been most vigorously studied and is considered the net result of histone acetylation by histone acetyltransferases (hats) and histone deacetylation by histone deacetylases (hdacs) [69] . acetylation neutralizes lysin's positive charge, thus leading to a looser chromatin structure that facilitates transcription. the transcriptional coactivator p300 belongs to hat proteins, whereas sirtuins belong to nad-dependent class iii hdacs. glucose-induced activation of p300 increases the transcription of carbohydrate-responsive element-binding protein (chrebp) resulting in the stimulation of lipogenic genes through histone acetylation and thus promoting the development of nafld [69] . tannic acid attenuated the effects of p300 leading to a decrease in the lipogenesis-related genes and to an improvement of nafld in mice [70] . similarly, inhibition of cdk4 protein reduces the formation of c/ebpα -p300 complexes reducing liver steatosis and correcting age-associated liver changes [71] . p300 may also be involved in the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts [72] . among the sirtuins, especially sirtuin 1 (sirt1) has been shown to participate in the regulation of hepatic metabolism and insulin sensitivity, with hepatic deletion of sirt1 resulting in steatosis and inflammation, whereas overexpression of sirt1 protecting from nafld [73] [74] [75] . similarly, sirt3 deficiency leads to insulin resistance, hyperlipidemia and steatohepatitis in mice [76] whereas a polymorphism of sirt3 is associated with metabolic syndrome and nafld in humans [77] . hdac3, a member of j o u r n a l p r e -p r o o f 20 human class i hdacs has been also implicated with circadian metabolic rhythm and its deletion leads to hepatic steatosis in mouse liver [78] . altogether, there is increasing evidence that both dna methylation as well as histone modifications play a crucial role in the development and progress of nafld. an important question is whether the numerous epigenetic changes observed in nafld in combination with the highly dynamic nature of epigenome can serve as diagnostic or prognostic markers of nafld or even as markers of treatment response. very few studies have addressed this point so far. one of them that included a small number of patients with nafld has shown that alterations in hepatic dna methylation may be sufficiently reflected in plasma. in this study, % of plasma dna methylation of pparγ was used to identify subjects with severe (kleiner 3-4) vs mild fibrosis (kleiner 1-2). the threshold of methylation was 81% and demonstrated 83% sensitivity and 93% specificity to differentiate between the two above categories [79] . although validation of the findings in largest and more heterogenous cohorts is needed, it is one of the first studies to suggest that changes in dna methylation can have beyond their pathophysiological significance also value as potential noninvasive biomarkers for the diagnosis and staging of nafld. a second study has also investigated differences in methylation of dna from peripheral blood leukocytes that may be able to discriminate between nash vs nafl. the study identified six genes that their methylation correlates with lobular inflammation and can be potentially used as diagnostic biomarkers (with the j o u r n a l p r e -p r o o f 21 best one showing 99% specificity and 71% sensitivity) for differentiating nash from nafld [80] . finally, in a large epigenome-wide association study consisting of 3,400 participants of european ancenstry, 22 cpgs were found in peripheral blood to be associated with hepatic fat as assessed by computed tomography or ultrasound imaging and among them one was also associated with risk for new-onset t2dm [81] . nevertheless, significantly more and larger studies are needed in order to decide whether epigenetic changes in the blood can have any diagnostic or prognostic value. targeting epigenomic changes for the treatment of diseases seems to be theoretically a very attractive option for a number of reasons. first, epigenomic alterations are involved in fundamental pathophysiological mechanisms of a disease through their impact on gene expression. second, epigenetic changes are also highly dynamic and reversible, thus correction of them holds promise for reversal to "normal status". for example, after bariatric surgery, robust changes in methylation have been reported. hypermethylation and consequently reduction of transcriptional activity of ptpre (encoding protein-tyrosine phosphatase epsilon) may improve hepatic insulin sensitivity [57] . exercise may also reduce methylation and improve mrna levels of mitochondrial genes, thus improving mitochondrial function [59] . third, it may be possible to combine treatments targeting epigenome with other drugs, having in that case a complementary role. such treatments are currently being developed mainly against cancer diseases and consist of pharmacological dna methylation inhibitors (dnmtis), hdac inhibitors (hdacis) or activators. in nafld very few of them have been tested so far. j o u r n a l p r e -p r o o f 22 among them, the sirt1 activator, resveratrol, improved dyslipidemia and steatohepatitis in a mouse model of atherogenic nafld [82] . several ongoing human clinical trials (two of them in phase iii) are currently investigating the efficacy of resveratrol in human nafld but results from the first studies have shown no significant effect on hepatic fat content [83] [84] [85] . other medications are currently also being developed and have shown promising results in animal studies, such as the 3-deazaneplanocin a (dznep) [86] or the hdac inhibitor saha but none of them is in a more advanced stage of clinical development [87] . epigenomics are one of the least investigated "omics" in nafld. the few studies performed so far have shown that the extensive hepatic epigenetic modifications observed in nafld are causally related to the disease. it is still unclear though how accurately the hepatic epigenome is reflected in peripheral blood and thus it is questionable whether epigenetic changes studied at the level of peripheral blood cells may have any significant diagnostic value. given also the limited amount of information from pathophysiological studies investigating the dynamic of epigenetic changes with nafld development and progression, it is probably premature to expect any drugs to be developed in order to target exclusively epigenome of nafld in the near future. genetic predisposition determines nafld transcriptomic signatures, as shown by recent investigations on the carriage of pnpla3 i148m on transcriptome variability [88] . as described above, transcriptomics are also linked to epigenetic modifications in nafld [56, 57] . through global microarray transcriptional snapshots, nafld and especially nash are characterized by overexpression of genes associated with lipid metabolism [89] [90] [91] [92] , acute phase regulators of insulin sensitivity [93] , cellular division [89] , dna [89] and tissue repair [56] , extracellular matrix organization [91, [94] [95] [96] , immune function [95] cellular adhesion and migration [91, 92, 96] , signal transduction [91] , p53 signaling [92] and cancer progression [96] among others ( figure 2 ). conversely, genes modulating mitochondrial function [93] oxidative, glucose, fatty acid and aminoacid or protein metabolism [56, 91-93, 96, 97] as well as insulin signaling and transcription factors [91, 94] are downregulated. and underexpression of pzp mrnas [92] . hepatic transcriptomes of nafld are distinct from those of normal and healthy obese subjects, yet a 132-gene signature, with extracellular matrix remodeling and immune system genes as its main components, distinguishes nash from simple steatosis [95] . more specifically, the transcriptional snapshot of nash is defined by upregulation of pdgf, stat, hnf-3 and smad-4 pathway-related genes [97] , as well as downregulation of bnip1, igfbp1 [97] , slc25a48, c4orf48 [95] sdc4, atf3, various inflammatory suppressors [94] and amino acid-metabolizing and reactive oxygen species (ros) scavenger genes [97] . interleukin-32 (il-32) was recently ascertained as the most significantly upregulated transcript in advanced nafld and nash (fold-change = 2.3 vs non-nash), correlating with lipid accumulation and disease severity [88] . additionally, several cancerrelated genes, such as akr1b10, krt23 and gpc3, are enhanced in nash compared with nafl or controls, by up to 155-fold and 460-fold respectively [96] . concerning fibrosis, cross-sectional transcriptome analysis of hepatic cells defines 3820 and 2980 differentially expressed transcripts for the inflammatory and fibrotic states, respectively, and further underlines 16 fibrosis-defining genetic routes, pertaining to cytokine and extracellular matrix receptor interaction, focal adhesion and the pi3k-akt signaling pathway [98] . differentially expressed microarray signals, such as ube2v1, bnip3l, rp11-128n14.5, linked to oxidative stress, inflammation, apoptosis and fibrogenesis, are upregulated in nas ≥ 5 and fibrosis stages 3-4 [99] . j o u r n a l p r e -p r o o f likewise, transcriptomics pinpoint dermatopontin, a propagator of extracellular matrix remodeling, which is robustly expressed (by 800-fold) in murine stellate cells, enhanced in human fibrotic livers and implicated in nafld fibrogenesis [100] . minor molecules such as micro-rnas (mirna) along with long noncoding rnas (lncrna) perform a variety of epigenetic and post-transcriptional functions, thus significantly affecting transcriptional activity in numerous diseases and among them also nafld [101] . for this reason, they are also considered important epigenetic modifiers. these processes are thoroughly delineated through large panels of differential mirna expression [102] [103] [104] while the distinct nafld-related roles of mirna subspecies were recently reviewed [101, 105] . of note, microrna-122 (mir-122), deriving from chromosome 18, represents 70% of hepatic mirna and has been implicated in cholesterol metabolism and liver cancer [106] . cheung et al. first mapped a 63% underexpression of liver mir-122 in nash patients compared with controls, through an evaluation of a 474-mirna panel that identified 46 differentially expressed mirna species and their corresponding targets [104] . mir-122 was subsequently associated with steatosis severity [107] . the (a,b), mir-125b (associated with metabolic syndrome, atherogenesis and circulation) [103] as well as mir-34a, mir-16 (associated with cancer) [108] , mir-21 and mir-451 [107] are upregulated in both nafld and nash. moreover, mir-301a, mir-34a and mir-375 alter lipid and carbohydrate j o u r n a l p r e -p r o o f 26 metabolism, reflect nafld severity and are implicated in hepatocellular carcinomas [102] . expression levels of lesser non-coding molecules have also been investigated [99, 109, 110] . global hepatic rna profiling has identified 535 lncrna and 760 mrna species overexpressed in nafld, whereas 1200 lncrna and 725 mrna species are underexpressed. these enriched rnas chiefly belong to small molecule and organic acid metabolic pathways [109] . functional transcriptome research of 4383 lncrna species by atanasovka et al. pinpoints elevated levels of hepatic lncrna rp11-484n16.1 in nash, and robustly associates it with nash grade, lobular inflammation and nas, while its knockdown in vitro aggravates hepatocellular growth and viability [110] . identified and validated through whole serum transcriptome analysis, lncrna rp11-128n14.5 has been linked to nash diagnosis and is upregulated in nas ≥ 5 [99] . furthermore, transport rna (trna) profiling pinpoints the differential expression levels of several anticodons for lysine, aspartate and glutamate in cirrhosis, in addition to various mitochondrial and amino acid trnas in cirrhosis versus nash and nash cirrhosis versus normal respectively [102] . the same study has also compared the abundance of 392 several small rna molecules (ribosomal, small nuclear and nucleolar) between healthy, nash and cirrhotic patients [102] . various predictive modalities involving gene expression parameters, targeted measurements and mirna panels with increased functionality are based on transcriptomics. in general, the reported accuracies for many of these tests j o u r n a l p r e -p r o o f 27 have been high for diagnosing nafld or nafl from healthy status but rather low for differentiating nafl from nash or for diagnosing advanced fibrosis. additionally, even the most promising studies, have not been further tested or have performed poorly in other cohorts. among them, predictive models based on mirnas have in theory higher chances to be robust and reproducible, since mirnas can withstand numerous freeze-thaw cycles and long-term storage before degrading [111] . several studies have identified mir-122 as a potential diagnostic biomarker. most of them have shown that mir-122 alone or in combination with other mirnas (e.g. mir-1290, mir-27, mir-192, mir-34, mir-99a) can accurately predict the presence of nafld or nafl, but they all perform inadequately when trying to differentiate nafl from nash [103, 108, [112] [113] [114] (table 2) . with other proteins in their algorithm. among them, nis4 tm , which includes mir-34a, alpha-2-macroglobulin, chitinase-3-like protein 1, has been recently tested in a population with type 2 diabetes (n=275), showing an auroc performance of 0.801 for diagnosing advanced nash (nas≥4) and significant fibrosis (f≥2), which was also higher than in other tests, such as fib4 (0.704), elf (0.704) or fibrometer (0.678) but probably still suboptimal [115] . additionally, the same test demonstrated 76% sensitivity and specificity in detecting nash patients at risk of cirrhosis [116] . another model integrating mir-122, mir-192, mir-21 and ck-18 fragments, can differentiate nash from nafl (auroc = 0.83) [117] (table 2) . new frontiers, such as circular rna (circrna) transcriptomics, are being investigated in animals. researchers have already mapped and correlated j o u r n a l p r e -p r o o f 28 circrna species and their corresponding transcriptional pathways in mouse models of nash, showcasing their associations with metabolism [118] and stellate cell activation [119] . yet whether these results could yield meaningful conclusions that can be extrapolated in human cohorts remains unclear. other prospective research questions in diagnostic nafld transcriptomics concern alternative rna splicing [120] and the scrutiny of unexplored transcriptional components, such as cd24 [92] . curative and bedside implications of transcriptomics in nafld are considerably limited. from an interspecies aspect, the human and murine nafld transcriptomes are largely dissimilar, yet their likelihood increases in murine models following high-fat diets [90] . hence the transcriptional signatures of nafld or nash as well as the organic transcriptomic responses to relevant treatments for liver disease can be evaluated by numerous available pre-clinical in vitro prototypes and animal models [121] . for instance, mapping of the murine nash transcriptome demonstrated an increase in the levels of tsukushi, a novel adipose-related thermogenetic and metabolic hepatokine. subsequent silencing of tsukushi improved nash and attenuated the hepatic transcriptional alterations associated with it [122] . transcriptomics were also implemented in recent murine models assessing the nafld-related role of poly rc binding protein 1, a cytosolic iron chaperone, whose deficiency exacerbates steatosis and inflammation, increases lipid biosynthesis and oxidative stress pathway expression levels, and is treated via reduced iron intake and vitamin e supplementation [123] . lastly, in view of the covid-19 pandemic, the expression levels of 4 sars-j o u r n a l p r e -p r o o f 29 cov-2 entry proteins were assessed by liver transcriptomics in human and murine nafld and nash cohorts, with no significant changes being detected [124] . future transcriptomic modalities should approach all different phases and components of transcription, kindle research for new remedies, and investigate the associations of liver disease with various pathologies precipitated by its metabolic milieu. transcriptomics have been widely used and have contributed significantly to our understanding of the pathophysiology of nafld. transcriptomics, with the exception of mirnas, do not add significant value in non-invasive diagnostic scores, since their hepatic profile is not adequately reflected in peripheral blood. with the rapid improvement of available technologies and data analysis, transcriptomics are now routinely used in order to identify potential mechanistic pathways affected by drug candidates for the treatment of nafld. proteomics refers to the investigation of "proteome", thus to all proteins expressed by a cell. the proteome, similar to transcriptome and epigenome, is highly dynamic. cell activation, trafficking, differentiation or transformation as well as tissue type have a major impact on the proteome. additionally, posttranslational modifications (phosphorylation, acetylation, methylation, glycosylation etc.) as well as alternative splicing render proteome much more j o u r n a l p r e -p r o o f 30 complex than transcriptome, with more than 120,000 proteins being expressed by a human cell. finally, the proteome is downstream of genome and transcriptome, and thus it is stronger associated with the final phenotype [125] . proteomic analyses have been performed for cell line -characterization or for protein investigation and identification in tissues under specific conditions or treatments, but their use has been in general rather limited, especially for biomarker or drug discovery [126] . there are several reasons for this. first, significant technical limitations exist that restrict the number of proteins that can be detected and can be identified with certainty. the first efforts involved two-dimensional gel electrophoresis and were able to detect some few dozens of spots that were subsequently matched to specific proteins with the use of ms. later approaches managed to detect more protein peaks without though being able to identify the corresponding proteins. advances in methods have enabled in the last years the identification of some thousands of proteins but still the largest part of proteome cannot be investigated with certainty with the available technologies. second, reliable quantification of the levels of proteins, especially in blood has been very challenging. in plasma, three classes of proteins mainly exist: a) the highly abundant proteins, that include human serum albumin (approximately 50% of protein mass), apolipoproteins, coagulation markers and acute phase proteins of the immune system, that are measured in mg to ug/ml level, b) the organ-tissue secreted proteins without functional role in the circulation such as alt, ast or troponins, measured in ug to ng/ml, c) the signaling molecules, such as cytokines or insulin, measured at ng to pg/ml level. this extreme range while keeping in mind the above limitations, we next present the efforts to identify pathophysiological pathways and develop diagnostic models for nafld with the use of proteomics. several studies have investigated hepatic proteome alone or in combination with blood proteome, either in animal models or in humans with nafld, aiming to answer fundamental pathophysiological questions [127] [128] [129] . in one recent study, the levels and cellular distributions of 6,000 liver proteins and 16,000 phosphopeptides have been assessed in the liver of mice developing hepatic steatosis due to high fat diet (hfd). this provided important j o u r n a l p r e -p r o o f 32 fundamental information about the reorganization of organelles, lipid accumulation and cellular dysfunction that occurs with nutrient overload [129] . in another study, proteomics in liver biopsies identified almost 220 proteins that their levels significantly differ in patients with nafld compared to obese metabolically healthy individuals. the proteins that their levels were increased in nafld were involved in ppar-signaling and extracellular matrix-receptor interactions whereas the ones that their levels were reduced, were mainly localized in mitochondria and participated in oxidative phosphorylation [128] . in a combined hepatic phosphoproteome and serum proteome analysis of 67 biopsy-proven nafld subjects, the ask1-mapk pathway that is activated by il-10 was recognized as important for liver fibrosis, indicated by its strong association with higher % of hepatic collagen. in serum, alpha-2 macroglobulin and coagulation factor v were strongly associated with hepatic collagen [127] . it has thus been suggested that these pathways can potentially serve as therapeutic targets. in another approach, proteomic differences in liver tissues of obese patients with vs without t2dm were investigated. the analysis identified 850 proteins, from which 27 were significantly different between the two groups. levels of proteins involved in methionine metabolism and especially of glutathione were reduced whereas levels of other proteins involved in oxidative stress were increased in the t2dm group [130] . expanding on complications of the disease, the proteome of liver tissues with nafld from mice developing hcc has been assessed. differences in tumor suppressor genes and oncogenes were observed, which were further investigated in human samples with nafld and hcc. among them s100a11, which is secreted by cancer cells and stimulates cell j o u r n a l p r e -p r o o f 33 proliferation and migration, was identified and associated with high-grade hcc and poor prognosis [128] . a small number of studies has aimed to develop diagnostic models with the use of proteomics (table 3) . when evaluating the results of these studies, we have to keep in mind the limitations and challenges related to such procedures, that are described below (8. conclusions -challenges -perspectives). in one of the early efforts, combined genomic/proteomic analysis involving liver tissues for assessing gene expression and serum samples for assessing proteins was performed in patients with different stages of nafld. a seldi-tof mass spectrometry (ms) identified approximately 300 protein peaks with 16 of them being significantly different between groups. at that timepoint, it was not possible to identify in which proteins these peaks corresponded to but their masses were compared with the masses of 1,440 serum proteins, which led to the identification of fibrinogen γ as one possible biomarker [131] . in another study including eighty morbidly obese patients, seldi-tof ms has identified in serum three protein peaks that their intensity significantly increased with the severity of nafld, i.e. when moving from steatosis to nash. none of these peaks correlated with liver function tests or metabolic parameters and all of them returned to normal after bariatric surgery. with immunoseldi assay these peaks were recognized as alpha-and beta-hemoglobin subunits [132] . one of the most comprehensive studies so far was able to identify 1,700 serum proteins in a population with nafld. among them 55 were significantly different between nalf and nash with fibrosis stage 3 or 4 (f3/f4) and 15 between nash vs j o u r n a l p r e -p r o o f 34 nash f3/f4 group. these proteins were involved in immune system regulation and inflammation, in coagulation cascades (including fibrinogen γ demonstrated by previous studies), in cell growth and proliferation (including igfbp2, igfbp3, also shown in targeted approaches to be relevant [133] ) and many of them were apolipoproteins (demonstrated also in [134] ) and blood carrier proteins. six (fibrinogen β chain, retinol binding protein 4, serum amyloid p component, lumican, transgelin 2, and cd5 antigen-like) were selected to create a panel for the non-differential diagnosis of nafld and its stages, ranging from healthy liver status to nafl, nash or nash with f3/f4 showing good to very good diagnostic accuracy (aurocs 1.0, 0.83, 0.86, 0.91, respectively). this was though a study with small sample size and its findings have not been replicated so far [135] . in another approach, diagnostic models for differentiating between nafld vs healthy controls among the obese population undergoing bariatric surgery were developed. these models combined genomic information and especially the pnpla3 genotype, phenomic data (i.e. selected biochemical measurements such as glucose, insulin, lipid profile, alt) and proteomic data. specifically, a proteomic analysis identified 1129 proteins with 30 of them being significantly different between the two groups. among them was aminoacylase-1, sex hormonebinding globulin, galectin-3, antithrombin iii and the hepatocyte growth factor. logistic regression models for differentiating between nafld and healthy status were trained by using each type of data individually (i.e. genomic, however, the utility of such algorithms is questionable given that ultrasound or other simpler algorithms (e.g. fatty liver index, etc.) show similar or higher sensitivity and specificity. additionally, diagnosing simply nafld does not initiate treatment decisions [136] . other studies performed first a proteomic analysis in a relevant condition or in another species and then tested their findings in patients with nafld. for example, in one study, an hcc pig model was used and a proteomic analysis in serum was performed. the subsequent analysis identified the serum interalpha-trypsin inhibitor heavy chain 4 as the most important protein corresponding with nafld progression and hcc development. this was further tested in human serum samples and showed that the levels of the proteins were higher in nafld patients with hcc compared to those without [137] . similarly, in another study a proteomic analysis in plasma of patients with liver cirrhosis vs healthy ones was performed. this identified 57 significantly different proteins which were involved in immune system regulation, inflammation, coagulation and fibrinolysis. several of these proteins were subsequently tested in nafld patients and were significantly different in people with nafld and normal glucose tolerance compared to nafld with t2dm [138] . proteomic studies have rather limited contribution to drug development against nafld so far. most of the treatment targets have been identified by animal studies supported by human physiology or epidemiological studies. proteomics have not been widely used in nafld research. the proteins and signaling pathways involved in the pathophysiology of the disease have been almost exclusively identified by targeted "hypothesis-driven" approaches based on data from mechanistic animal, observational human and in some cases large epidemiological studies. this is probably due to methodological challenges and technical limitations related to proteomics. we expect in the near future proteomics to be much more frequently applied in nafld studies. we expect that the main contribution of proteomics will not be in direct development of non-invasive diagnostic scores but rather in the identification of novel molecules and/or pathways with important role in the pathophysiology of the disease and with diagnostic and therapeutic potentials. metabolomics refers to the investigation of small molecules and metabolic products, such as aminoacids, fatty acids and carbohydrates. lipidomics are j o u r n a l p r e -p r o o f 38 considered part of metabolomics and refer to the investigation of cellular lipids. numerous studies have investigated the metabolome and lipidome in mouse models or patients with nash. the evidence linking bile acid homeostasis as well as gut microbiome with the development and progress of nafld have been recently reviewed elsewhere [156, 157] . in this section, we will focus particularly on the robust and extensive changes that are observed both in the hepatic (figure 3 ) as well as in the circulating lipidome, which have led to the development of numerous diagnostic models for nafld as well as to the identification of novel therapeutic targets. the development of nafld is strongly associated with obesity and is characterized by increased lipid accumulation due to higher uptake of elevated ffa by unrestricted adipose tissue lipolysis and due to increased [164] [165] [166] . in non-parenchymal liver cells, sfa stimulate the production and secretion of proinflammatory and profibrotic cytokines from kupffer cells and induce proinflammatory m1 polarization of macrophages [166, 167] . additionally, they stimulate the secretion of chemokines from hepatic stellate cells that recruit more macrophages in the liver [166, 167] . similar to sfa, lysophosphatidylcholines (lpc) have unfavorable hepatic effects. specifically, they stimulate er stress, cause mitochondrial dysfunction and increase apoptosis [168] . additionally, they stimulate the release of hepatic extracellular vesicles from hepatocytes that trigger inflammatory procedures [169] . moreover, the increased activity of the enzyme phospholipase a2 (pla2) that catalyzes the formation of lpc from pc, leads to the rapid depletion of pc which affects hepatocyte membrane integrity and results in hepatocyte apoptosis, high release of lipotoxic lipids and increased inflammation [170] . additionally, pc deficiency reduces vldl secretion resulting in higher intrahepatic lipid degradation and formation of toxic intermediates. furthermore, the low pc/pe ratio affects lipid droplet stability, leading to the formation of larger lipid droplets. finally, lpc is metabolized by the enzyme autotaxin to phospholipid lysophosphatidic acid which stimulates liver fibrosis and development of hcc [171] . ceramides are a type of sphingolipids whose hepatic levels are increased in nafld and correlate positively with disease severity [46, [159] [160] [161] [162] [163] . in hepatocytes, ceramide promotes insulin resistance by inhibiting akt-mediated insulin signaling, impairs beta-oxidation, induces ros production, er stress and proinflammatory cytokine secretion, enhances de novo lipogenesis, cholesterol synthesis and triglyceride accumulation and induces apoptosis [164, [172] [173] [174] [175] . finally, ceramide stimulates fibrogenesis and angiogenesis by increasing extracellular matrix deposition and secretion of pro-angiogenic factors by hepatic stellate cells [176] . [159, 162, 163] . the increased ω-6 concentrations lead to the synthesis of eicosanoids by their enzymatic oxidation (especially of aa but also of epa and dihomo-γ-linolenic acid) with proinflammatory properties such as prostanglandines, thromboxanes and leukotrienes which induce hepatic inflammation. this occurs in expense of the synthesis of spms that mainly act to restore normal cell function and thus reduce chronic inflammation and fibrosis [178, 179] . finally, the high ω-6 to ω-3 ratio is associated with an impaired fads1 activity that can affect cell membrane phospholipid composition resulting in membrane deficiency, cell necrosis and extracellular deposition of lipotoxic lipids that can aggravate hepatic injury [162, 163] . many studies have reported several diagnostic models based on metabolomics, lipidomics alone or combined with other biochemical and clinical parameters for the diagnosis and staging of nafld (table 4 ). the best studies use populations of biopsy-proven nafld to create non-invasive algorithms based on blood measurements. the models aim to diagnose advanced fibrosis (≥3) (in most studies), differentiate nafld from healthy status, distinguish between nash and nafl or nash vs non-nash (in many studies), or detect the presence of fibrosis independently of its stage (in few studies). challenges related to the development of these models are [180] . based on the above findings and methodology, the owliver® test was created, which includes 25 triglycerides and aims to discriminate nash from nafl patients. the test, which was initially trained in white patients without t2dm, performed poorly in a multiethnic cohort of patients with t2dm (auroc=0.69). this shows that patient heterogeneity is an important factor and that tests developed in non-diabetic populations may not be as accurate in patients with t2dm [181] . in another study, information from metabolomics and lipidomics were combined with biochemical and genotypic measurements to create an algorithm for differentiating nash from non-nash (i.e. combined healthy and nafl). 223 subjects were used to train the algorithm and 95 to validate it. the final model consisted of ast, insulin, pnpla3 genotype, glutamate, two aminoacids (glycine and isoleucine) and two lipids (lysopc 16:0 and pe 40:6) and was able to differentiate nash from non-nash with 85% sensitivity and 72% specificity [46] .however, the non-nash population consisted both of healthy subjects (53%) and of nafl (47%). it is though important to be able to distinguish accurately nafl from healthy status. additionally, sensitivity and specificity in most tests is high when healthy subjects are included, but the same tests may not perform as well when nafl is directly compared with nash. in a smaller proof-of-concept study a different approach was followed. a lipidomics, glycomics and targeted hormonal analysis was performed. results were analyzed with different machine learning methods and 19 different diagnostic models were reported for classifying the subjects simultaneously to healthy or nafl or nash group. the models consisted of varying number of j o u r n a l p r e -p r o o f 44 variables (from 10 up to 29) and demonstrated high sensitivities and specificities [144] . the variables included in the models were mainly lipids and often adiponectin, which its protective role against steatosis, inflammation and liver fibrosis is well established [152, 153, 182] . a validation cohort did not exist, but 2/3 of the study population was used to develop the models and 1/3 to validate it, with this procedure being repeated 100 times with several slices of data. this approach helps to control against random results but it still does not address population heterogeneity. nevertheless, the ability to diagnose between three and not two conditions (healthy, nafl, nash) can be attractive for large screenings of high-risk population in primary care setting and without the need for any additional imaging [144] and should be further evaluated in future studies. several studies have attempted to develop non-invasive algorithms for differentiating between advanced (stage 3-4) vs not advanced liver fibrosis (stage 0-2). in one of the most comprehensive studies so far, a cohort of 156 subjects was initially used to develop an algorithm after a metabolomic and lipidomic analysis, which finally consisted of 10 metabolites/lipids. among the 10 parameters, six were cholesterol-derived precursors of steroid hormones which were significantly reduced in advanced fibrosis, one was the primary conjugated bile acid, glycopholate, that was increased in advanced fibrosis and one the aminoacid taurine that is associated with bile acid conjugation and its levels were decreased in fibrosis. finally, the other two parameters were palmitoleate acid and fucose, which were both higher in advanced fibrosis. increased fucosylation, as described below (s. glycomics) is one of the main findings of studies that investigated alterations in circulating glycome sensitivity and 79% specificity for detecting advanced fibrosis, which was higher compared to fib-4 index (39% specificity) and nafld fibrosis score (59% specificity) [183] . finally, very few studies have tried to develop such models in pediatric population. the most comprehensive one so far included 222 children and adolescent with biopsy-or mri-proven nafld and 337 without the disease. metabolomics identified 11 metabolites of interest which together with waist circumference, whole-body insulin sensitivity index and triglycerides could detect the presence of nafld with 97% specificity and 73% sensitivity [184] . nafld is considered an unmet clinical need. several treatments that are currently in the pipeline restore many of the abnormalities observed in the lipidome. specifically, agonists of farnesoid x receptor (fxr) reduce lipotoxicity by promoting mitochondrial beta-oxidation, decreasing de novo lipogenesis and stimulating cholesterol excretion [142] . in phase 2 clinical trials and in an interim analysis of an ongoing phase 3 clinical trial, obeticholic acid reduced hepatic steatosis, inflammation and fibrosis compared to placebo [142, [185] [186] [187] . dual pparα and pparδ agonists (e.g. elafibranor) also stimulate mitochondrial and peroxisome β-oxidation as well as omega-oxidation [188j o u r n a l p r e -p r o o f 46 191 ]. pparγ agonists reduce circulating ffa and consequently the flow of sfa to the liver, they improve insulin sensitivity and guide macrophage and kupffer cell polarization from the proinflammatory m1 phenotype towards to the anti-inflammatory m2 one [142, 167] . various selective pparγ modulators, dual pparα/γ agonists (e.g. saroglitazar) or pan-ppar agonists (e.g. lanifibranor) have shown promising results in human studies and are currently under evaluation in phase 2 or phase 3 clinical trials [142, [192] [193] [194] . in this context, it was recently announced, that lanifibranor met the primary endpoint of a reduction in steatosis activity fibrosis score, including nash resolution without worsening of fibrosis in a phase ii clinical trial, thus being the first study that met both fda and ema regulatory endpoints for accelerated approval [195] acc inhibitors (e.g. firsocostat) attenuate the function of acetyl-coa carboxylase (acc) that catalyzes the conversion of acetyl-coa to malonyl-coa. consequently, malonyl-coa is reduced and hepatic de novo lipogenesis as well as mitochondrial fa beta oxidation are downregulated [142, 196] . in humans, acc inhibitors reduce hepatic steatosis and on-going clinical trials will evaluate their effect also on inflammation and liver fibrosis [142, 196] . regarding lpc accumulation, animal studies have shown that interruption of the generation of lpc from pc with inhibition of pla2 results in normalization of pc levels, prevention of nafld and reversal of nash and fibrosis [158, 197] . several treatments are targeting also ceramide levels by aiming to reduce ceramide synthesis (de novo or through hydrolysis from sphingomyelin) or j o u r n a l p r e -p r o o f 47 increase ceramide degradation. the reduction of ceramide synthesis can be achieved by inhibiting relevant enzymes (i.e. palmitoyltransferase) but it should be limited only to hepatic ceramides, since global reduction of ceramides can negatively affect nervous system [198] . alternatively, lower ceramide synthesis can be achieved by blocking intestinal fxr activation [199] . however, in that case, fxr blocking should be limited to the intestine and not affect fxr activation in the liver which acts beneficially. in other approaches, reduction of the accumulation of specific ceramides (i.e. c16:0 ceramide) is targeted with the use of several inhibitors (e.g. des-1 70) or degradation of ceramides is stimulated by enzymes (e.g. acid ceraminidase). these have till now been tested only in animal studies and not in humans. many treatments have targeted to restore the increased ω-6/ω-3 ratio by ω-3 supplementation. although the findings from animal studies had been very promising, and the first results from human studies had shown an improvement in liver steatosis, later studies could not demonstrate any benefit in steatosis, necro-inflammation or fibrosis in patients with nash [142, [200] [201] [202] . newer experimental strategies are focusing not on the supplementation of ω3 but on restoration of the ω-6/ω-3 ratio with the use of ω-3 desaturase that leads to the production of ω-3 from ω-6 pufas, showing beneficial effects in steatosis and necro-inflammation in animal studies [203] . finally, different experimental approaches are aiming to decrease proinflammatory eicosanoids and increase the concentrations of spms. specifically, some of them are targeting the inhibition of pla2α enzyme that catalyzes the cascade leading to aa and lysophospholipids generation, showing promising results in animal studies [197, 204] . others are targeting j o u r n a l p r e -p r o o f 48 the production of proinflammatory lipids by inhibiting the enzyme 5-lox [205, 206] which catalyzes the lipooxygenation of aa to leukotriene lipids or they aim to block the binding of leukotrienes to their receptors [207] . the 5-lox inhibitor tipelukast has been approved by fda for a phase iia clinical trial. finally, other approaches are aiming to increase the spms rvd1, rve1, protectin dx and maresin-1 leading to robust improvement of fibrosis in animal models of nafld [164, 208, 209] . given that spms are rapidly inactivated by oxidoreductases and that spm receptors are downregulated in obesity, recent efforts are also focusing on improving the pharmacologic properties of them by creating either resistant to deactivation analogues [210] or by enhancing their delivery on their site of function. altogether, most drugs in on-going clinical trials are targeting, among other mechanisms, hepatic lipidome in order to improve nafld. additionally, there is a significant number of medications that have shown beneficial effects on animal models of nafld by improving hepatic metabolome/lipidome and remain to be further clinically tested. metabolomics/lipidomics are one of the most investigated omics in nafld. glycomics refers to the comprehensive investigation of glycome, i.e. of glycan structures that circulate either as free glycans or they are bound to proteins (glycoproteins), lipids (glycolipids) or phospholipids (glycophospholipids). glycome is highly dynamic as it is affected by transcriptome, proteome, environmental factors (nutrition) and cellular secretory machinery [125, 211] . glycans can affect protein morphology and interaction with other proteins as well as regulate nutrient storage and sequestration [212] . additionally, they can protect cell stability and facilitate cell to cell interactions [212] . glycosylation is the process of the formation of the glycoconjugates (glycoproteins or glycolipids) [213] which can happen both intra-and extracellularly. in the extracellular glycosylation, enzymes secreted mainly j o u r n a l p r e -p r o o f 50 from liver hepatocytes and platelets (i.e. glycosyltransferases) are involved [213] . changes in glycan profile have been observed in numerous inflammatory diseases and in different types of cancer and have been often linked causally with the pathogenesis and progression of these diseases [213] [214] [215] . in nafld, few glycomic studies have been performed so far (table 5 ). these have either aimed to assess the circulating glycome in untargeted approaches or they have focused on the identification of glycans related to specific proteins (i.e. haptoglobin, transferrin, igg2, iga1, alpha-1 antitrypsin, ceruloplasmin). the findings of these studies suggest that in nafld and during its progression from nafl to nash and liver fibrosis higher concentrations in fucosylated, sialylated and agalactosylated glycans are observed. sialic acids in glycolipids or glycoproteins have diverse functions, including formation of a protective cell surface barrier, involvement in interactions of white blood cells with endothelial lining of blood vessels, recognition by pathogens and toxins and facilitation of cell migration by some cancers [216] . circulating sialic acid levels have been positively associated with metabolic syndrome and with nafld [217, 218] . fucosylated glycans are also involved in a variety of physiological and pathological procedures, including cell adhesion and migration, angiogenesis, malignancy, tumor metastasis as well as immune cell development and regulation [219] . consequently changes in fucosylation have been reported in numerous inflammatory conditions, such as in rheumatoid arthritis, chronic pancreatitis, sickle cell disease and crohn disease [219] . in the liver, fucosylation serves as a signal for the secretion of fucosylated glycoproteins from normal j o u r n a l p r e -p r o o f hepatocytes into bile [219] . in ballooning hepatocytes, which are observed in nash, the fucosylation-related sorting machinery is dysfunctional which may result in the secretion of the fucosylated glycoproteins in the sera instead of bile [219] . similarly, hypogalactosylation (lack of galactose in the formed glycoprotein or glycolipid) especially of igg has been associated with inflammatory response and with a number of autoimmune diseases [213] . most of the glycomic studies in nafld have tried to identify glycans or glycoproteins that can serve as blood biomarkers for differentiating between nafl and nash or for detection of the presence of liver fibrosis and its stage ( table 5 ). the diagnostic accuracy of most of these tests has been limited, with some reporting higher accuracy for diagnosing nash and others for diagnosing advanced fibrosis. this suggests that although changes in circulating glycome/glycoproteins are observed in nafld, these are not sufficient in order to be used alone for the development of diagnostic models of the disease and thus combination with other clinical or biochemical parameters are needed. in this context, fucosylated-haptoglobin showed below 70% accuracy at differentiating nafl from nash, but 76-81% when combined with mac2bp [220] . similarly, in our study, which detected with the use of mass-spectrometry the highest number of glycans so far, the concentrations of glycans in serum were able to differentiate between the presence of liver fibrosis or not with 76% sensitivity and 74% specificity [144] . glycans alone could poorly differentiate between nafl and nash but when combined with lipid species in models of 20 variables (18 lipid species and 2 j o u r n a l p r e -p r o o f 52 glycans) the sensitivity and specificity for discriminating between nash, nafl or healthy liver status increases significantly. regarding potential treatments, no study thus far has investigated whether targeting glycome can be a therapeutic option in nafld. treatments aiming to decrease fucosylation are currently under evaluation in autoimmune diseases and cancer. in this context, inhibition of fucosylation with a 2-deoxyd-galactose, a fucosylation inhibitor, reduces inflammation in rheumatoid arthritis by decreasing inflammatory macrophages and th17 cells in the lymph nodes as well as by reducing the levels of tnfα, interleukin-6 (il-6), and antibodies to type ii collagen in the serum [221] . similarly, inhibition of fucosylation by 2-fluorofucose suppresses the proliferation, migration and tumor formation of hepg2 liver cells [222] . whether similar interventions can be beneficial in nafld by reducing inflammation, fibrosis or hcc formation should be addressed in future studies. glycomics are one of the least investigated "omics" in nafld. given the strong relation between glycoprotein-glycolipid formation and liver function, more studies assessing the potentials of glycans in biomarker discovery and drug development are needed. performed. thus, these models often do not account for population heterogeneity, which is crucial for the accuracy all non-invasive diagnostic panels. this has been repeatedly shown in studies that focused on patients with overweight, obesity and especially with t2dm. several and diverse biomarker panels (including steatotest, actitest, nashtest-2, cytokeratin-18, fibrotest, owliver tests) were evaluated but none of them demonstrated optimal performance for diagnosing nash or liver fibrosis especially in patients with t2dm [5, 13, 181, 223, 224] . third, very few tests have been evaluated prospectively. this is a very important step, which even other simpler algorithms or imaging modalities have failed to pass [225] . additionally, depending on their mechanism of function, medications may have different levels of impact on the metabolic and lipid profile in the liver which may affect circulating metabolome/lipidome and its ability to reflect the stage of the disease through a diagnostic algorithm originally designed in untreated patients. altogether, diagnostic models based on omics have yet to reach exceptional levels of performance, while satisfactory study outcomes need to be cautiously scrutinized and repeatedly validated into large and diverse patient cohorts both cross-sectionally as well as prospectively. to this direction, fda has recently approved the parallel evaluation of diagnostic biomarker panels [239] metab j o u r n a l p r e -p r o o f (rs738409) impairs pufa transfer from dags to pcs, thus increasing pufa in tg and dag. tm6sf2 e167k (rs58542926) impairs pufa synthesis, increases polyunsaturated ffas and prevents pufa incorporation into tgs and pcs. both mechanisms lead to impaired vldl synthesis and lipid droplet hydrolysis. gckr p446l (rs780094) incites glycolysis, glycogen deposition and de novo lipogenesis by disinhibiting glucokinase. epigenetic modifications characteristic of nafld progression include cpg site hypermethylation, thus reduced expression, of genes pertaining to lipid and aminoacid metabolism and stellate cell inhibition. hypomethylation, thus increased expression, of genes pertaining to tissue repair, inflammation, carcinogenesis and fibrogenesis, increases insulin resistance and further propagates the disease. methylation levels of cytoskeletal, transcriptional, proliferation-related and metabolic genes are affected by age, fasting glucose levels and body weight. at the histone level, depletion of sirtuins 1 and 3 and hdac3 may propagate nash and increase susceptibility to mets, insulin resistance and hyperlipidemia. on the other hand, the glucose-activated hat p300 activates chrebp and thus precipitates stellate cell activation, elevates lipogenic gene expression and expedites steatosis, though these effects can be attenuated by tannic acid. finally, the nafld transcriptome is characterized by overexpression of lipid metabolism, cellular stress, division and adhesion, extracellular matrix production and repair, cancer progression and immunomodulatory genes, whereas several pro-metabolic and insulin signaling genes are downregulated. mirnas, especially mir-122, mir-192 and mir-34a, are linked to steatosis, cholesterol metabolism, liver cancer, atherogenesis and mets, whereas other noncoding molecules, such as lncrnas, are indicators of nash grade and hepatocellular viability. the uncontrolled lipolysis from adipose tissue, the increased dietary intake of tg and the upregulated de novo lipogenesis observed in nafld leads to elevated sfa, lpc, ceramides and ω6/ω3 pufa ratio. sfa stimulate the secretion of inflammatory cytokines via tlr4 and apoptosis via trail2, whereas they increase oxidative stress, er stress and impair β-oxidation in the mitochondria of hepatocytes. in stellate cells, they stimulate macrophage recruitment, whereas in kupffer cells and macrophages sfa induce their polarization to the m1 proinflammatory state. increased activation of pla2 enzyme leads to formation of lpc and depletion of pc. pc are important for lipid droplet stability and their deficiency leads to large droplet formation and inadequate vldl secretion. high lpc are also triggering mechanisms of impaired β-oxidation, apoptosis, fibrosis and hcc. high hepatic ceramide concentrations increase cholesterol synthesis and tg accumulation, promote insulin resistance by blocking akt-mediated insulin signaling, induce the secretion of proinflammatory cytokines and stimulate apoptosis by increasing ros generation, er stress and β-oxidation impairment. in hepatic stellate cells, they increase extracellular matrix deposition and pro-angiogenic factors secretion promoting fibrogenesis. finally, the high ω6/ω3 ratio leads to increased synthesis of proinflammatory molecules, such as prostaglandins, leukotrienes, thromboxanes in expense of the synthesis of anti-inflammatory spms, thus resulting in a pro-inflammatory complications, morbidity and mortality of nonalcoholic fatty liver disease the multiple-hit 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fragments related to fibrogenesis and fibrolysis in nonalcoholic steatohepatitis a gene-based model for staging liver fibrosis a biomarker panel for non-alcoholic steatohepatitis (nash) and nash-related fibrosis new discriminant score to predict the fibrotic stage of non-alcoholic steatohepatitis in japan the diagnostic accuracy of liver fibrosis in non-viral liver diseases using acoustic radiation force impulse elastography: a systematic review and meta-analysis magnetic resonance elastography for staging liver fibrosis in non-alcoholic fatty liver disease: a diagnostic accuracy systematic review and individual participant data pooled analysis key: cord-030369-4dn02a35 authors: peng, liang; gao, zhi-liang; wang, yu-ming; he, deng-ming; zhao, jing-ming; bai, xue-fan; wang, xiao-jing title: clinical manifestations and laboratory tests of aechb and severe hepatitis (liver failure) date: 2019-05-21 journal: acute exacerbation of chronic hepatitis b doi: 10.1007/978-94-024-1603-9_1 sha: doc_id: 30369 cord_uid: 4dn02a35 this chapter describes the clinical symptoms and signs of aechb and hbv aclf, classification, grading of hbv aclf and their features, diagnostic principles and standards in liver pathology, biochemistry, and virology of hbv aclf. 1. liver failure is defined as serious damage to the liver cause by a variety of etiologies, leading to liver function disorder or even decompensation, and clinical syndromes with coagulopathy, jaundice, hepatic encephalopathy, and ascites. 2. severe hepatitis b can be indicated pathologically by apparent hepatocellular necrosis, including extensive multifocal, confluent, bridging, sub-massive or massive necrosis. 3. laboratory tests during the course of severe exacerbation of chronic hepatitis b can reflect pathological changes and liver function in a timely manner, providing objective and informative reference data for evaluation of disease severity and treatment efficacy. among the most important laboratory tests are those for prothrombin activity, international normalized ratio, and increases in total bilirubin concentration. 4. severe hepatitis b is associated with interactions between the virus and host factors. detection of hbv dna, hbv genotype, quasispecies and hbv mutation can provide important theoretical bases for the prevention, control or mitigation of the progress of severe hepatitis b. 5. noninvasive imaging modalities can be used to visualize the entire liver and parts of it. measuring liver volume to evaluate liver size and liver reserve capacity is regarded as important in diagnosis, surgical approach and prognostic evaluation of patients with severe exacerbation of chronic hepatitis b and liver failure. 6. model for end-stage liver disease (meld) is the first quantitative method developed to assess whether a patient with liver failure requires a liver transplant. the predictive value of the meld model has been improved by the meld-na, imeld, and meso models. several other valuable prognostic models have been developed. for example, for patients with hbv-aclf, the established tppm scoring system was found to be more predictive than meld score. can reflect pathological changes and liver function in a timely manner, providing objective and informative reference data for evaluation of disease severity and treatment efficacy. among the most important laboratory tests are those for prothrombin activity, international normalized ratio, and increases in total bilirubin concentration. 4. severe hepatitis b is associated with interactions between the virus and host factors. detection of hbv dna, hbv genotype, quasispecies and hbv mutation can provide important theoretical bases for the prevention, control or mitigation of the progress of severe hepatitis b. 5. noninvasive imaging modalities can be used to visualize the entire liver and parts of it. measuring liver volume to evaluate liver size and liver reserve capacity is regarded as important in diagnosis, surgical approach and prognostic evaluation of patients with severe exacerbation of chronic hepatitis b and liver failure. 6. model for end-stage liver disease (meld) is the first quantitative method developed to assess whether a patient with liver failure requires a liver transplant. the predictive value of the meld model has been improved by the meld-na, imeld, and meso models. several other valuable prognostic models have been developed. for example, for patients with hbv-aclf, the established tppm scoring system was found to be more predictive than meld score. liang peng, zl huang, yy mei and zhi-liang gao currently, both clinical and pathophysiological diagnoses are made of severe hepatitis (liver failure) in china. according to the guideline for the prevention and treatment of viral hepatitis (2000), severe hepatitis is classified as acute severe hepatitis, subacute severe hepatitis, and chronic severe hepatitis. acute severe hepatitis is initially diagnosed due to acute jaundice that rapidly progresses to liver failure within 2 weeks. subacute severe hepatitis can be identified in patients with acute jaundice hepatitis that progresses to liver failure anywhere from 15 days to 24 weeks. chronic severe hepatitis often develops with pre-existing chronic liver diseases. the clinical manifestations of chronic severe hepatitis are similar to those of subacute severe hepatitis in some patients, or, in some patients, appear similar to decompensated cirrhosis at disease onset. the diagnostic criteria for severe hepatitis in china remain to be fully developed and hence have not been introduced internationally. to meet the clinical requirements and standardize the diagnosis and therapy of liver failure, the branch of infectious diseases and the branch of hepatology of the chinese medical association invited experts in china to develop the first guidelines for the diagnosis and therapy of liver failure in 2006. in those guidelines, liver failure refers to severe liver damage caused by multiple factors. that damage to the liver results in either the severe impairment or decompensation of synthesis, detoxication, excretion, and biotransformation in the liver and subsequent clinical manifestations characterized by coagulation disorder, jaundice, hepatic encephalopathy, and ascites. on the basis of pathological features and disease progression, liver failure is classified as acute liver failure (alf), subacute liver failure (salf), acute-on-chronic liver failure (aclf), and chronic liver failure (clf). alf is characterized by the rapid appearance of clinical manifestations. patients with alf usually develop a clinical syndrome of liver failure characterized by highgrade hepatic encephalopathy (he, >grade 2) within 2 weeks. patients with salf typically present with a clinical syndrome of liver failure anywhere from 15 days to 26 weeks. finally, aclf refers to the acute decompensation of the liver function in the presence of pre-existing chronic liver diseases, and clf refers to chronic decompensation of the liver function characterized by ascites or portal hypertension, coagulation disorder, and he due to progressive liver dysfunction in the presence of hepatic cirrhosis. the published guidelines systemically and extensively reflect the current status of the diagnosis and therapy of liver failure. in addition, the guidelines, for the first time, focus on liver failure rather than severe hepatitis, which broadens our horizons and highlights practicability. in china, acute severe hepatitis, subacute severe hepatitis, and chronic severe hepatitis correspond closely to alf, slf, and aclf, respectively, as illustrated in table 1 .1. in some patients, chronic severe hepatitis is similar to clf in other countries. on the basis of available guidelines for liver failure, we define severe hepatitis b as liver failure due to hepatitis b virus infection. clf is the most common, and alf and slf are rare. acute exacerbation of chronic hepatitis b (aechb) is a dynamic process, including mild, moderate, severe chronic hepatitis b and chronic aclf defined in above guidelines. the reference index of abnormality in laboratory examination is shown in table 1 .2. in addition to viral replication, other factors also contribute to the pathogenesis of hepatitis b-induced liver failure, such as concomitant infection of other hepatitis viruses (especially the hepatitis e virus), immune status, pregnancy, drug and/or alcohol consumption, concomitant bacterial infection, mental stress, and concomitant disease processes (e.g., hyperthyroidism). the liver is the largest solid organ in humans and has complex functions. hepatic parenchymal cells are responsible for metabolism, secretion, synthesis and bioconversion. factors that can cause severe damage to hepatocytes (i.e., parenchymal cells, kupffer cells) may result in disorders of metabolism, secretion, synthesis, detoxication and immunity. in turn, that damage can lead to jaundice, liver shrinkage, coagulation dysfunction, hemorrhage, secondary infection, hepatorenal syndrome, he, and other clinical entities described in detail here. the physical condition of patients deteriorates, and affected individuals usually develop weakness, extreme fatigue, and a severely diminished quality of life. they frequently require assistance to perform basic personal needs, such washing their face, brushing their teeth, and using the toilet. in the early stage of jaundice, in addition to developing extreme fatigue, gastrointestinal symptoms become evident, including extremely poor appetite, anorexia, intolerance of oily foods, nausea, vomiting, abdominal discomfort, and hiccups. in the jaundice stage, the gastrointestinal symptoms deteriorate further. patients can develop refractory vomiting, hiccups, evident abdominal distension, and reduced/ lack of borborygmus. clinically, patients initially note their urine color darkens, becoming a strong-tea like color. next, a yellowish pigmentation of the ski and conjunctival membranes develops. that jaundice progressively becomes deeper, characterized by hepatocellular jaundice. in this stage, serum bilirubin increases rapidly. in fact, the daily increment in serum bilirubin may be >17 μmol/l (>1 mg/dl). the sulfur-containing amino acids in the intestine are degraded into mercaptans that have the odor of rotting fruit. mercaptans cannot be metabolized in the liver and are therefore excreted from the respiratory tract. this distinctive odor is specifically noted in patients with he. the severity of hepatic foetor may, in some cases, reflect the severity of liver injury. the occurrence of coagulation dysfunction is primarily ascribed to the reduced synthesis of coagulation factors by the liver. a majority of the both coagulation and anticoagulant factors are synthesized in the liver. in addition, some coagulationrelated factors and their inhibitors are also metabolized in the liver. the outcome of coagulation dysfunction is dependent on the severity of damage to the hepatocytes. thus, even patients in an early stage of liver failure may present with coagulation dysfunction. prothrombin (pt) activity is often abnormal in the early stages of liver failure and may therefore serve as a sensitive indicator for the prognosis of liver failure. common clinical manifestations of coagulation dysfunction are mucocutaneous bleeding (i.e., spontaneous bruising, gingival bleeding, subconjunctival hemorrhage), ecchymosis at the site of injection/puncture, and purpura in more severe cases. gastrointestinal bleeding is also common in affected individuals, whereas bleeding into/from the genitourinary tract, lung, kidney, and retroperitoneum is rare but occasionally observed in some patients. if intracranial hemorrhage develops, it is frequently life threatening. in ahf, the incidence of bleeding and severe bleeding is as high as 73 and >30%, respectively. another cause of coagulation dysfunction is thrombocytopenia and platelet dysfunction. of the two, thrombocytopenia is more common. because platelets are derived from megakaryocytes in bone marrow, bone marrow fibrosis and either reduced bone marrow regeneration or invasion of lymphoma cells in the bone marrow can reduce the number of platelets. platelets perform multiple activities, including adhesion, aggregation, release, and shrinking blood clots. additionally, they play an important role in coagulation. platelet dysfunction may also increase capillary permeability and fragility, which may cause either spontaneous bleeding of the skin and mucous membranes or difficult hemostasis following vascular injury. in patients with slf, thrombocytopenia is mainly diagnosed in the latter stage of disease in which massive hepatocyte necrosis leads to posthepatic cirrhosis, portal hypertension, and hypersplenism. in clf patients, thrombocytopenia might be present, and hepatocyte necrosis may aggravate portal hypertension and hypersplenism, resulting in worsening thrombocytopenia. splenomegaly and splenic sinus hyperplasia increase the phagocytosis and destruction of platelets. further, splenomegaly can cause enlargement of the platelet pool within the spleen. as a result, the platelets in the spleen may account for >90% of platelets in the body. the above pathological changes may finally cause a reduction in the circulating platelets. the reason for thrombocytopenia in liver disease patients without hypersplenism is still poorly understood and might be ascribed to following factors (1) the hepatitis b virus may significantly inhibit the megakaryocyte system of the bone marrow, resulting in reduced production of platelets; (2) the thrombopoietin (tpo) level is reduced. the division of megakaryocytes into platelets in the bone marrow is controlled by both megakaryocyte colony stimulating factor (meg-csf) and tpo. meg-csf primarily regulates the proliferation of megakaryocyte progenitor cells, whereas tpo stimulates the maturation of megakaryocytes and production of platelets. tpo is almost exclusively produced by hepatocytes, and only a minority of tpo is produced in the kidney and other organs. tpo is a key factor affecting the production of platelets, and the synthesis of tpo is reduced significantly in patients with either severe hepatitis or hepatic cirrhosis, which affects the production of platelets. in patients with parenchymal liver diseases, abnormalities of platelets are present in both quality and quantity. for example, when the platelet membrane glycoprotein gpi6-ix is reduced, the aggregation of platelets following ristocetin treatment and the shrinkage of blood clots are markedly compromised; and (3) patients with liver diseases usually develop immune dysfunction and are therefore susceptible to infection. bacterial toxins and systemic inflammatory response syndrome may also cause thrombocytopenia. one published study of icu patients found that infection was an independent risk factor of thrombocytopenia. he he is both a neuropsychiatric syndrome, a type of central nervous system dysfunction, and metabolic disturbance due to hepatocellular dysfunction and portosystemic shunting. he is clinically characterized by mental and neurological abnormalities, such as abnormal personality and behaviors, irritability, sleep perversion, drowsiness, and complete loss of consciousness or coma. he is one of the major causes of severe complications and death in patients with liver failure and is typically classified into one of the four following stages: stage 1: the prodromal stage. this stage usually manifests with mild abnormal personality changes and behaviors, such as euphoric excitement, indifference, taciturnity, being sloppily dressed, and inappropriate defecation/urination. the affected individual can usually provide correct responses to questions but they are inarticulate and have slow speech. flapping tremor/hepatic tremor might also be present. to test for flapping tremor, patients are asked to close their eyes with their arms stretching straight, elbows flexed, palms in dorsal extension, with separated fingers. a positive response is determined when the metacarpophalangeal joint, wrist, elbow, and shoulder show irregular movements (jitter) when held in that position within 30 s. physicians may also ask the patients to hold the their hand for 1 min. if the physician feels the hand tremor, the test suggests a positive diagnosis of flapping tremor. the condition is caused by afferent dysfunction of joint-reticular formation of the brainstem and a characteristic neurological manifestation. that said, flapping tremor has no specificity fro he and can also be found in patients with either uremia or hypoxemia due to chronic respiratory disease/heart failure. the presence of flapping tremor in a patient with severe liver disease, however, is helpful for early diagnosis of he. patients with he usually have a normal electroencephalogram. stage 1 of he lasts anywhere from several days to several weeks. several patients with he in the prodromal stage may have no evidence of clinical symptoms; therefore, misdiagnosis is possible. stage 2: the precoma stage. patients with he in this stage usually presents with confusion, sleep and behavioral disorders, and symptoms as described in the prodromal stage further deteriorate. patients suffer from disorientation and understanding disorders as well as conceptual confusion over time, place, and person. patients are unable to perform simple intellectual composition (e.g., building blocks, arranging matchstick into pentagon), and have decreased computing capacity (e.g., 100-7 and continuing). slurred speech, writing disorders, and abnormal behaviors are also common. sleep perversion and daytime sleep and night awaking may be present. further, hallucinations, fear, and mania are also observed, and some patients can be misdiagnosed with mental diseases. patients with liver failure in this stage usually have evident neurological signs such as tendon hyperreflexia, increased muscle tone, ankle clonus, and presence of the babinski sign. flapping tremor and an abnormal electroencephalogram can also be observed. patients may also suffer from uncontrolled muscular activities and ataxia. stage 3: the lethargic stage. patients with he in the lethargic stage mainly manifest lethargy and insanity, and neurological signs continue and deteriorate. in the majority of time, patients are in a lethargic state, but can be waken up. patients respond to questioning, but may present confusion and hallucination. flapping tremor is also present. muscular tension increases, and there is resistance in the passive limb movements. pyramidal signs and abnormal waves in eeg can also be noted. stage 4: the coma stage. patients have complete loss of consciousness and are unable to be awakened. in a light coma, patients are responsive to painful stimuli and uncomfortable postures, have tendon hyperreflexia, and increased muscular tension. patients in this stage are usually unable to co-operate during an examination, and a flapping tremor may not be inducible. in a deep coma, various reflexes disappear; muscular tension reduces; pupils become dilated; and there are paroxysmal convulsions, ankle clonus, hyperventilation, and abnormalities on an electroencephalogram. stage of he is an important indicator of severity of disease. it may reflect not only the severity of brain damage but also the severity of liver disease. it is important to recognize that there is no clear boundary between two neighboring stages and that there might be some overlap between two neighboring stages (therefore missing the middle stage of he). when the disease condition either deteriorates or improves after therapy, the severity of he may be reduced by one or two stages. as mentioned above, the initial symptoms of he are personality changes. patients with extrovert personalities (i.e., lively, cheerful) may become depressed, whereas patients with introverted personalities (i.e., withdrawn, reticent) may become euphoric and garrulous. the second most common symptom is a change in behaviors. initially, patients have sloppy behaviors, such as meaningless behaviors like scattering garbage all over the place and defecating/urinating anywhere, looking at clothes, and touching the bed. those changes are usually only identified by close observation and careful experience. there are also changes in sleep habits. patients are often drowsy during the daytime but have difficult sleeping at night or show sleep perversion, which predicts imminent he. hepatic foetor is also an important feature of he. he patients usually have brain edema and present with nausea, vomiting, dizziness, headache, and either irregular breathing or even apnea. as blood pressure increases there might be a paroxysmal or sustained increase in systolic blood pressure. bradycardia may be also observed. muscular tension can increase or the patient can develop a decerebrate posture or even opisthotonus with severe he. the pupillary light reflex can become blunt/absent, the pupils can become mydriatic, and anisocoria can occur. achilles and knee tendon hyperreflexia may be observed. it is important to note that some signs might not be obvious in a patient with late-stage he. in clinical practice, clinicians may indirectly evaluate the severity of brain edema according to chemosis. accurate evaluation of brain edema is dependent on the subdural, epidural, or cerebral parenchymal measurement of intracranial pressure. the normal intracranial pressure is <2.7 kpa (20 mmhg), and brain edema is diagnosed once intracranial pressure is >20 mmhg. the most important sign of he is flapping tremor, which means the presence of he in stage ii. in addition, thinking and intelligence tests (such as number connection test, signature test, mapping test, and computing capability test) are abnormal in he patients. in some he patients (especially those with hyperammonemia due to he), slow waves with high amplitude may be observed on electroencephalogram, and positive-evoked potential is also a characteristic change. brain edema is a complication of alf. typical clinical manifestations of brain edema are sustained increase in blood pressure, abnormal pupils, irregular respiration, and papilledema. more than 80% of patients with he in stage 3 or 4 are likely to develop brain edema, and severe brain edema may result in cerebral hernia. brain edema has the clinical presentations of increased intracranial pressure and cerebral dysfunction, which can sometimes overlap with the manifestations of he. it is therefore sometimes difficult to differentiate the two, potentially resulting in misdiagnosis. he patients with brain edema may present dysphoria, irascibility, and increased muscular tension, which are more common than in patients with he without brain edema. if there are concomitant changes in pupils and respiration together with convulsions and/or seizures, cerebral hernia is suspected. in the late stages of liver failure, patients may develop intracranial hemorrhage, causing respiratory and circulatory arrest and even sudden death. thus, once cardiopulmonary arrest of unknown cause is present, intracranial hemorrhage should be considered. concomitant gastrointestinal bleeding in patients with severe hepatitis can be caused by multiple factors, including (1) decreased coagulation factor synthesis by hepatocytes and/or significant inactivation of active coagulation factors in the liver; (2) endotoxemia and disseminated intravascular coagulation consuming a large amount of coagulation factors; (3) hypersplenism causing abnormalities in the quality and quantity of the platelets; (4) portal hypertension causing the rupture of esophageal and gastric varices; and (5) stress response in severe hepatitis leading to diffuse gastric corrosive erosion. of the possible complications occurring in liver failure patients, bleeding is the most common and severe. in clinical practice, gastrointestinal bleeding with severe hepatitis seems to make the primary disease worse. it may worsen liver ischemia and hypoxia and aggravate liver dysfunction and ascites. blood in the gastrointestinal tract can be degraded into ammonia and increase the production of sulfur-like substance, resulting in he. in addition, bleeding may reduce immune function, which make infections difficult to control. the reduction in effective circulating blood volume may also induce hepatorenal syndrome. taken together, bleeding may cause multiple organ dysfunction, thereby complicating treatment and reducing the success rate of therapy. the causes of upper gastrointestinal bleeding are different among patients with different types of liver failure. in alf and slf, bleeding is related to reduced synthesis of coagulation-related factors and stress-induced gastric mucosal lesions. in clf, however, rupture of esophageal and gastric varices and gastric mucosal lesions secondary to portal hypertension are the main causes of gastrointestinal bleeding. in some cases, there is more than one cause of bleeding. in liver failure, the ability of the monocyte-macrophage system to clear intestinerelated endotoxins is reduced significantly, which may lead to intestine-related endotoxemia and deterioration of liver function. this clearly forms a vicious cycle and may cause multiple organ failure if it is severe enough. in addition, patients usually have compromised immune function and are susceptible to infection. invasive manipulations and use of broad-spectrum antibiotics and immunosuppressants further increase the possibility of secondary infection. concomitant infection in liver failure patients has the following characteristics: (1) a high incidence; (2) infection may occur at different sites either simultaneously or sequentially, and abdominal and biliary tract infection is the most common. once pulmonary infection is present, the disease condition will likely deteriorate, directly causing death; (3) a majority of infections are nosocomial infection, and pathogens are usually resistant to common antibiotics, making therapy challenging; (4) the pathogens causing infection are diverse but mainly gram-negative bacteria, although the incidence of gram-positive and fungal infections is increasing; (5) infection is closely related to the prognosis for liver failure patients. in sum, the more severe the disease, the higher the incidence of infection is and secondary infection may worsen the condition or cause death. the early diagnosis of secondary infections is based on clinical findings such as signs of infections (i.e., fever, increase in peripheral white blood cells, deterioration of primary disease, specific symptoms of infection of a particular organ). some patients may not present with an obvious fever and instead only show focal signs of infection. for example, in spontaneous bacterial peritonitis, examination could reveal abdominal tenderness and rebound tenderness and a slight increase in peripheral white blood cells and polymorphonuclear proportion (although they are in normal ranges). in contrast, pulmonary infections can present only with fever while the respiratory symptoms are not obvious/absent and thoracic radiographs fail to show abnormalities in affected patients. in such cases, computed tomography is required to identify the pulmonary lesions. in addition, liver failure patients are vulnerable to fungal infection, especially for those receiving long-term therapy with broad-spectrum antibiotics. gastrointestinal candidiasis is the most common fungal infection. oral candida albicans infection is characterized by thickening and a bean residue-like coating on the tongue, gastrointestinal fungal infection is characterized by increased stool frequency and stool with mucus, and pulmonary fungal infection (especially aspergillus infection) is a severe complication of liver failure that can progress rapidly and has a high mortality rate. once a pulmonary fungal infection is suspected, computed tomography of the thorax should be performed to confirm the diagnosis, and effective antifungal therapy should be initiated as early as possible. hepatorenal syndrome (hrs) refers to progressive functional renal failure in the absence of primary kidney disease in patients with severe liver diseases. hrs is most often diagnosed in the late stages of severe hepatitis and hepatic cirrhosis. the main clinical manifestations of hrs include: 1. late stages of liver failure; 2. renal failure after a reduction in effective circulating blood volume (e.g., water and electrolyte disorder, following paracentesis for ascites, excessive urination due to diuresis, gastrointestinal bleeding, secondary infection, vomiting, and diarrhea). however, hrs may present abruptly with no evident/discoverable causes; 3. hrs is often found in patients with moderate to severe ascites; 4. hrs has no significant relationship with jaundice and he; and 5. blood pressure reduces during hrs. thus, when patients are treated with propranolol for portal hypertension, physicians should pay attention to the baseline blood pressure because reduction in blood pressure after pharmacotherapy may reduce the blood supply to the kidney and decrease glomerular filtration rate, inducing hrs; 6. the abrupt decrease in urine output suggests the presence of hrs. diuretics usually fail to increase the urine output. patients often have a reduction in urine sodium and concomitant hyponatremia; 7. urinalysis shows similarities to prerenal azotemia but displays opposite features to acute tubular necrosis; 8. the symptoms of uremia may overlap with those of liver failure and cause the deterioration of original symptoms. in patients with progressive liver diseases, secondary renal dysfunction is closely related to the deterioration of their general condition, suggesting the aggravation of liver failure. in addition, the presence of uremia may contribute to metabolic complications. coagulation dysfunction in liver disease patients may be deteriorated due to compromised aggregation of platelets during uremia. uremia may also aggravate immune dysfunction. on the basis of clinical characteristics, hrs can be classified into two types. type i hrs is rare, has an acute onset, and is characterized by progressive renal dysfunction. serum creatinine may be either 2× that at baseline (i.e., >221 μmol/l or 2.5 mg/dl) within 2 weeks or creatinine clearance decreases by 50% within 24 h (i.e., creatinine clearance of <20 ml/min). patients with type i hrs have a poor prognosis, with 80% of patients dying within 2 weeks of diagnosis and only 10% of patients can survive for >3 months. the course of disease is short, and symptoms of uremia are not obvious. in type ii hrs, which is often found in clf patients with pre-existing hepatic cirrhosis, has a chronic onset. ascites patients with type ii hrs are usually nonresponsive to diuretics. in type ii hrs, renal failure shows a slow progression (lasting for several weeks to months), but the survival rate of patients is lower than that of hepatic cirrhosis patients with ascites. the main clinical consequence is refractory ascites nonresponsive to diuretics in patients with type ii hrs. a follow-up study of 234 hepatic cirrhosis patients with ascites showed the accumulative incidence of hrs was 18% within 1 year and 39% within 5 years. a retrospective study showed about 17% of patients with ascites on admission had hrs and hrs patients accounted for 50% of hepatic cirrhosis patients died. however, for hepatic cirrhosis patients, the 2-year and 5-year incidence of hrs is 32% and 41% after development of ascites. a majority of patients (80-95%) die within 3 weeks after development of azotemia. hps refers to a series of pathophysiological changes and clinical manifestations (including hypoxemia) due to abnormal pulmonary vascular dilation, gas exchange disorder, and abnormal arterial oxygenation. abnormal arterial oxygenation due to a gas exchange disorder may increase the alveolar-arterial oxygen pressure difference. hypoxemia is an important pathophysiological basis of hps, and hps is a severe pulmonary complication of end-stage liver disease that is clinically characterized by dyspnea and cyanosis. hps was first reported by rydell hoffbauer in 1956, but it wasn't until 1977 that kenned and knudson proposed the full concept of hps. hps per se refers to pulmonary vascular dilation and the shunting of venous blood with low oxygenation to arteries in the presence of severe liver disease. hps is mainly identified in patients with clf (child c hepatic cirrhosis). in addition, patients with either acute or chronic liver disease may present with a pulmonary vascular abnormality and arterial hypoxemia. hps occurs most commonly in patients with hepatic cirrhosis secondary to chronic liver disease, including hepatitis-induced cirrhosis, cryptogenic cirrhosis, alcoholic cirrhosis, and primary biliary cirrhosis, all of which have similar pathophysiological processes as hps. in hps, severe ascites, portal hypertension, and arterial hypoxemia (pao 2 < 10 kpa) may be related to the intrapulmonary vascular shunt, excessive production of nitric oxide, lung ventilation-perfusion imbalance, and interstitial fibrosis. the incidence of hps varies among studies. the incidence of hps is about 5-29% in chronic liver disease patients but higher in patients with hepatic cirrhosis. the most common clinical manifestations of hps are dyspnea, hypoxemia, and cyanosis caused by intrapulmonary vascular dilation and poor arterial oxygenation in the presence of primary liver disease: patients usually progressively develop respiratory manifestations (e.g., cyanosis, dyspnea, clubbed-fingers/toes, orthostatic hypoxia, supine breathing). progressive dyspnea is the most common pulmonary symptom of hps, and cyanosis is a unique and reliable clinical sign. supine breathing and orthostatic hypoxia are characteristic manifestations of hps. pulmonary examination often fails to identify clinically important signs, and hps is not associated with the either the cause or severity of liver disease. in a fraction of patients with stable liver disease, there is progressive lung dysfunction. research shows that hps is associated with esophageal varices and spider angiomas. intrapulmonary vascular dilation (i.e., pulmonary spider angiomas) is frequently found in liver disease patients with subcutaneous spider angiomas susceptible to hypoxemia. spider angiomas have been regarded as a marker of extrahepatic involvement. if patients have no primary heart and lung disease, concomitant lung disease (such as chronic bronchitis, emphysema, pneumonia, and pleural effusion) may coexist with hps. affected patients usually have obvious respiratory symptoms; therefore, physicians should differentiate between the conditions. hps is an independent risk factor for prognosis. specifically, studies have reported that the median survival time is 10.6 months after the diagnosis of hps. to date, no effective strategies have been developed for the therapy of hps. orthotopic liver transplantation should be performed as early as possible for hps patients. in liver failure, there is massive hepatocyte necrosis that may cause a reduction in glycogenolysis and abnormal gluconeogenesis. thus, patients are vulnerable to hypoglycemia, shock, coma, and impaired glucose tolerance. the synthetic function of the liver is impaired in such patients, and the serum level of cholesterol and triglycerides decreases. serum cholesterol has been used as an indicator for the prediction of prognosis of liver failure patients. the frequent use of diuretics can cause water and electrolyte imbalance, of which hypokalemia and hyponatremia are the most common. such imbalances may also induce he and brain edema. ap is a rare, but severe, complication of liver failure. the inciting cause(s) and pathogenesis of ap in patients with viral hepatitis remain unclear but might be associated with viral infection, biliary tract lesions, drugs (steroids and diuretics), and other factors. the reported incidence is 0.2-3%, but one autopsy study shows that the incidence of ap is as high as 33% in patients with severe hepatitis and hepatic cirrhosis. evidence shows that the incidence of ap is relatively high in patients with advanced liver failure. further, high serum bilirubin, low albumin, and a significant reduction in prothrombin activity may predict a poor prognosis for ap patients with severe hepatitis and a high mortality. two of the following three criteria are required for the diagnosis of ap: (1) patients have abdominal pain characteristic of ap; (2) serum amylase and/or lipase is ≥3× the upper limit of normal; and (3) there are characteristics of ap on medical imaging. that said, in cases of severe hepatitis with concomitant ap, the symptoms of ap are usually atypical, diverse, and easy to be masked by symptoms of severe hepatitis. thus, severe hepatitis is often considered the cause of abdominal distension, nausea, and vomiting, even in the presence of ap. in some patients, ap may be misdiagnosed as spontaneous bacterial peritonitis, cholecystitis, or gastritis, which may delay treatment and therefore worsen the patient's condition. the clinical manifestations of ap are usually atypical in patients with preexisting liver failure, therefore, physicians should highlight the diagnosis of ap in affected patients. when the following findings are observed, ap should be suspected and laboratory and imaging examinations should be performed as soon as possible for the confirmed diagnosis: 1. patients with severe hepatitis develop abrupt and persistent upper abdominal pain/peritoneal irritation that is nonresponsive to general antispasmodics; 2. patients present with severe vomiting, severe sialorrhea of unknown cause, and refractory hiccups; 3. patients manifest repeated and transient episodes of conscious disturbance, which are refractory and not caused by hepatic coma and hypoglycemia-like reaction; 4. patients have prior chronic cholecystitis or gallstones, receive treatment with diuretics or steroids, and have symptoms and signs described in (1) after exclusion of spontaneous peritonitis. for patients with severe hepatitis, routine blood testing and urine amylase detection should be performed dynamically. imaging examinations can be performed simultaneously. abdominal ultrasonography may be performed within 24-48 h after the onset of abdominal pain, which is helpful for the morphological change in the pancreas and the exclusion of biliary tract disease. however, gas in the gastrointestinal tract during ap may affect the performance of ultrasonography and make accurate diagnosis of ap impossible. thus, computed tomography is recommended as a standard imaging examination for the diagnosis of ap. computed tomography is helpful for the early diagnosis and subsequently timely therapy, which may improve the prognosis. to facilitate the determination of therapeutic efficacy and the evaluation of prognosis, the branch of infectious and parasitic diseases and branch of hepatology of chinese medical association published the guideline for the prevention and therapy of viral hepatitis in 2000 (xi'an conference). on the basis of those guidelines, severe hepatitis can be classified as early, intermediate, and advanced severe hepatitis. specifically, early severe hepatitis meets the diagnostic criteria for severe hepatitis (i.e., severe fatigue, gastrointestinal symptoms, deepening jaundice, serum bilirubin >10 × the upper limit of normal, prothrombin activation of ≤30-40%, or pathological characteristics), but patients have no evidence of he and no ascites. intermediate severe hepatitis patients have grade 2 he or obvious ascites, bleeding tendency (i.e., bleeding point, ecchymosis, and a prothrombin activation of ≤20-30%). advanced severe hepatitis patients develop refractory complications and hrs, gastrointestinal bleeding, severe bleeding tendency (i.e., ecchymosis at the injection site), severe infection, refractory electrolyte imbalance, he >grade 2 brain edema, or a prothrombin activation of ≤20%. currently, some investigators classify the natural history of liver failure into the following: prejaundice stage, bilirubin increase stage, bilirubin plateau stage, and bilirubin reduction stage. those stages are based on disease progression, serum bilirubin level, and recovery of liver failure patients. in the prejaundice stage, patients have fatigue, anorexia, and an intolerance of oil. they deteriorate gradually, the urine becomes yellow, liver function detection usually shows a significant increase in aspartate aminotransferase and alanine aminotransferase (higher than several thousand), and the prothrombin activity increases. serum bilirubin increases progressively (i.e., a daily increment of >17.1 μmol/l), and symptoms (fatigue, anorexia) deteriorate after the appearance of jaundice (which is different than manifestations of acute jaundice hepatitis). when the serum bilirubin peaks and remains relatively stable, the disease may be in the bilirubin plateau stage in some patients with no severe complications but present improved mental status and appetite. with the regeneration of hepatocytes, the disease progresses into the bilirubin reduction stage, in which the coagulation, mental status, and appetite improve. when the disease recommences its deterioration, it may progress from the so-called bilirubin increase stage directly to the end stage. in patients with alf, the bilirubin plateau stage is not obvious, and patients might die shortly after disease onset. if patients survive alf, the disease may be pathologically classified as a hepatocyte edema type, and liver function will improve in a short period. not all types of liver failure (including severe hepatitis b) have clear stages based on their natural history and characteristics, and the respective features are discussed in detail as described in the following sections. there is still no consensus on the definition of alf. in 2005, the us acute liver failure study group published guidelines for the management of acute liver failure. in those guidelines, they emphasized that liver failure within 26 weeks after onset can be diagnosed with alf in mother to child transmission of hepatitis b infection (or autoimmune hepatitis), although it has the possibility of progressing into hepatic hepatitis. in addition, some physicians propose that liver failure with an abrupt attack either secondary to chronic hepatitis b or in the presence of other hepatitis virus infection can also be classified as alf. the pathological basis of alf may be classified as necrosis-and degeneration-dominant (acute edema) type. in alf of the necrosis-dominant type, hepatocytes become diffuse and massive necrosis occurs soon after disease onset. in alf of the degeneration-dominant type, hepatocytes show diffuse and severe swelling. alf secondary to acute hepatitis b virus (hbv) infection is rare in clinical practice. patients with alf secondary to acute hbv infection usually have no history of hbv infection, are relatively young, and often have predisposing factors (e.g., stress, absence of rest after disease onset, malnutrition, alcoholism, use of liver damaging drugs, pregnancy, concomitant infection). moreover, it usually progresses rapidly, and patients may develop coagulation dysfunction before the jaundice becomes evident. such patients present with symptoms of liver failure characterized by he >grade 2 within 2 weeks, a prothrombin activation ≤40%, an obvious bleeding tendency (i.e., massive petechiae at an injection site), patients have no ascites, disease progresses rapidly and has a poor prognosis, and patients frequently die of complications such as brain edema or cerebral hernia within 3 weeks. some patients may recover rapidly after appropriate therapy and are usually diagnosed with liver failure of extensive hepatocyte swelling. after recovery, the risk for hepatic cirrhosis is relatively low. another situation is the presence of a history of hbv infection in which patients have a good liver condition and no evidence of/mild liver lesions. for hbv patients with alf, the liver condition is good (as in alf patients without prior hbv infection) and both alf patients with and without prior hbv infection share pathological basis, pattern of disease onset, and clinical course. alf usually progresses rapidly, and the four stages of alf (i.e., prejaundice stage, bilirubin increase stage, bilirubin plateau stage, and bilirubin reduction stage) are difficult to identify. alf may result in high mortality, and a majority of patients directly develop alf of the bilirubin increase stage or even terminal stage. pathologically, slf not only has extensive hepatocyte necrosis but also an obvious inflammatory reaction and formation of regenerative nodules in residual hepatocytes. slf usually has an origin of alf. when slf occurs in patients with or without mild liver lesions, it often shows an abrupt onset. in the early stages, slf is similar to acute icteric hepatitis and patients progressively deteriorate. affected individuals may also develop clinical symptoms of liver failure from 15 days to 26 weeks, including severe fatigue, loss of appetite, frequent vomiting, and deepening jaundice (i.e., a daily increment of >17.1 μmol/l or > 1 mg/dl and an increase in serum bilirubin of >171 μmol/l or 10 mg/dl). patients usually have hepatic foetor, refractory abdominal distension, ascites (susceptible to concomitant peritonitis), evident bleeding tendencies, and mental and neurological symptoms. in the late stages, hepatorenal syndrome may be present and patients often develop complications (such as gastrointestinal bleeding and hepatic coma) before death. the liver either shrinks or remains normal in size. the course of slf lasts for several weeks to several months. patients surviving slf following therapy usually develop postnecrotic hepatic cirrhosis. clinically, slf can be divided into two types. first, the ascites type results in profound jaundice (serum bilirubin of ≥171 μmol/l or > 10 × the upper limit of normal), ascites, and evident bleeding tendencies (i.e., a pta ≤40%). he might be absent or present in the late stages. patients often die of hrs, upper gastrointestinal bleeding, severe secondary infection, and intracranial hemorrhage. slf of the ascites type accounts for a majority of slf. second is the encephalopathy type. such patients have he as an initial symptom and present manifestations as in ash except for course of disease lasting for >14 days. patients usually die from either brain edema or cerebral hernia. slf of the encephalopathy type is also not rare. slf often has an abrupt onset, and the four stages (i.e., the prejaundice, bilirubin increase, bilirubin plateau, and bilirubin reduction stage) of liver failure are difficult to identify. it is usually associated with a high mortality rate. the pathological basis of aclf is similar to that of slf; therefore, they both share clinical characteristics. a majority of patients with aclf have ascites, spontaneous peritonitis, and biliary tract infection. in the late stages, patients may develop portal hypertension and other complications, repetitive he and hrs, and most die of gastrointestinal bleeding and hrs. according to the guideline for the prevention and therapy of viral hepatitis (2000), a fraction of patients with csh meeting the diagnostic criteria can be grouped with aclf. that is, patients have either chronic hepatitis or compensated hepatitis cirrhosis that remain stable, but some predisposing factors cause the deterioration of liver function, which, thereafter, progresses to liver failure. aclf refers to acute decompensated liver function in the presence of chronic liver disease. the previously mentioned guidelines emphasize pre-existing chronic liver disease and liver failure due to acute liver dysfunction. it is important to note that controversy regarding the basis of chronic liver disease persists. in 2002, an english physician proposed that aclf was diagnosed in chronic liver disease patients with compensated liver function presenting with acute aggravation of liver function within 2-4 weeks due to accidents characterized by jaundice, he, and/or hrs. german physicians subsequently proposed that the diagnostic criteria for aclf included (1) the liver has the histological, laboratory, or ultrasound evidence of hepatic cirrhosis; and (2) patients develop jaundice, ascites, coagulation dysfunction, and/or grade 2-4 he, meeting the definition of decompensated liver function. the guideline for the diagnosis and therapy of liver failure (2006) does not detail chronic liver diseases as a basis of liver failure. however, in general, hbv carrier status may not serve as a baseline liver disease for patients with either chronic hepatitis or hepatic cirrhosis. the term aclf also highlights that acute or subacute deterioration of liver function occurs, which rapidly progresses to liver failure. patients often display an abrupt onset and develop severe fatigue and evident gastrointestinal symptoms. in the early stages, there is acute liver damage; therefore, patients usually present with a significant increase in transaminase levels. thereafter, the disease condition becomes aggravated, and patients may manifest symptoms of liver failure. aclf can also be divided into the brain type and ascites type, of which aclf of the brain type has a higher incidence. further, the four stages (prejaundice, bilirubin increase, bilirubin plateau, and bilirubin reduction) of liver failure are very clear in patients with aclf. one goal for physicians and researchers is to determine individualized therapy for aclf patients according to the specific stage of aclf. patients with clf usually have decompensated hepatic cirrhosis that progressively evolves into chronic liver failure, resulting in clinical manifestations of chronic decompensated liver dysfunction characterized by ascites, portal hypertension, coagulation dysfunction, and he. the pathological basis of chronic liver failure is hepatic cirrhosis, chronic and progressive aggravation of hepatocyte injury, and reduction in hepatocytes that are unable to maintain normal liver function. physical examination usually shows signs of chronic liver diseases (such as liver palms and spider angiomas), imaging examination shows characteristics of chronic liver diseases (such as spleen thickening), and laboratory examination also supports the diagnosis of chronic liver diseases (increased gamma-globulin and reduced/inverted albumin/globulin ratio). of note, a majority of patients have no clear history of liver disease and may initially be misdiagnosed with alf. further examinations may provide evidence of hepatic cirrhosis. when patients with hepatic cirrhosis become decompensated, the liver dysfunction usually presents with acute deterioration due to complications or gradually aggravates in a small fraction of patients. on the basis of the above findings, liver failure secondary to decompensated hepatic cirrhosis can be divided into slowly progressive liver failure and acutely deteriorating liver failure. the former shows a chronic status of liver failure and is characterized by repetitive ascites and he. the latter shows an acute deterioration of liver function in the presence of chronic liver dysfunction, which is similar to aclf in the disease onset and clinical course. hepatopulmonary syndrome is often noted in acutely deteriorated liver failure, and patients usually die of heavy gastrointestinal bleeding, hrs, and severe infection. clf is generally characterized by slow progression of liver failure, and the course of clf is relatively long. the four stages (prejaundice, bilirubin increase, bilirubin plateau, and bilirubin reduction) of liver failure are difficult to identify. as such, finding ways to best preserve residual hepatic function reserve is one of the important therapeutic goals in affected individuals. yu-ming wang, deng-ming he liver failure is a clinical syndrome with high mortality by severe liver damage. it is caused by a variety of causes, results in serious obstacles or decompensation of liver synthesis, detoxication, excretion and biotransformation and appears with coagulation disorders, jaundice, hepatic encephalopathy and ascites as main manifestation. hepatic failure can be divided into acute liver failure (alf), subacute liver failure (salf), acute-on-chronic liver failure (aclf), and chronic liver failure (clf). although the incidence of liver failure is not high in western countries, the relevant papers, reviews, conferences and other exchanges have increased markedly in recent years. aasld, easl and apasl had established a thematic seminar and the definition diagnosis and classification of liver failure has been consistent. at the same time, there are different understandings. therefore, it is necessary to discuss the main differences of liver failure diagnosis and classification, so as to develop a more rational diagnosis and classification scheme. the classification of liver failure involves classification of hepatic injury. a variety of factors (drugs, virus, alcohol, etc.) can cause liver cell damage. although course and prognosis of liver cell damage are different, the most common mechanism is inflammation. wieland et al. found that there were two mechanisms of liver cell injury in the immune clearance of hbv; non-soluble cell damage occurring early and soluble cell damage, early mainly non-soluble cell injury, by the study of gorillas with hbv infected. in 1994, bonino et al. proposed the theory of nonsoluble liver cell damage in the study of fibrosing cholestatic hepatitis (fch) study. however, this theory was ignored because many scholars believed that it ignored the background of immunosuppression. at that time, fch was still considered as the injury of endoplasmic reticulum and golgi apparatus by the excess replication of hbv as well as overexpression of hbv antigen during immune inhibition. however, in 2008, masayoshi et al. reported that most effective antibodies had been detected in children after living donor liver transplantation who received chickenpox vaccine, attenuated vaccines in children, such as measles, rubella, and mumps. according to this, immune suppression cannot stop antibody production and in fch, non-cellular immune injury may be present. recently, we found that there were two types of hbv reactivation in immune-suppressed; high alt type (>10 × uln) and low alt type (<5 × uln) and both with bad prognosis. we proposed that there may be different injury mechanisms, both immune and non-immune damage. in 2000, rolando et al. found that 56.8% of acute liver failure patients with systemic inflammatory response syndrome (sirs), and there was significant correlation between the progress of hepatic encephalopathy and infection and between the degree of hepatic encephalopathy and the occurred rate of infection. infection aggravates degree of illness and fatality rate in liver failure patients. accordingly, we speculate that serious primary liver injury can cause injury to other organs by cytokines, while injury to other organs can aggravate liver injury. corresponding with this, we summarized relevant literature and referred that alf can occur secondary in the development process of multiple organ failure induced by non-primary liver damage. this suggests that this type of liver failure is a special type of liver failure, which is a result of rapid changes in internal environment and inflammatory factors induce liver damage. therefore, liver failure can be divided into one with primary injury and with secondary injury. liver failure mainly caused by non-soluble cell injury is extremely rare; as "paralyzed type", "stunned type" or "edematous type", and with the good prognosis. in 2005, small-for-size syndrome after partial liver transplantation was defined by dahm et al., which is a hepatic failure due to less of liver tissue. actually, this syndrome can also be seen as a special type of liver failure caused by non-soluble liver cell damage. soluble cell injury of liver is relatively common, such as the early hepatic failure in hepatocellular carcinoma after interventional therapy, some drug-induced alf, etc. liver failure caused by immune injury is more common in liver failure caused by autoimmune liver disease. liver failure caused by non-immune injury is more common in liver failure patients with severe cellular immunity damaged (hiv/ aids patients, chemotherapy patients) or immunity inhibited (in patients after organ transplantation). primary type is common in fulminant hepatic failure (fhf), the secondary type is seen in severe systemic diseases, such as severe sepsis and acute hemorrhagic. in fact, clinical liver failure is the result of combination of different proportions of various types of damage factors. based on the role of various factors in liver failure, it can be categorized. according to acute and sustained, the clinical course and the image changes, liver failure can be divided into different stages with some or certain factors dominated. we have divided it into two categories, necrosis type and the decompensation type. although this classification is based on practice and clinical management, large-sample analysis is needed in the future. no matter what the cause, liver failure may be divided into two major categories on the pathophysiology. one type is necrosis induced by hepatic inflammation; the other type is decompensation of liver cells. in particular, alf and salf are types of necrosis, aclf and clf are decompensated type. mixed type, both necrosis type and decompensation type, is possible, which treatment should be considered. the relevance of these two type of liver failure mainly reflects in treatment. necrosis type mainly focuses on treatment of the cause (as antiviral treatment for hbv infection and corticosteroid treatment for autoimmune hepatitis) and symptomatic support treatment (as anti-inflammatory treatment and integrated symptomatic support treatment). decompensation type mainly focuses on intensive treatment (as control infection for sirs and control bleeding for gastrointestinal hemorrhage). some pathophysiological processes such as hepatic encephalopathy (he) are in both type, but are different in different liver failure type. for example, cerebral edema is more prominent and progressive and less to do with high protein diet in he patient of necrosis type of liver failure, while is just the opposite in decompensation of hepatic failure. based on pathologic features and speed of progression, liver failure can be divided into four categories: alf, salf, aclf, and clf (tables 1.3 and 1.4). alf occurs liver failure syndrome characterized with varying degrees of hepatic encephalopathy (9):643-6 (article in chinese) [1] in 26 weeks except for liver cirrhosis. salf occurs liver failure syndrome in 15 days to 26 weeks. aclf is acute hepatic decompensation on the basis of chronic liver disease. clf refers to chronic hepatic decompensation characterized by ascites, portal hypertension, coagulation disorders or hepatic encephalopathy based on cirrhosis. diagnosis and classification of liver failure are the most controversial part, but has tended to unify in recent years. in 2007, professor roger william from the university of london proposed the same type criteria as chinese. the only difference is limited to 8 weeks for alf and no aslf. due to aslf belonging to alf, this part is not contradictory for two criteria. in recent years, a discussion of aclf proposed many times by sarin from india made aclf more valued and accepted. clf is also getting more recognition. recently, to clarify meaning and avoid misunderstandings, it was recommended that clf be converted into end-stage liver failure (eslf). although issues related to classification of liver failure have largely agreed, there are some differences in practical application. dispute over whether to set up subtypes (namely aclf/saclf) of aclf occurred during drafting the new version of guide. two suggestions of modification are: (1) with existence of cld, clinical manifestation of acute (within 2 weeks) and sub-acute (2 ~ 26 weeks) liver function decompensation occur; (2) with existence of cld, clinical manifestation of acute (within 4 weeks) liver function decompensation occur (this actually is the original version). the reasons are: (1) the classification of 2006 guide has only been published for 6 years, and it was not easy for it to be widely recognized home and abroad. it requires more time to accumulate experience in this field, so frequent revision are inappropriate; (2) as is generally accepted home and abroad, aclf mostly occurs within 1 month (4 weeks) after onset, while those as late as 26 weeks after onset are rare (the document of our department indicates the same result); (3) it still requires medical evidence and extensive clinical summary and proof to have the new diagnostic term "saclf" in english and chinese established and accepted; (4) clinical significance and importance of salf classification are neither prominent nor urgent. after multiple discussions, diagnostic classification in the 2012 guide applied the latter one (table 1.5). despite the differences, academia has become unified about the classification of liver failure in the world. the differences towards the convergence of: (1) in terms of naming and classification. naming has simply become to acute liver failure (including acute and subacute) and clf (include acute-on-chronic and chronic decompensation), and tend to be more simplified. aasld guidelines clearly stated that nouns used to differentiate the length of the course (such as hyper acute, acute, and subacute) had claimed not to use. (2) in terms of clinical diagnosis. because of many cause of liver failure, it is very difficult to achieve unity. the only way is to combine the clinical diagnosis (such as acute hepatitis) and the pathophysiologic diagnosis (such as alf). (3) if hepatic encephalopathy as a prerequisite for liver failure. currently, it is a prerequisite for alf, and not necessary for clf because hepatic decompensation is the main clinical manifestations. according to the severity of the clinical manifestations, liver failure can be divided into early, middle and late stage. 1. extreme weakness, severe gastrointestinal symptoms such as significant loss of appetite, vomiting and abdominal distension; 2. progressive jaundice (serum total bilirubin ≥171 μ mol/l or increased by ≥17.1 μ mol/l daily); 3. bleeding tendency, 30% < prothrombin activity (pta) ≤40% (or 1.5 < inr ≤ 1.9); 4. no hepatic encephalopathy or other complications. based on the early stage of liver failure, further develop to one of the following two: 1. below grade ii hepatic encephalopathy and/or obvious ascites and infection. 2. obvious bleeding tendency (bleeding point or ecchymoses), and 20% < pta ≤ 30% (or 1.9 < inr ≤ 2.6). based on the middle stage of liver failure, further aggravating, severe bleeding tendency (such as ecchymoses on injection site), pta ≤ 20% (or inr ≥ 2.6), achieve one of the following four: hepatorenal syndrome, upper gastrointestinal bleeding, severe infection and above ii° hepatic encephalopathy. considering the notorious difficulty to treat hepatic failure and its high mortality rate, special attention has to be paid to and active treatment has to be performed on patients showing the following early-stage clinical features of hepatic failure. 1. extreme weakness, severe gastrointestinal symptoms such as significant loss of appetite, vomiting and abdominal distension. 2. progressive jaundice (51 μ mol/l ≤ t.bil ≤ 171 μ mol/l), and increased by ≥17.1 μ mol/l daily; 3. bleeding tendency, 40% < prothrombin activity (pta) ≤50% (or 1.5 < inr ≤ 1.6). clinical practice has shown that, with or without history of chronic liver disease, there are patients with grade ii hepatic encephalopathy in a short period, with rapid development, poor prognosis. these patients should be regarded as fulminant type. meanwhile, in asia, including china, there are some patients with severe jaundice, ascites and bleeding as the main presentation, with relatively slow development and very poor prognosis, but without hepatic encephalopathy. these patients should be classified as subacute type. fulminant type must have a hepatic encephalopathy. however, it is not necessary for subacute type, which mainly characterized by severe jaundice and ascites. compared with severe hepatitis in china, fulminant type amounts to acute severe hepatitis and chronic severe hepatitis with acute onset, subacute type amounts to subacute severe hepatitis and chronic severe hepatitis with subacute onset. currently, a large divergence of these two types is about time, from 10 days to 8 weeks. according to clinical features of alf, alf can be further divided into fulminant type and subacute type interval for 4 weeks. however, according to more researches, fulminant hepatitis, characterized by massive necrosis of liver, brain edema, and hepatic encephalopathy, concentrated in 2 weeks, most of them in 10 days or less. taking into account the subacute type belongs to acute category, subacute are not be established in international classification. alf are defined as liver failure in 26 weeks. on the difference between acute and chronic liver failure, most chinese scholars depend on the past history, which be ignored internationally. the difference lies in this onset. acute inflammation, necrosis and chronic decompensated were classified as acute and chronic processes, respectively. most typical example is that patients with acute heavy syndrome of onset in hbv carrier were divided into alf by the scholars from hong kong, macao and taiwan. similarly, liver failure caused by hepatitis flares in chronic hbv carriers, reactivation of chronic hepatitis b, super infection with hdv and hbeag seroconversion are included in alf. it is inconsistent with classification methods in china. the reason lies in greater emphasis on the continuous development processes of hepatitis chronicity and severity in china, and focused on the acute effects this time abroad. some scholars suggest that considering the significant difference between clf and the other three types in clinical manifestation, it is worth discussing whether to list clf as a type of hepatic failure. we believe that significance and importance of clf classification are: (1) clf are similar to crf (uremia) in nephrology and chronic cardiac failure (congestive heart-failure) in cardiology. although their clinical manifestation differ significantly, the "coexistence of acute and chronic failures" is shared by failures of all those organs; (2) clf classification has been generally recognized at home and abroad, and the necessity of classification are further proved by the difference between clf and the other three types; (3) clf cases are relatively large in proportion (nearly 30%), which is still increasing (since the proportion of alf/salf are lowering); (4) complications of clf are common and are found in various forms, with bad prognosis; (5) in clf patients with correlation to hbv, virus replication are commonly found, which is closely related to decompensation. the efficacy of nucs are satisfying, which, if taken for a long term, can reverse decompensation, avoiding liver transplantation; it also increases support means in a fast rate, creating more chances for treatment. if the strict definition of acute and subacute liver failure as "no past history of liver disease" is executed, how to name the patients who had a history of chronic liver disease (caused by hbv from mother to child transmission in china)? as for alf and salf, rigorous definition for the past history of liver disease (including hbv carrying history) is necessary in china, and more interested is in this attack instead of the latent infection in the past, even a dominant attack in europe and america. in clinical practice, past history should not be ignored, because patterns of chronic hepatitis b reactivation vary. we summarize them into four types: (1) burst type: suddenly attack based on immune tolerance state, eventual liver failure; (2) recurrent type: repeated unequal flares, finally developing into liver failure; (3) occult type: no obvious attack, presenting with symptoms of decompensation; (4) document type: compensated cirrhosis, acute decompensation in certain situations (mainly due to sepsis and other infections). in burst type absence of history, or only carrier state, the past history can be ignored. in recurrent type, history of recurrent injury is important, which the significance lies in the extent of the occurrence, duration and consequences. because these factors determine the basis of injury to the patient's liver, the patients with mild liver disease have mild or even have no hepatic fibrosis and liver cirrhosis, otherwise, the symptoms will be serious and obvious. the former attack often leads to liver necrosis, the latter often lead to decompensation. the difference between occult type and document type is in the speed of decompensation; the former is slow and the latter is fast. in summary, although history has the certain reference value, pathophysiological changes in attack is main of necrosis or decompensation, or a combination of both. whether hepatic encephalopathy should be considered as a complication of liver failure is controversial, because many scholars have listed it as a prerequisite for liver failure, but in recent years, some patients do not necessarily have encephalopathy. from the complete course and early prevention and treatment of liver failure, it is necessary to incorporate non-encephalopathy type, but the effect and prognosis of the rescue treatment should be divided into the encephalopathy type and nonencephalopathy type, because they are different. hepatic encephalopathy is divided into a, b, c type in international guidelines. type a is acute hepatic encephalopathy (alfa-he), which does not include acute hepatic encephalopathy associated with chronic liver disease. some patients with a long-term hbv carrier were diagnosed with aclf or clf on the first time severe, especially in china. in fact, this type of patients with acute or subacute liver necrosis caused by alf. the mechanism of hepatic encephalopathy in this type liver failure is different from alfa-he, as well as treatment. liver failure classification has the greatest impact on treatment of hepatic encephalopathy, based on the following facts: (1) in acute phase of alf, fasting protein diet on the first day, unnecessary in the short term (4 days); however, chronic hepatic encephalopathy of clf don't have to fast; (2) the metabolism of branched chain amino acids (bcaa) in alf was reduced and increased in clf. this suggests that the former should not be added bcaa, and the latter can supplement the bcaa. (3) high blood ammonia in clf is more than in alf. the effect of clf was better than that of alf patients, but the effect was not good for deamination drugs. (4) because cerebral edema in alf is more than in clf, it is better to reduce the intracranial pressure in alf treatment, and in clf with poor efficacy; (5) as for type a and c according to the international consensus of hepatic encephalopathy are equivalent to the current alf and clf. severe cerebral edema has been found in alf and its mechanism is not clear. study on cerebral edema treatment have been found that hypothermia therapy can reduce the cerebral blood flow and brain edema. in 46th easl, larsen from affiliated hospital of university of copenhagen in denmark reported that therapeutic hypothermia did not support application in the treatment of patients with alf in a prospective, multicenter randomized controlled experimental study (the study was carried out in 2004-2010). the study results are different from previous studies. we believe that the reason is likely from the bias of group selection. in addition, there is a great difference of the mechanism of hepatic encephalopathy in patients with chronic hepatic failure and alf, so the response to hypothermia therapy will vary greatly. based on the existing research, hypothermia therapy has better effect on hepatic encephalopathy in alf patients caused by cerebral edema, and has bad effect on hepatic encephalopathy in the chronic decompensated liver failure caused by metabolic abnormalities. as for hepatic encephalopathy in aclf patients, the effect depends on the roles of cerebral edema in pathogenesis. therefore, it is recommended that the patients should be carefully screened to obtain a comparable result in the study of the hypothermia therapy in hepatic encephalopathy. we believe that, with the gradual elucidation of the pathogenesis of liver failure, the treatment measures will also be more targeted, the efficiency may be further improved. glucocorticoids (gcs) therapy in chronic active hepatitis b began in the 1960s to 1970s. however, compared with the control group, gcs did not show better effect. there have been reports that, after stopping the treatment of immunosuppressive therapy, early re-given long-term high-dose gcs can prevent severe hepatitis in hbv reactivation patients. however, this result had not been affirmed in the future clinical practice. although more application of gcs before 1980s, each effect is different. there is a negative trend in 1990s. we have analyzed, the effect may involve two major factors: one is the choice of indications, dose and duration of treatment; another is the prevention of adverse reactions and complications of gcs. according to the guidelines for diagnosis and treatment of liver failure (2006) in china, liver failure without viral infections, such as autoimmune hepatitis and acute alcoholism (severe alcoholic liver disease), etc. are gcs indications. at the early stage of liver failure caused by other reasons, in the patients with developed rapidly and no serious infection, bleeding and other complications, gcs may be appropriate to use. gcs can improve the survival rate of patients with autoimmune hepatitis and severe alcoholic hepatitis, and has been recognized by most scholars. for hepatitis flares in chb patients, if on the basis of combined application of nucs, gcs will inhibit excessive hyperactivity of host cellular immunity and excessive release of cytokines, and help preventing liver cell death. at the same time, the role of gcs in the treatment of chb and the specific usage, as well as the effect of combined treatment is still controversial. the reason is that the advantages and disadvantages of gcs are very prominent. the key of success or failure lies in the clinical skills. in recent years, the reports of gcs for the treatment of hbv related liver failure increased. there are three main reasons: (1) nucs can effectively resist hbv replication due to gcs; (2) application of proton pump inhibitors can effectively prevent gastrointestinal bleeding due to gcs; (3) increasing of infection prevention and treatment can effectively fight infection due to gcs. even so, the above three aspects of the problem have not been satisfactorily resolved. therefore, we put forward several viewpoints on the current application of gcs: first, to fully analyze the advantages and disadvantages, to consider the main function and purpose after the gcs application and the risk of adverse events before expanding the indications of gcs. secondly, both short course treatment (3-5 days) and long course treatment has drawbacks. the former may not be sufficient to adequately inhibit a strong immune response, and induce a stronger immune response after a sudden stop; the latter can induce bleeding, infection or viral resistance. finally, in a common clinical liver failure induced by hbv reactivation under immunosuppression, the mechanism is often unrelated with immune activation. the typical representative is fch, and the prognosis is extremely bad. it should be vigilant, focus on prevention. our department has treated a chronic hepatitis c (chc) patients after kidney transplant and taking large doses of gcs. severe fch was induced, resulting in liver failure and eventually died. in recent years, the hepatic stem cell therapy of liver failure was concerned, human stem cell transplantation in the treatment of clinical study on severe hepatitis/liver failure were carried out and the results were satisfactory. however, due to the difference of the patient's condition or stage, and most of which are case report, it is difficult to draw an objective conclusion. we have repeatedly reported that the human umbilical cord blood stem cells and bone marrow stem cells in vitro and in vivo can be successfully transformed into liver cells, but the biggest obstacle to its application to clinical practice is the limited number. it is difficult to repopulate the whole liver or at least part of liver compensatory function. in addition, considering the part liver failure patients with cirrhosis background, which clinical manifestations as aclf and clf in the practical application, there are the following issues: (1) it is difficult to provide effective growth and functional support for implanted cells in a diseased liver; (2) the original portal hypertension can be aggravated by portal vein implanted cells; (3) input cells by vein can obstruct the pulmonary vein, thus affect the pulmonary circulation; (4) if the implanted cells for allogeneic or xenogeneic tissue, may have compatibility problems; (5) when using gcs to prevent rejection, severe hepatitis patients are easy to infection and other related adverse reactions. finally, it is pointed out that there is a tendency of the academic circles to the understanding of liver failure for decades, that is, the existence of the chronic process is often neglected in the process of analysis, and vice versa. a typical example, until nearly a few years ago, many scholars in the world still do not recognize the existence of aclf and clf. chinese research papers on liver failure published very little in the international, the main reason is that the aclf and clf is not clearly defined. the papers are often rejected because the diagnosis of type does not conform to the international. in contrast, as previously mentioned in the definition of aki and hrs, the existence of alf (including salf) is ignored. another example: in the report of antiviral treatment of hbv related liver failure, some foreign scholars put forward that the liver failure should be changed to the decompensated liver cirrhosis, the reason is that acute hbv infection is self-limited. however, at present, there has been a clinical recognition that the hbv related alf is actually an acute episode of chronic carrying process and should be referred to as aclf. in chinese guidelines for the diagnosis and treatment of liver failure (2006), liver failure was divided into four types, mainly because of their different mechanisms and treatment (sometimes even the opposite). due to limitation of the space and data, we have not discussed infection, bleeding and other complications in relation to liver failure. these pathological processes are closely related to the classification, and should be studied further in relation to liver failure. in future, we should develop classification of liver failure according to the mechanism of liver injury with different causes, and provide the basis for clinical types of hepatic failure. in order to improve the level of diagnosis and treatment of liver failure, the mechanism based classification should be carefully assessed and evaluated. standard' of definite diagnosis, severity evaluation and treatment effect assessment, and it is irreplaceable compared with other examinations. histopathological evaluation of aechb and liver failure not only contribute to the definite diagnosis of severity of aechb and provide pathological evidence for effective clinical treatment of hepatitis b, but also is helpful to the early detection of histopathological proof of aechb by pathological examinations and of pre-warning function for clinical treatment of aechb. this section mainly focuses on pathological features of aechb and other types of liver failure. aechb, clinicopathological manifestation of aggravation of liver necroinflammation and disease deterioration of chronic hepatitis b, tends to be of poor prognosis without positive and effective intervention. its pathological characteristics mainly include: ballooning degeneration of diffuse hepatocytes, significantly increased focal necrosis, confluent necrosis, bridging necrosis, extensive and intensive interface hepatitis, massive or submassive necrosis, many neutrophil infiltrations in hepatic lobules and portal areas and moderate intrahepatic cholestasis. prominent hepatocyte necrosis is the pathological foundation of aechb and manifests extensive multifocal necrosis, confluent necrosis, bridging necrosis and other forms of necrocytosis. severe ones can even occur submassive and massive necrosis leading to the extreme form of aechb, severe hepatitis (liver failure). ballooning degeneration of diffuse hepatocytes is one of pathological characteristics of aechb ( fig. 1.1 ). the hepatocytes manifest sparse and granular cytoplasm, sometimes can be micro-bubble like. the degenerated hepatocyte is 2-4 times bigger than the normal hepatocyte. sometimes the ballooning degenerated hepatocytes can fuse and transform into multinucleated cells, and this lesion is similar to that of neonatal giant cell hepatitis when it is relatively extensive. the general performance of the liver is increased volume, tense capsular and cutting edge eversion due to tension. ballooning degeneration of hepatocyte is not specific histological manifestation of hepatitis band also can occur in liver tissues of hepatitis caused by factors such as alcohol or drugs. extensive and diffuse ballooning degeneration of hepatocyte can make the hepatocytic plate wider, and hepatic sinusoid is pressed to be narrower, causing microcirculation disorder of liver tissue and exacerbation of disease. liver lobular inflammation activity is enhanced, and apoptotic bodies and focal necrosis increased significantly when aechb occurs. hepatocyte apoptosis is the programmed necrosis and one of the major forms of hepatocyte death in hbv infection. histopathology manifests cell membrane shrinkage, deepened cytoplasmic staining, eosinophilic degeneration. free apoptotic bodies in liver sinus are large or the apoptotic cell fragments, sometimes containing nuclear fragments. focal necrosis is another form of liver cell necrosis and manifests as an interruption of the hepatocytic cords or replacement by focal lymphocytes and macrophages, with hepatic regeneration that often causes irregular arrangement of hepatocytic cords. this necrosis often inferred from the disappearance of the hepatocytes and the infiltration of inflammatory cells rather than what is actually seen under a microscope. swelling hepatocytes presented increased size and loosing cytoplasm, further develop to ballooning degeneration showed almost spherical in size and transparent cytoplasm, predominantly in the lower right confluent necrosis and bridging necrosis are the common histopathological changes, play important roles in the progression of aechb and are closely related to the adverse prognosis of hepatitis b. due to the larger necrosis range of confluent necrosis and bridging necrosis, even during repair stage after going through the active stage, the liver tissue often undergoes fibrous repair, resulting in liver fibrosis and hepatic lobule reconstruction, thereby causing liver cirrhosis. statistics show that about 18% of the patients with viral hepatitis who had bridging necrosis progress to cirrhosis. confluent necrosis is regional lytic necrosis of hepatocytes on a larger scale and is common in active stage or aggravation of viral hepatitis, or drug-induced liver injury, which often occurs around the central veins and inflammatory cell infiltration is not obvious. specific confluent necrosis can also be seen in other parts. take ferrous sulfate poisoning for example, confluent necrosis is more common in zone 1 of liver acinus, and when confluent necrosis expands to connecting vascular or portal area, bridging necrosis occurs ( fig. 1.2 ). bridging necrosis is large area hepatic lytic necrosis that connect the portal area to central area (p-c), portal area to portal area (p-p), and central area to central area (c-c). it can be caused by the expansion and confluence of interface hepatitis, or a one-time large-scale translocular necrosis. p-c necrosis: it starts on the periphery of the lobules, affects the central hepatic lobules when it expands, and forms bridging necrosis phenomenon. the currently acknowledged mechanism is as follows: the initial pathological change is serious periphery necrosis of hepatic lobules. with the aggravation of the disease, microcirculatory disturbance occurs in the lobules and causes the hypoxia, degeneration and necrosis of central area liver cells. p-p bridging necrosis: most scholars think it is caused by the expansion of interface inflammation, especially based on the fibrous septum, hbv load increasing significantly, immune response enhancing, activating the signal pathway of liver cell death. with the enhancement of the lesion activity, fresh and severe necrosis occurs. c-c bridging necrosis: it is usually seen in serious disease with bridging necrosis of hepatocytes, but mononuclear cells infiltration is rarely seen in the necrosis area and serum transaminase increases significantly (up to 1000 iu/l above). histological manifestations of bridging necrosis can vary due to the different stages of the disease. in the early stage, the liver parenchymal cells necrotize and then disappears, reticular framework residue accompanied by the infiltration of lymphocytes and macrophages. with the time extending, reticular framework collapses and forms the sparse interval crossing the liver tissues. when bridging necrosis is accompanied with reticular framework collapse, with hepatic necrosis and regeneration, disorder of hepatic lobules occurs. at this time, it is difficult to distinguish the fibrous septa between bridging necrosis and chronic hepatitis, and elastic fiber staining can help to solve this problem. the elastic fiber staining of bridging necrosis is negative, because elastic fiber formation often takes several months. interface hepatitis, formerly known as piecemeal necrosis, is one of the symbolic histological manifestations in the chronic activity of chronic hepatitis b. it mainly refers to single or small clusters of liver cells around the portal areas necrose and shedding, leading to worm-eaten defect of limiting plates. significant lymphocytes infiltration is commonly seen in and around the portal area. mononuclear cells extend to the hepatic lobules along the destructive limiting plates and encase the necrotic liver cells, resulting in the enlargement of portal areas ( fig. 1.3) . interface hepatitis increases significantly and extensive interface hepatitis occurs in aechb. the interface hepatitis area can exceed 50% of portal areas periphery and be more than a third of the depth of hepatic lobules, even causing bridging necrosis (p-p and p-c). because the limiting plate of the lobules is an important structure to maintain a whole hepatic lobules, interface hepatitis destroys the integrity of hepatic lobule structure. extensive and intensive interface hepatitis can often cause bridging necrosis and bridging fibrosis, and is an essential part of poor prognosis of aechb. massive necrosis and sub-massive necrosis are considered to be the basic pathological changes of severe hepatitis (liver failure) and major substratum for histologic diagnosis of liver failure. massive necrosis is the diffuse lytic necrosis of the liver parenchyma involving more than 2/3 of hepatic lobules. thorough and rapid liver tissue necrosis is shown with invisible necrosis process, and only reticular framework remained and is filled with red blood cells ( fig. 1.4) . sub-massive necrosis is diffuse liquefaction necrosis of the liver parenchyma involving 1/2-2/3 of hepatic lobules. reticular framework collapses and forms reticular fiber bundles, residual liver cells and bile ducts proliferate. massive necrosis and sub-massive necrosis will seriously affect the prognosis of patients with high fatality rate once they occur. the cause of massive necrosis and sub-massive necrosis remains unclear, and the possible causes include excessive virus replication, virus mutation, overlapping with other virus infection and microcirculation, etc. when extensive confluent necrosis, massive necrosis and sub-massive necrosis involve the entire hepatic lobule and even several adjacent hepatic lobule, causing a lobular or adjacent several lobular hepatocytes lytic necrosis, then the panacinar or multiacinar necrosis occur, which is the most severe form of necrosis of aechb. in panacinar and multi-acinar necrosis, with large range of liver cell necrosis, only a small amount of liver cells remain. residues of clump, rosetting, island or glands-like arrayed liver cells are commonly seen around the collapsed reticular framework after necrosis or loose fibrous connective tissue. due to distortion, normal structure cannot be recognized and sometimes can only be identified by portal area range around the necrotic area. cells proliferation can be observed in periportal area, arraying like bile duct structure and these cells can express hallmarks of hepatocyte and bile duct epithelial cells at the same time, which is considered to be the histological manifestation of liver stem cells (hepatic stem cell) activation and proliferation. infiltrating inflammatory cell types tend to be multiple, and the quantity varies. when there is less infiltrating inflammatory cells, the main cell type is macrophage, often containing brown pigment particles. notably, the liver biopsy might be error in diagnosing the necrosis of wide range of diffuse dissolved necrosis of liver parenchyma (above 2/3 lobule), with only the mesh stents remained the whole lobules and multi-lobules, and that is due to limited amount of liver biopsy specimens. for example, multi-acini necrosis occurs only in the area under the liver capsule, and liver biopsy pathological examination may overestimate the severity of illness. in aechb, types of infiltration inflammatory cells in the liver tissue are various, and most of them contain many neutrophils. it is different from obvious lymphocyte accumulation within liver parenchyma and portal area common seen in hepatitis b. usually, cd8 + t cells/cytotoxic t lymphocytes (ctls) are the major effector cells of the inflammatory response in hepatitis b. but when exacerbation occurs, the neutrophils in inflammatory cells infiltrating liver acinus and portal area increase significantly. neutrophils play an important role in innate immunity, and are the first inflammatory cells migrating to the lesion during inflammatory response. these neutrophils kill the invading pathogenic microorganisms through releasing protease and anti-microbial proteins, and producing reactive oxygen. meanwhile, neutrophils have important functions in activation and regulation of innate immunity reaction and adaptive immunity reaction and could release cytokines such as il-8 to participate in regulating adaptive immunity reaction. although cytokines produced by neutrophils are less than mononuclear macrophages, since neutrophils are the first inflammatory cell moving to inflammatory lesion, the immune regulator function of neutrophils is more important during the early or acute stage of immunity reaction. evidently increased neutrophils in liver tissues of aechb might be beneficial for eliminating infected cells, but it is also a "double-edged sword". accumulation of neutrophils may cause extreme immune response and excessive inflammatory reaction might lead to deterioration. therefore, when increasing neutrophils appear in liver tissues of hepatitis b patients, more attention should be paid to identify whether virus mutation, overlap infection, drug-induced liver injury occur and cause aechb. intrahepatic cholestasis (cholestasis), especially moderate or severe cholestasis, is one of the common histologic manifestations of aechb. intrahepatic cholestasis takes shape from the bile thrombus within the cholangioli around the central vein which is hard to be identified, forming cholestasis in expansive interlobular bile duct and large "bile lake" in hepatic tissue ( fig. 1.5) . microscopically, bile can be characterized by dark brown, green or yellow color, occasionally also can present the gray which is difficult to recognize. bilirubin is revealed as green in van gieson staining, which is helpful to pathological diagnosis of intrahepatic cholestasis. moderate and severe intrahepatic cholestasis often cause feather-like degeneration of hepatocytes, even intrahepatic cholestasis infarction. with the extension of duration of intrahepatic cholestasis, the inter-hepatocyte structure relation of 2-3 normal liver cells surrounding the capillary bile duct also changes. the number of liver cells around the bile duct increases, and the bile canaliculi expands, causing the cholestasis related rosette structure forms and the fusing multi-nucleus giant hepatocyte appears. kupffer cells with brown bile pigment can be seen in the hepatic sinusoid. remarkably, although intrahepatic cholestasis is often associated with clinical symptoms such as jaundice and risen serum bilirubin, the severity of intrahepatic cholestasis is not consistent with clinical symptoms and serum bilirubin level. severe hepatitis b (liver failure) is the most severe liver syndrome complex, which is characterized by poor clinical course and high mortality rate. over the years, scholars have continued to explore the definition, classification, diagnosis and treatment of liver failure, but so far no consensus has been reached. according to histopathological features and progression of the disease, china released guidelines on diagnosis and treatment of liver failure in 2006, which divided liver failure into four categories: acute liver failure (alf), sub-acute liver failure (salf), acute-onchronic liver failure (aclf) and chronic liver failure (clf). hbv infection is the most common cause of liver failure in china. acute liver failure is characterized by acute onset, and hepatic encephalopathy (above stage ii) often develops within 2 weeks of the onset, which results in high mortality rate. former knowledge about acute severe hepatitis mostly comes from the autopsy. nowadays, with the development and universal application of biopsy technique, and the further study on acute liver failure, we have better understanding of the development and process of necrotic lesions, and can predict prognosis and outcome according to the necrotic area and type. in alf, liver atrophy is significantly present in gross pathology inspection, especially for the left lobe. coverings shrinkage, thin edge, soft liver texture, section may be yellow or reddish-brown, and some area is red alternating with yellow, and the weight of the liver drops sharply to 600-700 g. histopathology emphasizes extensive and consistent liver cell necrosis caused by one-time strike, and most patients die in the short term. the morphology of liver tissues is relatively simple, manifested as massive or sub-massive necrosis of liver cells, dissociation of liver cords and hepatolysis. the regeneration of liver cells is not obvious, and surviving liver cells show clear ballooning degeneration. hepatic sinus expand and, congest with blood and occur hemorrhage. kupffer cell proliferates and sinusoidal mesh stent does not collapse or completely collapse. quantity of liver cell necrosis is closely correlated with prognosis. if the amount of necrosis is over 70%, mostly the patient will not survive. if the amount is less than 50%, the patient is expected to resume with rapid regeneration of hepatocytes. if there is diffuse small steatosis, the prognosis is often poor. concerning the hepatic regeneration of acute liver failure, former pathology emphasizes on liver cell and bile duct cell proliferation of sub-acute liver failure, and relatively neglects cell regeneration of acute liver failure. based on the authors' knowledge, in some acute liver failure cases, liver tissue demonstrates obvious bile duct-like or acinar-like regeneration within 4 days after onset. the regenerated liver cells were co-expressing albumin and ck18, ck19, indicating these cells have double markers of hepatocytes and biliary epithelial cells, which presumably come from liver pluripotent stem cells. the regenerated liver cells in acute liver failure have their unique characteristics that degenerated and regenerated cells co-exist in the liver. as the time of liver biopsy is different, the morphological change is also varied because of the rapid restoration. the usual dual-core, large nuclear or nuclear fission are rarely seen in regenerated liver cells; liver cell body swelling, transparency of cytoplasmic periphery and slightly basophilic center are commonly seen in regenerated liver cells. because of cell enlargement and transparent cytoplasm, it is often difficult to distinguish from serious ballooning degeneration; in some cases, it shows bile granules within the cytoplasm, bile thrombus within bile capillary with cell swelling and transparency symptoms, which is also similar to the feather-like degeneration caused by bile salt siltation. however, unlike feather-like degeneration which was scattered by small groups or disorderly arranged severe ballooning degeneration, cell enlargement often shows the pole adenoid arrangement, which is known as a sign of liver cell proliferation. the outcomes of continuous liver biopsy also prove its rapid regeneration. nayak et al. also proposed that hepatocyte swelling during the acute liver failure recovery stage indicates good prognosis. the continuous liver biopsy of 12 liver transplant centers in europe also confirmed that the appearance of liver cells enlargement after 12 days of partial liver transplant of acute liver failure with massive necrosis, cells linked to sheets, and lobular structure are basically recovered in 2 to 3 months. the vacuolation, cholestasis and duct-like structure presented by these regenerated enlarged liver cells will be gradually disappeared. the onset of salf is acute, and liver failure syndromes appear within 15 days to 26 weeks, mostly caused by delay of acute liver failure. in salf, liver atrophy is mainly presented as in gross inspection, and variable sizes of regenerative nodules, the yellow-green cutting surface due to cholestasis. histopathology manifests the new and old sub-massive necrosis of liver tissues, or bridging necrosis. in older necrosis area, reticular fibers collapse, or collagen deposit. survived liver cells may have varied degrees of regeneration, and are arranged in nodular. fine, small bile duct proliferation and cholestasis are commonly seen in the periphery lobe. the sinusoids congest in the early stage, collapse in mid-stage, and occlude in late stage. the histopathological distinction of salf and alf is based on the consistency of necrotic lesions. alf emphasizes consistency of necrotic lesions, that is, 'onetime strike', while the necrotic lesions of salf are mixed, caused by 'multiple attack'. in addition, differences also exist in aspects such as cell regeneration and extracellular matrix (ecm) expression between alf and salf. liver stem cells play an important role in the liver cell regeneration process of alf. the regenerated liver cells express dual markers of liver cell and bile duct cell, and are often orderly proliferated along mesh stents. whereas salf presents unipolar regeneration of liver cell and bile duct cell, and the regenerated liver cells is disorderly arranged. due to the different length of disease course, there is no obvious ecm deposit in alf, while salf presents in iii collagen-based ecm deposit. this type of liver failure often develops into post-necrotic cirrhosis. acute-on-chronic liver failure (aclf) refers to acute liver function decompensation occurring on the basis of chronic liver disease. liver gross manifestation of acute-on-chronic liver failure differs with the different stages of chronic liver disease. for instance, aclf occurring in the stage of cirrhosis is accompanied by hepatic cirrhosis nodules besides liver atrophy. the main histology of aclf is new and varied degrees of liver cells necrosis, hepatocyte focal and spotty necrosis, bridging necrosis, confluent necrosis, massive necrosis and sub-massive necrosis on the basis of chronic liver injury. the common chronic changes are as follows: fibrosis in collapsed reticular framework or periportal area with obvious extracellular matrix deposit, forming of large number of fibrous septa, sparse scars, or bridging fibrotic septa when distortion of lobule structures associated with disproportionate numbers of central veins and portal area, pseudolobule formed; twin-cell or multiple-cell of liver plate is commonly seen and the liver plate lose the radiated array, activated regeneration of liver cell caused the occurrence of tumor-like cell. chronic liver failure is chronic liver function decompensation caused by progressive deterioration of liver function on the basis of liver cirrhosis, with ascites, portal hypertension, blood coagulation dysfunction and hepatic encephalopathy as main symptoms. liver gross appearance of clf is significant liver atrophy and nodular liver cirrhosis. histopathological changes are mainly those of liver cirrhosis, including diffuse liver fibrosis, nodular liver cirrhosis with unevenly distributed liver cells necrosis. progression of hepatitis b is an interaction between hbv infection and body response. development of aechb is mainly caused by obvious increased viral load and/or decreased immune clearance. large number of hbv replication can activate hepatocyte death pathways, leading to serious liver inflammation, necrosis and aggregation of disease. additionally, hbv infection overlapping with hcv/hiv, or with etiological factors like drugs and ethanol, could also affect disease progression. fibrosing cholestatic hepatitis (fch), a new clinicopathological type, develops in stages of severe immunosuppression caused by various reasons, especially in hepatitis virus-infected patients lots of immunosuppressant after organ transplant. due to immunosuppressor, hbv replicates rapidly in the patients, leading to quick progression of hepatitis and progressive failure of liver function. the histopathological features of fch are as follows: fibrosis straps starching from the portal area to hepatic sinusoid and circumvoluting basal plates of biliary epithelial; obvious intrahepatic and hepatocytes cholestasis, bile embolism forms in small bile duct; hepatocytes ballooning degeneration with disappearance of cells; mass ground-glass hepatocyte; mild to moderate mixed inflammatory reaction ( fig. 1.6) . fch could quickly proceed to liver failure with blood coagulation dysfunction and hepatic encephalopathy, and mostly die in several weeks to months. due to common transmission, coinfection with hbv and hcv or hiv is not rare clinically. 5-20% of the chronic hbv infected patients carry hcv antibody and there are 7-20 million coinfectious patients all over the world. studies showed that hbv and notable intrahepatic cholestasis (black arrow) and ballooning degeneration and/or feathery degeneration (red arrow), a large number of ground glass hepatocytes (blue arrow), associated with inflammatory response (green arrow) hcv coinfection could promote the synthesis of collagen and promote disease progression to liver fibrosis. compared to the single hbv infection, hbv and hcv coinfection presents more severe liver fibrosis and inflammatory necrosis. studies demonstrated that hbv and hcv coinfection could promote chb progression, cause severe damage of the liver function and then exacerbation, increasing the probability of liver fibrosis, liver cancer and liver failure in chb patients. in hbv and hiv coinfection, hiv infection can affect the natural history of hbv and accelerate the development to end-stage liver disease and liver cirrhosis. immune deficiency induced by hiv infection fosters hbv replication, and even fibrotic cholestasis hepatitis in severe cases. histologically, chb caused by hbv and hiv coinfection had a severer fibrosis than that by simple hbv infection. cases of hepatitis b overlapping drug or alcohol induced liver damage are not rare. even antiviral drugs can cause aechb, and there is previous case report on hepatitis b patient died of acute liver failure induced by anti-hepatitis b virus medication lamivudine. abuse or nonstandard drug use and alcoholism have become the common causes of aechb. pathology manifests features of overlapping drug or alcohol induced liver injury on the basis of hepatitis b changes. for instance, in aechb caused by overlapping drug-induced liver injury, liver tissues present histological characteristics of hepatitis b accompanied with drug-induced liver injury, such as evident increased percentages of infiltrated eosinophils and neutrophils in liver tissues, confluent necrosis with less inflammatory cell infiltration in acinus three area, cholestasis of bile canaliculi and so on. in summary, aechb has its relative histopathological features. understanding of these pathological characteristics can not only help with clinical diagnosis and effective treatment, but also aid to prevent aechb. it is important to note that despite the value of histopathological examination in diagnosis, classification and prognosis assessment, considering the significantly decreased coagulation function of liver failure patients and liver biopsy examination has certain risk, hence more attention should be paid to indications of liver biopsy in clinic. laboratory tests for liver diseases is the important basis to help and ascertain the clinical diagnosis, and the important reference to evaluate disease severity, make classification, predict outcome and guide therapy. the laboratory tests may reflect the pathological change and the functional status of liver in time, and may provide the objective and detailed data as reference for clinical classification and evaluating therapeutic effects, so that clinical intervention and effective treatment can be performed successfully. the liver is a complicated organ and the laboratory test items of relevance to severe hepatitis b are many, there are various biochemical items reflecting liver function, including coagulation function, immune and inflammatory cells and genetic markers. in this section only those laboratory tests that are relevant to acute exacerbation of chronic hepatitis b and severe hepatitis b will be described. for nonspecific laboratory tests, the reader is referred to other more general pathology books and literature. serum bilirubin is not a sensitive parameter of hepatocellular injury, but a significant increase (commonly ≥ ten times of upper limit of normal value) is usually a specific manifestation of acute exacerbation of chronic hepatitis or liver failure, which is also necessary condition to diagnose severe hepatitis or liver failure. in the course of acute exacerbation of hepatitis both direct and indirect bilirubin rise markedly due to the disturbance of bilirubin metabolism and secretion because the injury and hypofunction of hepatocytes, paracholia, and the rupture of bile capillary and biliary duct. the main enzymes reflecting liver function are alanine aminotransferase (alt), aspartate aminotransferase (ast), γ-glutamyltransferase (γ-gt, ggt) and cholinesterase (che). enzyme protein content in the liver account for 66.7% of total liver protein. because the aminopherase content of the liver is 100 times that of blood, in a pathological condition, as long as 1% of the enzyme in the liver is released into blood and keep active, this will be enough to keep the activity of enzyme in the serum increasing at rate of double. alt is an enzyme with the highest increasing amplitude and highest positive incidence when acute liver damage is occurring, with activity in the liver 3000 times that of serum. there is a large range of activity in daytime, commonly higher in the afternoon than the morning. although the activity of alt is almost coincident with the degree of liver damage, the activity of the enzyme decreases rapidly when liver failure or hepatocyte necrosis becomes widespread, with significantly increasing levels of serum bilirubin, manifesting the disassociation of enzyme and bilirubin. alt mainly resides in the cytoplasm of the hepatocyte, whilst ast is found more in the mitochondria. when liver cellular necrosis and change of cell membrane permeability appear, there is more release of alt than ast, but in very severe damage of liver, mitochondria damage is witnessed with elevation of ast and significant elevation of ast/alt. che in the serum is mainly produced by liver, with its activity and synthesis decreasing when liver damage occurs. although the change of che is less compared to the aminopherase when liver cell damage occurs, it will drop dramatically when there is severe liver necrosis and liver decompensation, especially in liver encephalopathy. the half life of human serum albumin is 20-26 days. because the occurrence of low levels of serum albumin is usually a late marker, the albumin level cannot accurately reflect acute liver cell damage, especially during the acute exacerbation of chronic hepatitis b. serum globulin, especially the γ-globulin, is only elevated in particular chronic hepatitis situations such as decompensated cirrhosis and autoimmune liver disease. the half life of serum prealbumin is only 1.9 days, and its reaction is more rapid and sensitive, which can reflect liver cell damage earlier. serum prealbumin has special diagnostic value with acute exacerbation of liver especially acute and subacute severe hepatitis. total cholesterol is composed by cholesterol ester and free cholesterol in healthy people. when hepatocellular damage appears, its cholesterol esterification noticeably decreases. it has been reported that α lipoprotein decreased significantly in patients with liver failure and this indicates a poor prognosis, while the changes of other lipoprotein and serum triglycerides were not specific for the severe type of patients. it has been reported that patients with severe liver dysfunction usually have bile acid metabolism abnormalities, indicating that serum total bile acid is a sensitive indicator reflecting functional recovery of liver cell and improvement of pathogenetic condition, just like alt. detecting serum total bile acid plays an important role in predicting disease prognosis of severe liver failure and evaluation of therapeutic effect. plasma coagulation factor xii (hageman factor) can produce activating factor xii by surface activation, which can further activate kallikreinogen (also called prekallikrein, pk) to produce kallikrein, kallikrein then makes prokinin release bradykinin, which cause vasodilatation, increasing capillary permeability with decreasing blood pressure. because of the short plasma half-life of pk, its plasma content decreases rapidly during liver failure, so pk has important diagnostic value with acute severe type hepatitis and liver failure. it was reported that its content was 92 ± 20% in healthy controls, 30 ± 6% in survivors with decompensatory liver cirrhosis, while only 16 ± 6% in non-survivors. a content level below 23% predicts poor prognosis, with patients usually dying of liver dysfunction within 30-45 days. gst is a protein rich in the liver, the renal tubules and intestine cells in mammals. its major function is detoxication by combining with multiple metabolistic organics such as bilirubin, bromphenol, cholecystographic agent and epoxide. because of the high content of gst in the hepatocyte, its small molecular weight and short half-life (only 90 min), it can be released into the blood after hepatocyte necrosis with high concentration. thus, gst has become a good predictor of observing hepatocyte necrosis especially in patients with acute liver failure and fulminant hepatitis, not only for early diagnosis of hepatocyte necrosis, but also for predicting prognosis. other factors such as multiple circular lysosomal enzyme and serum hyaluronate are all significantly elevated during a course of liver failure. these markers can also be used for monitoring and diagnosis of the disease. severe hepatitis and liver failure caused by hbv infection and other pathogenic factors are not obviously different under laboratory examination, although they have different etiological factors, pathogenesis and clinical manifestation. the major liver function, including protein (especial various kinds of coagulation factors) synthesis, metabolism and detoxication, are the first to lose function, and so result in severe hemorrhage and hepatic encephalopathy. because of the difference on the production of coagulation factors and the steps they involve in, not all coagulation factor detections are suitable for detection of severe type hepatitis and acute exacerbation of hepatitis b. vitamin k dependent factors mainly includes prothrombin (factor ii), pre-convert in (factor vii), christmas factor (factor ix) and fibrin stabilizing factor (factor x). patients with severe type hepatitis can manifest vitamin k deficiency caused by bile accumulation exterior and interior of liver, ingestion reduction or diarrhea. factor vii with the shortest half-life (4-6 h) is influenced firstly, which cause prolonged prothrombin time (pt). factor ii is not sensitive on vitamin k deficiency, while factor ix and factor x are moderately sensitive. factor vii can be considered as a reliable indicator, and it has important clinical value on prediction of prognosis of acute liver failure because of its short half-life and less influence by other factors, such as inflammation, dic, fibrinolysis, etc. it was reported that when the level of factor vii was below 20% of normal controls, the probability of death increased significantly, with predicting value of 100% sensitivity and 77% specificity. human fibrinogen-like protein 2/fibroleukin prothrombinase is a mediator of inflammation produced by activated macrophages, which belongs to fibrinogen superfamily and can catalyze and convert prothrombin to activate thrombin directly, thereby starting the clotting process and promoting thrombogenesis. some studies have demonstrated that the level of human fibrinogen-like protein 2/fibroleukin prothrombinase, if expressed highly and specifically on pbmcs and liver tissue in patients with acute-on-chronic liver failure, can be correlated with disease severity. other clotting factors such as factor vii, xi, xii, i, c protein, plasminogen and platelet count are all decreased in various types of liver diseases, with no special changes on the course of severe-type hepatitis or acute exacerbation. so they are not suitable for evaluation of acute exacerbation of chb. some scholars considered that combination detection of antithrombin iii (at iii), hepaplastin test (hp) and thrombin time (tt) has important value on early predicting fulminant hepatitis. pt is mainly used to detect activity of factor vii, x, ii, v and i. it has three measurable methods: one is the prolonged pt, normal pt is 12 to 16 s, with abnormality above 3 s of normal control value; another is prothrombin activity (pta), which can be calculated by mathematical formula. normal pta is 80-120%, usually below 40% in liver failure. the third is international normalized ratio (inr), which can be calculated by certain correction factor: pt in patients/pt in healthy. inr is above 1.2 in abnormality, and it is generally over 1.5 in liver failure. pta and inr detection values have been included in diagnostic criteria of international and domestic liver failure. ptt is a screening test of intrinsic coagulation system. ptt can prolong when it is faced with factor vii, ix, xi and xii deficiency or factor i, ii, v and x reduction and increment of anticoagulant substances. because ptt can be prolonged in a variety of liver diseases, demonstrating that ptt is not necessarily specific for the diagnosis of liver failure. tt tests for the activity of plasma fibrinogen. tt can be prolonged when fibrin degradation product (fdp) is increasing, fibrinogenolysis activity increasing, or fibrinogen (fib) decreasing, or heparin-like anticoagulant substances occurrence. apart from tissue-type plasminogen activator (tpa) and plasminogen activation inhibitor-1 (pai-1), other proteins and molecules involved in the course of fibrinolysis are all synthesized in liver. so plasminogen, α2 antiplasmin and thrombin activation fibrinolysis inhibitor (tafi) all decreased significantly in severe liver disease such as decompensated cirrhosis. as a result of dysfunction of liver clearance, the tpa level elevates inversely, while with normal or less elevated pai-1 in patients with cirrhosis, can cause proportion disequilibrium and final hyperfibrinolysis. in patients with acute liver failure, because of large elevation of pai-1 as acute phase reactive molecule, the activity of fibrinolysis decreases. on the contrary, taf-1 decreases by almost 50%, which induces elevation of fibrinolysis activity. although synthesis of partial coagulation factors and clotting dysfunction usually appear during the course of acute exacerbation of hepatitis b and development of severe-type hepatitis/liver failure, with manifestation of prolonged pt/decreased pta/elevated inr, some investigators had observed conflicting results recent years. specifically, although the mean inr was 3.4 ± 1.7 in acute liver injury/acute liver failure patients complicated by hepatic encephalopathy, concentrations of factor v and factor vii also decreased, the mean values of the indicators mentioned above detected by thromboelastography (teg) were normal, and also five teg parameters were normal in 63% patients. we think that the reason of normal clotting function detected by teg in patients with ali/alf might be the normal value of platelet count and fibrinogen quantitation. furthermore, more platelet and factor vii can be produced, and the levels of anticoagulant protein (protein c, protein s and antithrombase) decrease, which also compensates for the defect of the other coagulation factors. in total, clotting parameters such as inr, etc. can be considered as valuable indicators for predicting prognosis, although they cannot be used to reflect hemorrhage severity in patients with ali/alf. ammonia has been considered as a precipitating factor of hepatic encephalopathy for more than 100 years. during severe liver dysfunction, carbamide synthesis is injured, and brain tissue becomes a major organ of ammonia detoxication. with glutamine synthetase, astrocytes in brain can convert glutamate to glutamine to remove accumulated ammonia in vivo by the amidation of ammonia. because the synthesis of glutamine consumes energy, the large consumption of atp can cause energy exhaustion. excess accumulation of glutamine in astrocytes induced by high blood ammonia can cause increasing osmotic pressure and brain cellular edema. this has been confirmed by mri, while with the recovery of liver antidotal function after liver transplantation, previous hepatic encephalopathy can be reversed. in patients with acute liver failure, the risk of cerebral hemorrhage increases rapidly when arterial blood ammonia is above 150 μmol/l. about 55% patients can have acute intracranial hypertension when arterial blood ammonia exceeds 200 μmol/l. bernal w, et al. in their study which involved 165 patients with acute liver failure, observed that the level of arterial blood ammonia on admission was an independent risk factor of hepatic encephalopathy and intracranial hypertension, and the sensitivity was 70% for predicting severe hepatic encephalopathy with arterial blood ammonia above 100 μmol/l. combining with the meld score can further increase specificity and sensitivity. the toxicity of ammonia is multiple. the accumulation of ammonia not only influences brain metabolism, injuries brain cellular organelle directly and indirectly, causes disequilibrium of brain internal inhibitory and excitatory neurotransmitter and injuries brain energy metabolism, but also changes a series of gene expressions that are important proteins maintaining brain function. autoregulation of cerebral blood flow is affected and the blood brain barrier broken down. latest studies have revealed that ammonia can injury many important functions of neutrophil, such as chemotaxis, phagotrophy and degranulation, and can also stimulate and produce large reactive oxygen species (ros), cause sirs, which then further aggravates the toxic effects of brain cells from ammonia resulting in a vicious cycle. the liver is an important site of amino acids metabolism. except for bcaa (leucine, isoleucine and valine) metabolism in skeletal muscle, almost all essential amino acid metabolism occurs in liver. because of this, patients with liver failure or cirrhosis, have large amounts of amino acids accumulate in the blood. fischer, et al. demonstrated that disequilibrium of plasma amino acids might be reason of encephalopathy, and further indicated that the molar ratio of valine+leucine+isoleucine and phenylalanine +tyrosine (bcaa/aaa) was closely related with the severity of hepatic encephalopathy. the analysis of plasma amino acids in animals with hepatic encephalopathy demonstrated that other concentrations of amino acids increased significantly except the concentration of arginine, which declined. analysis of brain homogenates from cases of fatal hepatic encephalopathy demonstrated that the concentration of aspartate, arginine and glutamate decreased significantly, and this was closely related with the severity of hepatic encephalopathy. other amino acids, especially the aromatic amino acids, such as tryptophan, phenylalanine and histidine, increased but with carrying amounts; thus, the concentration of the aromatic amino acids was closely related to the severity of hepatic encephalopathy, implying that these aromatic amino acids may play important roles in the pathogenesis of hepatic encephalopathy, although perhaps not as the primary driver. jiang y, et al. conducted a clinical study which enrolled 22 patients with acute hepatitis, 65 patients with chronic hepatitis, 22 patients with severe hepatitis and 47 cirrhosis patients. they observed the ratio of bcaa and aaa was normal in acute hepatitis, mildly lower in the chronic hepatitis (p > 0.05), significantly lower in the severe grade of chronic hepatitis (p < 0.001), and the lowest in the patients with severe-type or cirrhosis (p < 0.05). as for the child-pugh grading, the ratio of bcaa and aaa: c grade <b grade <a grade, with significantly different among the groups (p < 0.001 or p < 0.02). of the patients with severe-type hepatitis and cirrhosis, the ratio of bcaa and aaa in the patients with hepatic encephalopathy was lower than that without hepatic encephalopathy (p < 0.001); the ratio of bcaa and aaa was significantly lower in the non-survivors compared with survivors (p < 0.01 or p < 0.005). they concluded that detection of bcaa/aaa can reflect the degree of liver damage, the lower the ratio was, the worse the liver injury was, and with high risk to develop hepatic encephalopathy; the ratio might also have certain predictive value for prognosis. elevation of plasma free tryptophan has been correlated with aggravation of hepatic encephalopathy; while in patients with acute liver failure, serum methionine concentrations clearly increase, there is no overlap between acute hepatitis and acute liver failure, and this can be used for differential diagnosis between the two kinds of liver diseases. endotoxin is produced by gram-negative bacteria, and has many biological activities, which can cause endotoxemia, with multiple pathological changes, such as fever, shock, dic and granulopenia. critically ill patients even have acute respiratory distress syndrome (ards), acute renal failure (arf) and multiple organ dysfunction syndrome (mods)/multiple organs failure (mof). wang mr, et al. revealed that the incidence of endotoxemia is 64-100% in severe-type hepatitis patients, 46.5-75.9% in decompensated cirrhosis, 23.5% in compensated cirrhosis, and 36% in acute viral hepatitis. it has been considered that the main mechanism of endotoxemia in patients with cirrhosis and severe-type hepatitis is: (1) intestine mucosal nutritional disturbance, epithelial atrophy, shedding and ulceration, which cause injury of intestine mucosal barrier; (2) intestine mucosal congestion, edema and increased intestinal wall permeability; (3) alteration of intestinal flora resulting in translocation of bacteria from the intestinal tract and increasing endotoxin production and absorption; (4) low ability of clearance of endotoxin absorbed by intestine or endotoxin into systemic circulation from bypass circuit. most studies have demonstrated that endotoxemia and liver injury are interrelated; the liver injury can be aggravated when accompanied by endotoxemia. the detection of endotoxemia is beneficial for predicting clinical course, disease severity and prognosis. medium molecule substance was discovered in patients with uremia accepting hemodialysis, and these molecules have molecular weight of 300-1500 d. thereafter, it was confirmed to play an important role in hepatic encephalopathy. medium molecule substance can inhibit na + -k + -atp enzymatic activity of brain cells, which result in na + retention and cause cerebral edema. studies have confirmed that medium molecule substance in the blood increased when acute liver failure patient presented with hepatic encephalopathy, which indicated that an increasing amount of medium molecule substance was closely correlated with hepatic encephalopathy and could be used for predicting clinical severity and prognosis. as for the property of medium molecule substance, it has not been totally characterized. the clearance of human medium molecule substance is mainly by secretion and filtration from the glomerulus. according to clinic observations, the contents of medium molecule substance can be detected in the plasma when plasma creatinine (cr) concentration is above 397.8 μmol/l or creatinine clearance rate (ccr) is below 15 ml/min. lactic acid is an intermediate product of glycometabolism. the concentration of lactic acid can reflect glycometabolism, peripheral circulation, tissue blood-supply and oxygen-supply indirectly. large studies has demonstrated that total blood lactic acid levels are correlated with disease severity and prognosis. the higher the lactic acid is, the worse the clinical condition is, and also for predicting a poor prognosis. some studies have revealed that the arterial blood lactic acid has a low predictive value for liver failure caused by hepatitis virus infection, toxin and immunity, but a better predictive value for acute and super-acute liver failure caused by acetaminophen over-dose. however, this is controversial. schmidt le, et al. showed that the predictive specificity of lactic acid was not high, even for liver failure caused by acetaminophen. furthermore, lactic acid cannot be used as an indicator for of liver transplantation, and if used, must be done in association with the current king's college hospital (kch) criteria. procalcitonin (pct) is a precursor of human calcitonin, with composed of 116 amino acid residues, 13kd of relative molecular weight, and also has no hormone activity as a glucoprotein. pct mainly originates from the c cells of the thyroid. during pathologic conditions, multiple tissue and organs can become production areas, such as liver, lung and glandular tissue. the production of pct can be regulated by bacterial toxin and multiple inflammatory factors, and bacterial toxins are a major stimulating factor inducing production of pct. pct is the best ideal indicator for early diagnosis of systemic infection, and has high specificity. serum pct concentration is positively related with the degree of bacterial systemic infection, but can also descend to normal level gradually accompanied with control of bacterial infection and improvement of clinical condition. as for the cirrhosis patients with ascites, detection of serum and ascitic pct can be of benefit to the clinician for predicting early diagnosis and the curative effect and evaluation of spontaneous bacterial peritonitis (sbp). the pct levels can decrease significantly in cirrhosis patients with sbp upon effective antibiotics treatment. so, pct can be considered as a good predictive factor on diagnosis and differential diagnosis of cirrhosis ascites complicated by bacterial infection. it has been reported that the blood lactic acid and pct levels were elevated to different levels when facing patients with either acute liver failure or acute exacerbation of hepatitis b complicated by intestine endotoxemia. a persistently elevated compared to an or not decreasing level of lactic acid and pct are usually indicative of poor prognosis and therapeutic efficacy. combined detection of blood lactic acid and pct does have certain predictive value for acute exacerbation of hepatitis b. in patients with chb, peripheral blood tregs were positively related with serum virus load, while in patients with acute hepatitis b, peripheral blood tregs experienced process from low to high, and usually are normal in the acute, convalescence, and recovery stages respectively. for the patients their treg were rejected, the ability of ifn-γ secretion in the pbmcs increased significantly upon antigen stimulation. while the tregs isolated from the patients with hbv infection could inhibit proliferative response of hbv antigen treated with autologous pbmcs obviously, which might indicate that the peripheral blood and liver in the patients produced hbv antigen specific tregs. so, tregs not only play an important role on regulating immune response of hbv infection, but also influence the prognosis of the patients with hbv infection. natural killer cell (nk) and natural killer t cells (nkt cells) construct the first line of defense of body to defense pathogen invasion, which account for 13% and 4% respectively in peripheral lymphocytes, while higher with 37% and 26% in the liver. the content of nk cells account for 90% in liver lymphocytes. the accumulation of nk and nkt cells in the liver demonstrates that they have important function, which can directly eliminate virus by killing infecting cells or secreting cytokines. under the condition of virus specific igg antibody production, nk cells can mediate surface igg fc receptor (fcγriii), identify and kill target cells with complexed igg specifically by antibody dependent cell mediated cytotoxicity (adcc). furthermore, nk cells can also play a role in immunoregulation by secreting cytokines, such as ifn-γ, il-2 and tnf. nkt cells are a group of inherent t cells which can recognize lipoid and glycolipid antigen presenting by mhc-i molecules associated with antigen presentation, with cd1d molecule restriction. nkt cells manifest phenotype of both t cells and nk cells (tcr and nk1.1 [nkrp1c], ly-49). nkt cells have two subtypes: type i nkt cells, also called inkt cells, and type ii nkt cells, also called non-inkt cells. the important characteristic of inkt cells is to produce th1 and th2 type cytokines rapidly by activation of tcr signal, and also activate multiple other cell types, such as dc, nk cell, b cells, and conventional t cells rapidly by activation of tcr. so, inkt cells can influence adaptive immunoresponse and series of host defense reaction and pathological course effectively. tian et al. demonstrated that nk cells exist abundantly in normal liver, which account for 33% of lymphocytes within liver and can play important roles on natural immunoresponse of anti-hbv infection. nk cells within the liver can be directly activated by virus or indirectly activated by other cells, such as nkt cells and antigen presenting cells (apc), and exert antiviral effects by their natural cytotoxicity and production of high level of antiviral cytokines. also they can regulate the function of lymphocytes, such as t cells, b cells, apc, and co-ordinate an adaptive immune response. after the binding of fasl expressed by ctl and nk cells and fas expressed by liver cell, the intracellular region of fas protein binds with fas associated death domain (fadd) protein by its c-terminal death domain (dd), then fadd protein activates caspase and lead to apoptosis. malhi h et al. showed that the role of nk cells and nk t cells was a major factor leading to mass death of hepatocyte during the clinical course of viral hepatitis. dendritic cells (dc) are antigen presenting cells with the most powerful function not only playing an antiviral role by secreting cytokine, such as il-12 and ifn-α, but also by promoting immunoclearance by t lymphocytes activated via antigen presentation. furthermore, dc also can regulate the equilibrium of th1 and th2. so, the abnormity of number, phenotype and function of dc is bound to influence the outcome of hbv infection. some studies illustrated that during liver failure the number of pdcs and mdcs in the liver increased significantly; ifn-α produced by pdcs in the liver was correlated with the production of il-12 and il-10; the ability of ifn-α production by pdcs in the peripheral blood decreased and the degree correlated with disease severity, which indicated that the dcs in the peripheral blood had accumulated in the liver and activated when hbv associated acute-on chronic liver failure occurred. after dc activation, the role of antigen presentation was enhanced, and the immune response activated and even to a peak level. zh qian et al. observed that the dc of peripheral blood in patients with chronic severe hepatitis b manifested dysmaturity, dysfunction of cytokine excretion, especially the low secretion of il-12 which mediated cellular immunity, a high level of secretion of il-6 and tnf-α which mediated inflammatory reaction, and finally become an important factor of aggravating liver inflammatory reaction leading to severe hepatitis. th17 cell is a new discovered subgroup of cd4+t lymphocytes, with significantly different biological function, cytokine expression and differentiation compared with th1 and th2 cells. th17 cells can mobilize, recruit and activate neutrophil leukocytes by secreting cytokines such as il-17, il-6 and tnf-α, in order to mediate inflammatory reactions more effectively. zhang et al. detected the th17 cells in the peripheral blood and liver of chronic hepatitis b (chb) and hbv associated acute-on-chronic liver failure (aclf) patients. they observed the level of th17 cells and its associated cytokines increased in chb and aclf patients, and the increasing of th17 in the peripheral blood and liver is significantly positively associated with hbv dna load, serum alt level and histological index of activity. in vitro, il17 could promote mdcs and monocyte activation, increase its secreting ability of proinflammatory factors, such as il-6, tnf-α and il-23, which demonstrated that th17 might lead to aggravation of liver injury in chb patients, and th17 can be considered as one of the immunologic indicators reflecting clinical prognosis of hbv associated aclf patients. wu, et al. observed that the ratio of peripheral th17 in patients with ahb and severe hepatitis increased significantly compared with general patients with chb and health volunteers. th17 cells were significantly positively associated with serum alt increasing in severe hepatitis, while there was no correlation with hbv dna quantitation. furthermore, serum il-10 levels were significantly negatively associated with the ratio of th17 cells, which indicated that th17 cells correlated with the course of hbv and the severity of liver injury. ye et al. also observed that the ratio of secreting il-17 and secreting ifn-γ in the liver of the child-pugh c patients with hbv infection was significantly higher than that of child-pugh b; the numbers of the cells secreting il-17 in the patients with severe liver injury were more than the cells secreting ifn-γ; the degree of cell infiltration with il-17 secreting were significantly positively correlated with liver inflammation grading, expressing of il-8 in the liver and neutrophilic leukocyte infiltration. chen et al. observed that increasing serum ifn-γ levels was correlated with disease progression of chb. ifn-γ might be involved in inherent and acquired immunity after hbv infection; ifn-γ can be considered as a potential biomarker for predicting clinical severity of hepatitis b, especially for predicting the tendency of liver failure. il-10 is a suppressive cytokine which can be produced by th2 cells, kupffer cells and liver cells, with major functions of suppressing antigen presentation of macrophage, production of multiple proinflammatory cytokines and th1 response. zheng et al. observed that high expression of il-10 promoted active viral replication in chb patients, induced excessive immune response. il-10 can be used to evaluate disease severity and prognosis of severe type b hepatitis. serum il-10 levels decreased significantly in severe hepatitis b patients complicated by bacterial infection or overlap with other hepatitis virus infections, which indicated endotoxemia and concurrent viral infection play important roles on promoting occurrence of severe hepatitis b. tnf-α is mainly produced by active mononuclear macrophages. the concentration of tnf-α is low under normal conditions, with the function of regulating the immune response. persistent high level of tnf-α can induce liver apoptosis or necrosis. tnf-α has two surface receptors: tnfr1 and tnfr2. du et al. observed that levels of serum tnf-α, tnfr1 and tnfr2 increased significantly in patients with acute fulminant liver failure compared with subacute fulminant type, acute type patients and health volunteers, and levels of tnfr1 were even higher in nonsurvivors. another study showed that expression of tnf-α and tnfr was obviously positively correlated with apoptosis liver cells in patients with fulminant liver failure; tnf-α might lead to widespread liver apoptosis by inducing fas gene transcription and other signal transduction system. except for inducing apoptosis, tnf-α can also induce production or release of other cytokines, such as il-6, il-8, and so on, to promote intrahepatic inflammatory reaction and aggravate liver cell necrosis by cascade amplified action. ip-10 is a recently discovered cxc chemokine, mainly induced by ifn. ip-10 has strong function of chemiotaxis and activation on t cells expressing specific receptor cxcr3, and also plays important role on lymphocytic accumulating to inflammatory reactive site, which may be an important factor of inflammatory cell migration toward hepatic tissue and causing large area hepatic cell necrosis. detecting serum il-10 concentration can indirectly reflect expression of ip-10 in local hepatic inflammatory response. luo et al. observed that the levels of serum ip-10 in patients with severe hepatitis b were higher than that of chb group, indicating the higher degree of liver inflammation and injury, the higher serum il-10 levels. so, serum levels of il-10 might reflect the degree of inflammation in hepatitis b patients. trail is a new member of tnf α superfamily. a large number of studies indicated trail-r2/dr5 play an important role on apoptosis. the trail induced apoptosis pathway may play an important role in the pathogenesis of severe type hepatitis. trail receptor/ligand system can specifically kill cells infected by virus in hbv infection. vander sloot am et al. observed that affecting hepg2 cell strain by soluble trail produced by cultural monocyte after lps stimulation can cause apoptosis; higher levels of soluble trail, more obvious apoptosis, which demonstrated that trail induced hepg2 apoptosis was dose dependent. further studies have shown that serum soluble trail increased significantly in patients with severe type hepatitis and was positively correlated with serum lps concentration and severity of liver injury, which illustrated that decrease of lps degradation accompanied with the degree of liver injury can stimulate mononuclear macrophage and dc to express more trail, shedding strail also increased, cause liver apoptosis and aggravated liver injury after its binding with receptors on liver cells. interleukin-22 (il-22) is one of the major cytokines secreted by th17, and is a member of the il-10 cytokine family. th17 is one of the major cells secreting il-22. osteopontin (opn) is a secretary phosphorylated glucoprotein which contains rgd (arginine, glycine and aspartic acid) protein with an integrin binding region discovered in 1979. it was isolated from bone matrix by herring in 1983 who found that opn was a key cytokine which accumulates in immunocytes and initiates th1 cell immunity, and is involved in multiple pathological process of inflammatory reaction. some studies demonstrated that levels of peripheral blood opn were significantly high in patients with severe hepatitis b. it also confirmed that nkt cells can secrete opn, and opn also can enlarge activation of nkt cells, to further trigger neutrophil infiltration and activation. further studies on pathogenesis of opn in hepatitis demonstrated that the opn transgenic mice, after been treated with cona, manifested large necrosis of liver cells and monocytes infiltration, while with only minor liver injury on the controls. so it presumed that opn might cause immune disequilibrium of th1 and th2, induce large hepatonecrosis. in patients with fulminant hepatitis, opn increased significantly, indicating that opn can initiate the effect of th1 cytokine network such as il-18 and ifn-γ by an autocrine pathway, to further cause macrophage activation and severe hepatonecrosis. as a specific receptor of endotoxin, toll-like receptor 4 (tlr4) can recognize serum endotoxin, bind with lps/lbp/cd14, and recruit myd88, activate irak (il-1r correlated kinase) and mapkkk (mapk kinase kinase) family, induce activation of nf-κb, activate target genes transcription, release a series of cytokines, and cause hepatocytes damage. in patients with severe hepatitis, because of intestinal microbial population derangement, bacterial overgrowth and intestinal mucosa congestion, the ability of endotoxin production and absorption increases, which is accompanied by severe hepatonecrosis, immune dysfunction and a decrease in kupffer cells clearance, which further causes a reduction of exdotoxin and exdotoxin immunocomplex clearance, and finally gives rise to endotoxemia. lipopolysaccharide (lps) is a major component of gram-negative bacteria, with important physiopathologic function. tlr4 is a key receptor for lps via signal transduction. regulating expressing of tlr4 should control lps related inflammatory reaction. xu, et al. designed and constructed tlr4 sirna expression vector, evaluated mouse phagocyte raw264.7 gene silencing efficiency by transfection, and evaluated therapeutic efficacy of tlr4 gene silencing in the mouse hepatic injury model. it demonstrated that the expression of tlr4 mrna and protein decreased significantly in raw264.7 cells; tlr4 sirna can inhibit obviously the up-regulation of tnf-α and mip-2 treated with lps; the activation of p38-mapk and erk1/2 by lps can down-regulate by tlr4 sirna. further studies demonstrated pretreatment of tlr4 sirna can be of benefit to control lps inflammatory reaction, reduce hepatic lesion of g57bl/6 mouse treated by d-ga1 n/lps, and reduce mortality of mice with acute hepatic injury. some studies also showed that the tlr4 protein expression on the surface of pbmcs and mrna were obviously higher in patients with chronic hepatitis b compared with the healthy controls; levels of tlr4 in severe-type patient of chb were significantly higher than that of chb, which indicated tlr4 played important roles during the course of liver injury with hbv infection, and with obvious increasing accompanied with clinical severity. fan jg, et al. observed the expression of tlr4 increased accompanied with the degree of endotoxemia and the prolong of stimulus duration; lps can positively up-regulate expression of tlr4 and enlarge biological effect of lps. so the severe hepatonecrosis and weakening of kupffer cell deactivation can accelerate the occurrence of endotoxemia, while endotoxin also can regulate liver cell metabolism or influence tlr4 mediated immune reaction to aggravate the clinical situation. the two aspects interact quite closely. all the studies mentioned above indicated high expression of pbmcs tlr4 in patients with chronic hepatitis, especially with severe-type presentation. monitoring dynamic changes of tlrs might be benefit for treatment. nitric oxide (no) is a biological regulatory factor with an extremely short halflife, produced by l-arginine catalyzed by nos (no synthetase), with extensive biological functions. it is now believed that no has potential antiviral inductive activity, and also might be one of the factors mediating liver injury. the studies of mouse model with inos defection demonstrated that the mouse model can tolerate ctl induced immunologic injury during the course of inhibiting virus replication because of no dyssynthesis, and also opposed against injection of fatal anti-fas antibody, further caused no obvious hepatonecrosis after treated with anti-fas antibody. in severe-type patients with hepatitis, serum no levels significantly increase during the early stage, then no levels decrease significantly with extenuation of inflammatory reaction, liver function recovery in the convalescence phase. these studies revealed that no is related to the degree of inflammation and clinical severity, and can play an important role in the clinical course of severe-type hepatitis. some studies demonstrated that inos gene was related closely with viral hepatitis. the expression of inos gene was extremely low in normal hepatic cells. once invasion of hepatic cells by virus appeared, it can induce hepatic cells to activate inos gene expression and produce large amounts of no, not only to kill liver tumor and pathogens such as virus, bacterium, parasites, play broad-spectrum antiviral function, but also injury to adjacent normal liver tissue by cell toxic action, and cause liver cell death. gong fj, et al. presumed that pre-s2 protein can downregulate inos transcriptive activity by its trans-regulation, further inducing the decline of no production and blocking the clearance of hbv in hepatocytes by no, and finally accomplishing long-term hbv chronic infection. so, expression of inos can be considered as an important factor of inflammatory injury. neutrophil gelatinase-associated lipocalin (ngal) was considered as a novel troponin-like biomarker, which was discovered in 1993 by kjeldsen, et al. who studied neutrophil matrix metalloproteinase 9 (mmp-9) at that time. it was believed that ngal could be used to monitor acute kidney injury/acute renal failure. renal dysfunction is extremely common during the clinical course of severe liver disease such as decompensated cirrhosis and liver failure. serum cr levels can increase rapidly when acute renal failure occurs and is easily detected, while it is not easy detected or diagnosed when complicated by chronic kidney injury. furthermore, other diagnostic methods based upon glomerular filtration rate (gfr) are needed to detect clearance rate, and are not suitable for routine application. the detection of serum cr is simple, while is not accurate on detection of kidney injury based upon cirrhosis. gerbes al, et al. observed 22 cirrhosis patients with ascites whose serum cr below 1.5 mg/dl, compared the levels of serum ngal and cr. the patients was divided into two groups, the gfr was 69 ± 15 ml/min and 29 ± 10 ml/min, and with serum cr of 0.8 ± 0.2 mg/dl or 1.1 ± 0.3 mg/dl respectively, with no obvious difference. the estimated gfr was 106 ± 34 ml/min and 65 ± 17 ml/min respectively, also with no difference. while in the study, serum ngal was 50 ± 15 ng/ml and 136 ± 61 ng/ml, demonstrating significant difference (p < 0.01). furthermore, levels of urinary ngal is similar with the blood ngal, especially the patients in the group 2, with urinary ngal above 100 ng/ml. area under the curve (auc) analysis also revealed that ngal was better than serum cr assay when gfr < 50 ml/min, with auc = 0.98 (0.96 ~ 1.00) of the former, and 0.79 (0.66 ~ 0.92) of the latter (p < 0.05). macrophage inflammatory protein (mip) is a novel protein first discovered in 1988 by wolpe, et al. which can be divided into five subtypes: mip-1, mip-2, mip-3, mip-4 and mip-5. mip-2 has a binding site for heparin, which can interact with heparin dextran sulphate in endothelial extracellular matrix, and enhance adhesion of leucocytes and vascular endothelial cells. mip-2 is involved in the whole process of inflammation by chemical chemotaxis and activation of inflammatory cells, with neutrophils as specific target cells. mip-2 can also activate neutrophils specifically and has been considered as an important proinflammatory cytokine during the early stage of inflammation. chen z, et al. observed that the levels of mip-2 in severe-type patients with hepatitis b was significantly higher than other groups. persistent high expression of mip-2 can be used to predict acute exacerbation of hepatitis b and clinical severity of patients with hepatitis b. thymosinβ4 (tβ4) is one of the important actin regulatory molecules in human body. it can bind with globular actin (g-actin), inhibit production of fibrous actin (f-actin), reduce formation of microthrombi and microcirculation dysfunction in patients with liver failure, and inhibit occurrence and development of mof. tβ4 can also reduce the levels of free radicals, slow lipid peroxidation and inhibit production of proinflammatory factors. patients with liver failure usually manifest a large amount of mononuclear macrophage infiltration, secretion, releasing proinflammatory factors and aggravate systemic inflammatory reaction. liu y, et al. observed that the levels of serum tβ4 decreased significantly in patients with liver failure compared with the cirrhosis group, chb group and healthy controls. dynamic changes of serum tβ4 can be considered as a novel predictive indicator for prognosis in patients with liver failure. t cell immunoglobulin mucin 3 (tim-3) is a surface molecule correlated with function of t cells, which was discovered in 2002. the studies on mouse model revealed that tim-3 can be expressed on the surface of th1 selectively as a negative regulatory molecule; the interaction of tim-3 and its ligand plays a down regulatory role on th1 cellular mediated immune response. sabatos ca, et al. observed that the levels of peripheral tim-3 in chb patients increased significantly, and it indicated the expression of tim-3 can be induced by regulating treg whose activity was modulated during the chronic infection period in patients with chb, and then inhibit specific th1 cell responses. zhou xq, et al. observed that the levels of tim-3 was significantly higher than other groups in severe-type patients with hepatitis b, which can be used to predict and monitor the clinical severity of hepatitis b patients. with the progression of chb, serum inos and ngal all increased, with the highest levels in severe grade of chb, while decreased inversely in the serum of the severe-type patients with hepatitis b, which can predict acute exacerbation of chronic hepatitis b. it was reported that the crosspoint of persistent elevation of tim3 and mip2, and declining of inos and ngal, demonstrated important clinical significance on predicting acute exacerbation of chb. recent studies have demonstrated that genetic variation of hormone receptor gene was significantly different between chb patients with acute exacerbation and hbv carriers, which could explain the sexual difference on different clinical course of hepatitis b. clinical studies further demonstrated that male patients with hbv infection had higher incidence and ratio of chronicity; male patients with hbv infection had higher activity frequency, which indicated the sex hormone and its receptor might influence the process of hbv infection by regulating host immuno response and level of viral replication. it is now believed that immunoresponse of viral clearance might down-regulate male sex hormones and up-regulate female sex hormones. furthermore, steroid hormones might regulate replication of hbv mediated by androgen receptors. it has been proved that androgenic hormone can inhibit cellular and humoral immune reaction; estrogenic hormone can inhibit cellular immunoresponse and enlarge humoral immunoresponse inversely; progestogen can promote conversion from cellular immunity to humoral immuno reactivity. several studies have reported the action of estrogen receptor (esrs) in the diseases of multifactorial inheritance, and its roles have also been observed by geneticists and virologists. large number of studies have demonstrated that esr2 played important roles on regulation in patients with persistent hbv infection, and esrs genes were correlated with host hereditary susceptibility. because of low expression of esrs in hbv carriers, the reaction of immune system to sex hormone was insufficient, which make clearance of hbv difficult and can lead to hbv persistent infection. human esrs are divided into two types: estrogen receptor a (esr1) and estrogen receptor b (esr2). all the genetic variations can cause degression of estrogenic function, which may further cause different kinds of host hereditary susceptibility after hbv infection. of them, esr1 gene can mutate, with tendency of sex hormonal resistance. sex hormone mainly plays the role of binding esr1, and esr1 gene is the true minor gene with host hereditary susceptibility after hbv infection. deng gh, et al. observed the relationship between esr1 gene polymorphism and chronic persistent hbv infection in a large sample study. they found that the susceptibility increased significantly compared with the individual of esr1 29t/t genotype and the individual containing at least one 29c isoloci site (p < 0.001). linkage disequilibrium plotting analysis indicated that t29c polymorphism included a linkage disequilibrium area sited from esr1 promoter to intron 3, which indicated that the esr1 t29c convention originated from esr1 itself, and esr1 gene polymorphism in the chinese population was positively related with hbv persistent infection. the negative feedback regulation of hypothalamus-pituitary gland-genital gland axis can be performed by serum testosterone (t) and estradiol (e2) through central nervous system feedback, and control releasing of luteinizing hormone (lh) and follicle stimulating hormone (fsh) . some studies demonstrated that the levels of serum t and e2 decreased in male patients with hepatitis b cirrhosis, and the levels of lh and fsh increased correspondingly, which indicated liver injury might be an important reason of sex and hypophyseal hormone abnormality. until now, the studies on changes of sex and hypophyseal hormone in acute exacerbation of hepatitis b and severe-type hepatitis are still limited, while it is certain that precise detection of sex hormone and correlated hypophyseal hormone can reflect the severity of liver injury to a certain degree, and can be considered as a useful indicator evaluating the clinical course of hepatitis b, cirrhosis and its complication, hepatocellular carcinoma. it is now believed that the priming or control point of severe-type hepatitis occurrence and development is influenced by two aspects: virus (such as viral variation, viral protein and viral genotype) and host (such as cell immunity, cytokines and apoptosis), and of them, viral replication is the necessary condition and cause of severe-type hepatitis. some studies demonstrated that viral replication was relative vigorous during the early stage of severe-type hepatitis, which indicated viral factor occupy a central position in the occurrence and development of acute exacerbation of hepatitis b. the changes of host transcription factors and hbv genetic variation all possibly cause the changes of hbv transcription and replication, resulting in acute exacerbation of hepatitis b. hepatocyte nuclear factor 4 (hnf4) is a protein with the function of genetic transcription and modulation, and is mainly found in liver. long ce, et al. observed that the hnf4 binding sites, located in enh i/xp and enhii/c promoter, is an important factor regulating c promoter activity and 3.5 kb mrna transcription, and has an important role on hbv replication and expression with high tissue specificity. previous studies demonstrated the expression of hnf4 increased in patients with chb, and the level of hnf4 is positively correlated with hbv replication. furthermore, from a study on liver biopsy specimens using gene array in patients with hbv infection, honda m, et al. observed hbv infection can cause more gene transcription of hnf4, rxr/ppar and c/ebp, and possibly related with liver injury. so, the interaction of hnf4 and hbv genetic transcription and control might play an important role on the pathogenesis of severe-type hepatitis b. deng et al. studied cxcl10 snp variation on th1 immune response pathway, and observed that the polymorphism site g-201a existed in cxcl10 gene promoter region was significantly related with disease progression in male hbv carriers. an emsa study revealed that the g-201a site can change its binding ability with nucleoprotein; and reporter gene assays also revealed that this site can influence the transcriptional activity of cxcl10 gene promoter region; mrna real time quantitative experiments further revealed that the expression of susceptible allelotype -201g in pbmcs was significantly higher than allelotype -201a; elisa and immunohistochemical assays revealed a higher expression of cxcl10 protein in plasma and liver tissue of patients with chb that progressed than that of nonprogressive patients. all the studies mentioned above demonstrated that g-201a is a new functional polymorphism site, cxcl10 protein involved in the process of inflammation and necrosis in liver tissue of patients with hepatitis b, and influence the progression of chb. furthermore, yan z, et al. reported that mutation of ifn-γ gene can influence expression of ifn-γ, which is a predisposing factor of chronicity of hbv infection; fulminant hepatitis patients with poor prognosis had high frequency of occurrence of -1031c, -863a and tnf-βb2 allele in tnf-α gene promoter region; snp in il-10 gene promoter region was related with progression of chronic hbv infection; the frequency of haplotype expressed by down-regulated il-10 in the il-10 gene promoter region in patients with fulminant hepatitis was higher than the controls, while the frequency of haplotype gene occurrence by up-regulated il-10 expression was even lower. yan et al. also revealed the relationship between polymorphism of il-10 gene promoter in th2 immuno response pathway and chronic severe-type hepatitis, which provided new evidence on natural selection theory of il-10 promoter and sirs pathophysiology theory of acute liver failure. in a study of human fibrinogen-like protein 2 (hfg12) gene regulation mechanism and network, han et al. observed that hbc protein and hbx protein all had the function of hfg12 activation, while hbs protein cannot activate the gene. a series of promoter deletion experiments demonstrated that the regulation sequence of activating hfg12 gene existed between the sits of hfgl2 gene promoter −712 ~ −568, transcription factor c-ets-2 can bind with cis-acting element on hfg12 gene promoter, then activate the expression of the gene under the action of that viral protein; the phosphorylation level of p-jnk and p-erk was enhanced in patients with severe hepatitis b than healthy controls, which indicated jnk and erk signal pathways were activated. some studies in vitro revealed that jnk and erk signal pathways were activated respectively under the effect of viral protein hbx and hbc, and c-ets-2 expression and transposition were correlated with the activity of jnk and erk. all the studies mentioned above indicated that viral protein hbc and hbx can activate jnk pathway and erk pathway respectively, further activate transcription factor c-ets-2, shift into the nuclear, and finally up-regulate the expression of hfg12 gene by binding with cis-acting element of hfg12 gene promoter. tnf-α is an important proinflammatory factor induced during liver failure. mfg12/prothrombinase plays an important role on mouse fulminant hepatitis and acute on chronic liver failure. gao et al. constructed eukaryotic expression vector of mfg12 gene and tnfr1 gene, green color fluorescence fusion protein and its shrna interfere plasmid. it was more effective to improve the survival rate of mouse with severe type hepatitis using combination of fg12 and tnfr1 genes compared with single gene intervention, and improve serology and pathological changes of liver, which indicated that fg12 and tnfr1 genes might produce synergistic effect during the progression of severe-type hepatitis. hbv infection is a necessary condition and cause of occurrence and progress of severe-type hepatitis, while hbv gene transcription and replication are a key step of the hbv life cycle. beginning from the new point of genetic transcription, further exploring the effect and mechanism of acute exacerbation of hepatitis b on hbv genetic transcription and regulation, will provide important clue of host transcription factor, which can be useful to predict and monitor the occurrence, development of severe-type hepatitis, and to provide new targets for effective methods of treatment of severe-type hepatitis. genome-wide association study (gwas) has been available since 2005, and have become the main aspect of complicated genome association study. the strategy does not need to select candidate genes, but to make genome-wide associations and analysis by selecting directly ten thousand single nucleotide polymorphism (snp) sites and copy number variation (snv) sites over the total genome. compared with the strategy of a candidate gene, gwas was a significant improvement in statistical efficacy, and also avoided the bias of group stratification and randomness of gene selection. in recent years, some studies on the aspects of viral hepatitis and hbv related hepatocyte carcinoma has gained great breakthrough using these approaches. several independent and large sample gwas in usa, europe and japan demonstrated that only rs1297980 site near the il-28b gene had relationship with curative effect of interferon in the study on genetic correlation of ifn response in patients with hepatitis c (with p value of 10 −25 ). guo et al. observed that gene variation of hlp-dp gene can play great influence on clinical severity of persistent chronic hbv carriers. karlsen et al. also reported the relation between hla-ii gene and hbv infection. he fc and zhou gq made gwas of hbv related hepatoma, and they discovered a related site 1p36.22, where one of the three genes (kif1b, ube4b, pgd) might be predisposing gene of hbv related hepatoma. the studies on genetic predisposition of severe-type hepatitis b are relatively less. only a minority of studies have examined a candidate gene-disease associated study strategy, and their study populations were all from asia. recently, under the support of the national 973 program and key state science and technology project of infectious disease, chinese experts have taken affymetrix snp 6.0 array (including 900,000 snp sites and 900,000 cnv sites in the range of human total genome-wide) to make gwas in 1600 severe-type patients with hepatitis b and asymptomatic carriers. the project is now being analyzed, expecting to explore common genetic variance sites influencing severe-type hepatitis b under the point of host total genome. epigenetics mainly studies the change of herediable gene expression with no change of dna sequence. currently, epigenetic mutation mainly includes dna methylation, microrna (mirna) and chromatin remodeling. several functional studies also demonstrated that mirna participated and regulated the expression of multiple inflammatory signal factors. it was found that mir-150 and mir-223 might be linked with liver inflammatory activity of viral hepatitis. mir-150 mainly participated in regulating maturation of b lymphocytes, and also regulate the differentiation of th1 and th2 cells. mir-223 located in human x chromosome, and highly expressed specifically in granulocyte series cells. johnnidis, et al. observed the granular leukocytes increased twice in mir-223 gene knock out mouse, and these leukocytes were more sensitive when treated with antigenic stimulation outside (especial fungi); compared with mir-223 gene non-knock out mouse, the lung inflammation of mir-223 gene knock out mouse was more obvious, and the tissue injury induced by exdotoxin stimulation even more serious. the latest studies have discovered seven susceptible molecules correlated with acute exacerbation of hepatitis b: mir-478a (down-regulation in severe-type twins patients with hepatitis b), mir-7a (up-regulation in severe-type patients with hepatitis b), mir-16 (up-regulation in severe-type patients with hepatitis b), mir-122 (providing early warning signal on acute exacerbation of hepatitis b and disease severity), mir-1187 (has obvious decline tendency with the development of liver failure, and can be considered as biomarker for predicting clinical severity and acute exacerbation of hepatitis b), mir-155 (its expression decreases gradually accompanied with viral clearance, can be considered a biomarker of turnover of acute exacerbation of hepatitis b, and provide early warning signal on acute exacerbation of hepatitis b and treatment), mir-197 (down-regulation has predictive value to liver inflammation). it has been finally shown that detection of specific mirna correlates with liver inflammation, by using mirna array technique in severe-type patients, can provide novel target on early prediction of severe-type hepatitis. qi zx, et al. observed the difference of th1 and th2 cells chromatinization status in il-10 gene promoter region, which interfered with the binding of promoter and transcription factors, and influenced gene transcription of il-10. the distribution of methylation in patients with liver failure showed obvious differences between chb patients and healthy controls; concentrations of serum il-10 were negatively related with promoter methylation status, which indicated the methylation of il-10 gene promoter region might involve in the pathogenesis of liver failure, and it is also a reason of il-10 change. furthermore, some studies revealed that methylation of glutathione-s-transferase p1 promoter region can promote acute exacerbation of hepatitis b. of hepatitis b toll-like receptors (tlrs) mediated signal transduction pathway includes two forms: myeloid differentiation factor-88 (myd88) dependent form and myd88 independent form. tlr7, tlr8 and tlr9 mediated signal pathway belongs to myd88 dependent form pathway; tlr3 mediated signal pathway belongs to myd88 independent form or trif independent form signal pathway. in the myd88 independent form pathway, tlrs recognized ligand, then activate nf-κb and mapk, induce release of multiple cytokines, up-regulate costimulatory molecules such as cd80, cd86, et al., then finally activate specific immune system. tlrs and tlr4 in particular, play important roles on the mechanism of acute exacerbation of hepatitis b, which can better predict tendency of the disease, and can be considered as indicators for evaluating prognosis of severetype hepatitis b. tlr4 is a key receptor in endotoxin transmembrane signal transduction; it is also a rate limiting factor in lps transmembrane signal transduction of mononuclear cell and macrophage. some studies demonstrated that the expression of tlr mrna in severe-type patients with viral hepatitis was higher than the healthy controls; and it is higher during the aggressive stage than the critical stage in the nonsurvivors and it is lower during improvement than the critical stage in the survivors. furthermore, expression of tlr4 mrna is positively correlated with the levels of endotoxin. deng m, et al. observed that the severe-type patients with viral hepatitis had tendency to endotoxemia, with endotoxin binding with tlr4, then transmembrane signal activation of nf-κb through intracellular structure of the tlr4, further induced release of inflammatory factors, such as tnf-α and il-1, and produce secondary intrahepatic microcirculation disturbances. tlr2 is a pattern recognition receptor with extensive pathogen recognition abilities. tlr2 can recognize cell walls from multiple bacteria such as gram-positive bacteria, gram-negative bacteria, fungi, spirochaetes, mycobacterium tuberculosis and mycoplasma, and play important roles on acute and chronic infection. yan cg, et al. observed the expression of tlr2 of mouse with fulminant liver failure induced by d-gal/lps. they observed the high and persistent expression of tlr2 mrna, and accompanied with high expression of tnf-α, which indicated that tlr2 can up-regulate expression of downstream inflammatory response genes and release of cytokines, to involve in acute liver failure induced by d-gal/lps. metabolomics is a new technology, which studies various changes of endogenous metabolic products and biologic conditions in humans by observing biosystem changes qualitatively and quantitatively under various stimulations and changes of internal and external environment. the study object is the micromolecule compound with relative molecular mass < 1000 in the metabolic network. at present, the major techniques include nuclear magnetic resonance spectrum (nmr), gas chromatogram (gc), liquid chromatography (lc), capillary electrophoresis (ce), mass spectroscopy (ms), infrared spectrum and ultraviolet spectra. of them, nmr, gc-mc and lc-mc are used more extensively. yang et al. investigated the serological profile in acute hepatitis b patients with sudden aggravation of liver function by hplc-ms and pls analytical techniques. they observed the changes in individual atoms in lysophosphatidyl choline (lpc) and gcdca, which can predict early disease aggravation. feng b, et al. analysed the metabolic product in serum of rats with fulminant liver failure by gc-mc and pca software, and observed high content of 5-hydroxyindole acetic acid, glucose, β-hydroxybutyric acid and phosphate, which indicated the changes of these substances might become potential biomarkers of fulminant liver failure. yu, et al. set up a research platform based upon liver failure metabolomics by using gas chromatographic mass spectrometry (gcms) and super-high performance liquid chromatogram mass spectrometry technology, constructed a model for evaluating clinical severity of liver failure, and constructed serum specific metabolism spectrum by partial least squares discriminant analysis, which can be used to predict the prognosis, with sensitivity of 91.3%. mao et al. also derived similar results with a high positive diagnostic rate of 93.62% for liver failure, and also found that the changes of climax concentration of plasma citrate had potential diagnostic significance. li lj, et al. pointed out the low level of lysolecithin, high level of fatty amide and nonelevation of bile acid by using metabolomics were risk factors for predicting poor prognosis in liver failure patients with pretreatment and post-treatment by artificial liver support systems. by observing the trend changes of serum metabolic spectrum in patients with pretreatment and post-treatment with an artificial liver, we can predict the prognosis and assess curative effect of artificial liver. electrolyte disturbances were usually complicated in patients with liver failure because the patients typically have low renal blood flow and glomerular filtration rate (gfr). a majority of patients manifest hypokalemic, hyponatremia, hypomagnesiumemia and hypocalcemia. the occurrence of hyponatremia and its severity are positively related with prognosis in patient with liver failure. severe hyponatremia can cause acute low sodium syndrome and encephalopathy; and hyponatremia can also cause renal injury and promote or aggravate development of hepatic encephalopathy and hepatorenal syndrome (hrs), and increase mortality. some studies indicated that the mortality of the patients with severe hyponatremia (<115 mmol/l) is extremely high and up to 93.88%. in patients with acute liver failure, hypochloraemia can be caused by insufficient intake of chlorine, vomiting, large excretion of aldosterone and acidosis, while water intake insufficiency, renal failure, renal tubular acidosis and potassium chloride overdose can cause hyperchloraemia inversely. furthermore, hypophosphatemia is more readily detected than hypocalcemia in most patients. acid base disorders (abd) are very common in patients with acute liver failure, especially alkalemia. li xm, et al. presumed that arterial carbon dioxide pressure depression and respiratory alkalosis are frequent manifestations of acid base disorders. abd is not related with basic pathological changes of severe-type hepatitis, whilst blood ph value is an important factor influencing the survival outcome of severe-type patients with hepatitis. in the latest years, some scholars think that pre-estimation of compensatory formula (pcf) for abd has practical value on differential diagnosis of partial simple and duality abd. partial patients with acute liver failure complicated by abd can be diagnosed by anion gap (ag), potential hco 3 −· and cl − . elevation of ag is a reliable indicator of appearing potential metabolic acidosis, and is common in patients with hepatic encephalopathy, lactic acidosis caused by hypoxemia. during hepatorenal syndrome since reduction of acidum excretion of kidney ketoacidosis may appear. potential hco 3 −· is the hco 3 −· value exclusive of the masking hco 3 −· by high ag metabolic acidosis, which can raise the existence of ag metabolic acidosis and metabolic alkalosis with triplicity abd. a mixed metabolic acidosis can be discerned by combination of ag and blood ci. series of primary abd can overlap, even to develop duplex and triplication abd. for example, if the respiratory alkalosis prolongs, accompanied with increasing of potassium excretion by renal, and with iatrogenic factors to develop metabolic alkalosis, and further develop multiple complications combined with metabolic acidosis, which finally develop triplicated abd. triplication abd is usually the end stage of acute liver failure, with worst prognosis. so, prevention and active treatment of abd is an important parameter for rescuing patients with acute liver failure. liver biopsy is a good method to acquire pathologic changes of liver in multiple liver diseases including liver failure, which have important value on directly evaluating the severity of liver damage. liver biopsy is generally divided into percutaneous biopsy, transvenous biopsy, surgical/laparoscopic biopsy and plugged biopsy. of them, percutaneous biopsy is safe and simple, without special instrument or equipment, and being used extensively. transvenous biopsy is more suitable to the patients with large amount of ascites, clotting dysfunction, diminution of liver volume and increasing of liver hardness because of cirrhosis, difficulty to determine hypochondria because of obvious obesity, and with high hepatic vein pressure. the contraindication of liver biopsy are as the follows: (1) excessively prolonged pt, excessive declining of pta, which might cause haemostasis difficult even mild injury; (2) obvious cavity bleeding or large area of petechia; (3) patients with unconsciousness, who might not be able to fit the operation; (4) medium dose ascites, especially patients complicated by abdominal infection; (5) patients with obvious pleural effusion and severe heart and lung disease. it has been proved that liver biopsy is safe to a great extent under careful preparation, attentive operation, even in the liver failure patients with obvious clotting dysfunction. what we need to point out is that although liver biopsy has important value on recognizing liver tissue degeneration, necrosis and degree of inflammatory infiltration, the pathologic change of the biopsy tissue might not be able to reflect entire pathologic change of the liver because of the limited liver tissue obtained. so, it is very important for clinicians to collect and combine various forms of clinical data, laboratory and pathogenetic results from the patients, for getting an accurate judgment. the incidence of cerebral edema in severe-type patients is high, with ratio of 50 to 80%, and a quarter of them can appear cerebral hernia with poor prognosis. so, measurement of intracranial pressure plays important role on diagnosis and treatment of cerebral edema. traditional lumbar puncture manometry can measure intracranial pressure only one time; it cannot be observed that the changes of intracranial pressure dynamically and exactly. on the other hand, lumbar puncture on patients with acute intracranial hypertension might cause or aggravate cerebral hernia. furthermore, under the condition of cerebral hernia, cranial cavity and spinal cord cavity can not intercross, so the pressure detected by lumbar puncture cannot represent actual intracal pressure. so, persistent detection of intracranial pressure retrieves shortage of lumbar puncture, and cerebral ventricle catheterization is still an extensively used method, which is considered as gold standard of intracranial pressure monitoring by clinicians. intracranial hypertension is defined as persistent elevation of intracranial pressure with levels above 15 mmhg. according to the difference of intracranial pressure, intracranial pressure can be divided into four stages: (1) normal: 5 ~ 15 mmhg; (2) mild: 15 ~ 20 mmhg; (3) moderate: 20 ~ 40 mmhg; (4) severe: above 40 mmhg. if intracranial pressure is close to mean arterial pressure (map), which illustrates almost no brain perfusion, with nearly close to brain death. currently, 20 mmhg is defined as the critical value of applying treatment of anti-intracranial hypertension. complications of intracranial pressure measurement include infection, intracranial hemorrhage, iatrogenic acute intracranial hypertension and brain parenchyma injury. although laboratory parameters can reflect pathological changes and liver functions during the clinical course of severe-type hepatitis and acute exacerbation of hepatitis b sensitively and in time, and can provide detailed reference data for clinical typing, grading and curative effect evaluation, in total, the extensive and large numbers of indicators listed above are still under study and experimental evaluation, and their availability and utility need to be checked by more clinical practice. until now, laboratory parameters have been extensively used and applied clinically but are still limited. the laboratory parameters conventionally tested in the majority of hospitals are mainly pta, inr, serum bilirubin, alt/ast, pre-protein and blood ammonia. furthermore, some studies also reveal that serum afp, serum sodium and phosphate are closely related with prognosis of liver failure. although some indicators such as arterial blood lactic acid, pct, nagl and inos are closely related with severe-type hepatitis or liver failure, they also score as abnormal in other severe diseases or conditions such as septic shock and mods, so these parameters are not specific for severe-type hepatitis b and liver failure. it is the same on the indicators related with immunity or inflammatory cell or factors, with high sensitivity, while without specificity. other indictors related with heredity, metabolomics, genomics, proteomics and glycomics are still in research, or needing expensive instruments, and still difficult to the routine clinical practice in the short term. what we need to point out is that the various kinds of prognosis scoring systems and mathematical models constructed and tested in the latest years, such as royal school of medicine standard in united kingdom, model for end-stage liver disease (meld) and child-pugh scores, have higher accuracy and utility on predicting prognosis of severe-type hepatitis and liver failure, although some of them still need additional supplement and improvement in the future. because of diversity and individualization for particular causative factors, clinical types, clinical course, complications and clinical treatments, until now, there are still many challenges reflecting prognosis of liver failure. it is believed that more and more novel biomarkers will come into the clinic in the near future, and with development of studies of mechanism and clinical characteristics on acute exacerbation of hepatitis b, therapeutic efficacy of severe-type hepatitis and liver failure will be improved. pathogenesis of aechb is generally considered to include viral factors (hbv genotype, viral replication and viral mutation, etc.), host factors (host genetic characteristics, immune injury, cell apoptosis and necrosis) and interaction between these two aspects. currently, there are at least ten different hbv genotypes and several subtypes distributed all over the world (see table 1 .6) based on the differences of whole genome sequences >8% for the major genotypes or 4-8% at the sub-genotype level. genotype a is mainly distributed in northern europe (a2), west africa (a3), and sub-saharan africa (a1). in asia, genotype b and c are most common. genotype b have six subtypes (b1-b6), in which, b1 was found in japan, b2 ~ b5 were found in east asia, and b6 was found mainly in the arctic regions, such as alaska, northern canada and greenland. genotypes b2-b5 are also regarded as recombinants with genotype c. genotype c, including five subtypes (c1 ~ c5), is mainly distributed in east asia and southeast asia. genotype d, including subtypes d1 ~ d5, prevails in africa, europe, mediterranean and india. genotype e is only found in west africa. genotype f, having four subtypes, is found in central and south america. genotype g has been reported popular in france, germany and the united states. genotype h is found in central america [2] [3] [4] . genotype i was recently newly found in vietnam and laos, but this new genotype is under controversial [5] . the newly found genotype j in japan has a close relationship with the orangutan's genotype and human genotype c. population migration, promotion of antiviral therapy and host immune selection pressures result in increased risk of hbv gene recombination or mutation [6] . with the development of genetic testing methods, even more new genotypes could still discovered. due to the distribution differences of hbv genotypes, the study on the relationship between hbv genotypes and severe hepatitis b is quite limited. comparisons have only been conducted in a few genotypes. a study from united states on patients with hbv related acute liver failure suggested that outcomes of these patients were not associated with genotypes [7] . a multicenter study from japan involving 301 patients with acute hbv infection has compared 5 genotypes (ae [a2],ba [b2-5],bj [b1],ce [c2],cs [c1],dandg). multi-factor regression analysis indicated that subtype bj(b1) is one of the independent predictors for fulminant hepatitis. subtype ae(a2) is more related to hbv persistence but not self-limiting hepatitis b [8] . another multicenter study from japan showed that patients with genotype c accounted for 80 and 69% in patients with acute-on-chronic liver failure and acute liver failure, respectively. these rates are much higher than that in patients with chronic infection, suggesting that patients with genotype c are more likely to suffer from fulminant liver failure [9] . a study from china included 26 hbv carriers, 61 patients with chronic hepatitis b, 20 patients with aclf, 12 hbv related liver cirrhosis and 7 patients with hbv related hcc. data showed that genotype b (38.1%) and c (54.8%) were the main genotypes in these patients. compared with genotype b, genotype c was seen more frequently in those with severe liver diseases, was accompanied with high levels of hbv replication, indicating that genotype c is associated with high hbv replication and severe liver disease [10] . however, results from another study showed no difference in genotype composition among patients with chronic hepatitis b and those with chronic severe hepatitis [11] . in a study involving 487 hbv-infected pediatric patients, in which 217 patients had been treated with nucleos(t)ide analogues, genotype c2 and b2 were found to be the most prevalent subtypes (73.7 and 21.1%). compared with genotype b2, genotype c2 is more likely to cause severe hepatitis in hbeag positive pediatric patients [12] . the association between various genotypes and the pathogenesis of severe hepatitis needs further studies. hbv uses reverse transcription to copy its dna genome and lack of proof-reading capability permits the emergence of mutations in the genome. every day, approximately 10 11 viral particles are produced and released to maintain a stable level of virus in blood. the average mismatch rate of hbv polymerase is from 1:10 5 to 1:10 4 , potentially resulting in a large amount of mutants in the circulation [13] [14] [15] [16] . for single individuals infected with hbv, hbv genome mutation either naturally occurs naturally or is selected out by antiviral drugs or the change of internal host environment. the probability of hbv mutations varies in different regions of the whole genome. generally, mutations are more likely to occur in the basal core promoter (bcp), pre-c region and neutralization determinants of the viral envelope, but they can also be found in other regions. some of these mutations have important clinical significance, but most of them are silent mutation without biological significance. pre-c region encoding hbeag, is composed of 29 amino acids. the most common mutation is a guanine (g) to adenine (a) substitution at nucleotide 1896, which creates a premature stop codon at codon 28, and prevents the translation of the hbeag [17] . although synthesis of hbeag is inhibited, the hbv replication still continues, manifesting as hbeag negative hbv infection. hbeag expression is not necessary for viral replication, its role in the hbv life cycle remains unclear. in the immune system, hbeag may act as bait, which induces immune tolerance, especially in the newborn babies whose mothers have high level viremia. the hbeag-induced immune tolerance can prevent the attacks on the virus-infected hepatocytes by ctl on hbcag, thus the hbv-infected hepatocytes are not able to be cleared. the hindered synthesis of hbeag may facilitate the ctl to damage infected liver cells, which might be one of the mechanisms of severe hepatitis. it is reported in japan that patients, especially children, infected with this mutant, are more likely to suffer from severe hepatitis. other pre-c mutations include point mutations generating other termination codons, for example g1897a generating uga codons. point mutations may change the initiation codon of p25 or the specific amino acid for cleavage and insertion of key signal peptide, particularly between 1838 and 1839. all of these mutations can affect the hbeag production. the pre-c mutation can reduce hbeag expression, increase hbv replication and aggravate liver damage [18] . a number of studies have suggested the association of pre-c mutation and development of severe hepatitis. the hbcag encoded by c region, contains a t-cell-dependent/independent epitopes and induces the host humoral and cellular immune responses. the c gene mutation may cause deletion of the cell surface antigen, and consequently the lack of humoral immune response by the host against hbv. however, the cytotoxicity of ctls was not affected resulting in potentially massive necrosis of liver cells and eventually leads to severe hepatitis. core promoter directly activates pgrna transcription, and plays a central role in hbv replication. core promoter is composed of basic core promoter (bcp) and its upstream regulatory elements. the bcp region partly overlaps with the 3 'end of x gene and the 5' end of pre-c gene and can independently start the transcription of pre-mrna and pgrna. the core promoter mutation, which is associated with nt1758-1762 that are at the upstream of the starting point of pre-c mrna, reduces the synthesis of hbeag, but has no effect on hbcag. the development of severe hepatitis is often accompanied with hbv core promoter mutation, especially the 1762,1764 double set of mutations [19] . other common mutations in the core promoter region of patients with severe hepatitis include nt1768 c-t, nt1770 t-a and cluster mutations, including nucleotide insertion, deletion and substitution. the bcp mutations can induce changes of two codons (l130m and v131i) in x protein, and generate the hnf1 (hepatocyte nuclear factor 1) binding sites. insertion of 11 bases into the core promoter produces a new binding site of hnf1, which can enhance viral replication and lead to severe hepatitis [20] . in-vitro studies have confirmed that bcp trans-activating transcription causes x protein replacement, which downregulates the transcription of pre-c region and pgrna [21] . however, the double-mutant can upregulate the transcription of pgrna, increase hbcab production, thereby enhancing viral replication. previous studies have shown that a1762t and g1764a mutation are more prevalent in hbv genotype c, which partially explains the stronger pathogenicity of genotype c than genotype b. recent studies have shown that a1762t and g1764a mutations in hbv genotype b may be associated with severe hepatitis, but in hbv-infected pediatric patients, a1762t and g1764a mutations show no significant difference in genotype b and c. overall though, compared with wildtype hbv, bcp double mutation is more commonly associated with severe liver diseases, especially liver cirrhosis and hcc [22] . the mutations in pre c/bcp region may alter the biological characteristics of the virus, and induce the development of severe hepatitis through impacts on host immune responses, being more vigorous in hbeag-negative chb [23] . however, the biological significance of mutations in different sites or forms, the dynamic interaction between virus mutation and host immunity, the influence of different genotypes and viral quasispecies on mutation are still not fully elucidated. immune evasion or vaccine failure related pre-s point mutation or deletion mutation does not affect the viral replication. the pre-s1, s2 recombination, including deletion mutation and promoter mutation, have been regularly found in patients either with chronic or fulminant hepatitis. these mutations frequently occur after hbeag seroconversion or interferon treatment, suggesting the host immune selective pressure during their selection. the pre-s region mutations may play a role in hbv persistent infection, and may also cause liver damage. it has been reported that the pre-s2 mutation is related to fulminant hepatitis, and the pre-s mutation is a strong risk factor for the development of hbv-related liver cancer [24, 25] , presumably as a result of an accumulation of viral envelope proteins inside the cell.mutations of the surface antigen protein, particularly aa145 mutation (sg145r), result in conformational change of the major antigen epitope 'a'. this change disables the immunological recognition and surveillance of the host immune system for the mutant can also result in failure of the clinical vaccination and might be one of the precipitations for the exacerbation of hepatitis b [26] . the p gene mutation in key catalytic domains indicates the hbv resistance against nucleos(t)ide analogues (na). the current five nas in clinical application include lamivudine, adefovir, telbivudine, entecavir and tenofovir. the clinically extensive application of nas leads to rapid selective drug resistance. the selection of drugresistant mutation depends on the following factors: (1) the long half-life of hepatocytes and intrahepatic cccdna; (2) the capacity of hbv replication and mutation; (3) antiviral drug pressure; (4) genetic barrier to resistance. for example, the lamivudine resistance is closely associated with the hbv reverse transcriptase gene ymdd motif mutation, presenting as rt m204i and rt m204v mutation, with or without rt l180m mutation. the replication capacity of rt m204i/v mutants is weaker than the wild strains in the absence of drug. the rtl180m mutation can restore the hbv replication capability. in addition, the single or combined mutation of rt l80i, rt l82m, rt f166l, rt v173l, rt a200v and rt v207i may compensatorily restore the replication capability of rtm204 i/v mutant [27] . the resistance rate is up to 40% after 2-years of lamivudine therapy, and more than 76% after 5-year mono-therapy. the 1-year resistance rate of telbivudine is about 7%. the rtn236t site mutation (threonine substitute asparagine) is mainly seen in adefovir resistance, and the 5-year resistance rate is about 29%. the rta181t/v (valine/threonine substitution alanine) site mutation is found in all the above three antiviral drugs. entecavir resistance mutations include 204 and 180 substitutions, combined with substitutions at codons 184, 202 and 250. the 4-year resistant rate in patients initially treated with entecavir is less than 1%. for patients that have been previously treated with lamivudine, the resistance rate of entecavir increased significantly. so far, there have been no reports on the primary drug resistance for tenofovir mono-therapy. the rta194t mutation was found in the combination therapy of tenofovir and lamivudine [28] . the entire s gene is included in the p gene, and the rt region overlaps with s gene, thus the rt mutation may cause the s gene mutation. double mutations in these two regions can change the viral replication capacity. the hbv drug-resistance mutation occurs in patients with chronic hepatitis b may reduce the hbeag seroconversion rate, reverse the histological improvement, increase the disease progression rate, aggravate liver cirrhosis, and increase the death risk of liver transplant patients. rt mutations may also cause the s protein epitope changes and affect the hbs antibody and the function of ctl, suggesting its role in the development of liver failure [29, 30] . as salvage therapy for lamivudine resistance, lamivudine combined with adefovir dipivoxil has higher rate of viral suppression and lower rate of adefovir resistance compared with switching to adefovir dipivoxil monotherapy [31, 32] . tenofovir is a potent antiviral drug for lamivudine-resistance salvage therapy, and showed a better effect than switching to adefovir dipivoxil monotherapy [33] . in contrast, switching to entecavir is not an optimal choice for lamivudine-resistance [34] . telbivudine resistance is associated with the rtm204i mutation, and has crossresistance with lamivudine. therefore, telbivudine could not be an alternative for lamivudine-resistance [35] . treatment for telbivudine resistance is similar to that for lamivudine resistance [36] . treatment for adefovir-resistance is determined by the virus mutation types and antiviral medication history [37, 38] . lamivudine has been proved to be effective on inhibiting rtn236t adefovir resistance mutations [39] . in vitro data has also suggested the effectiveness of telbivudine. additionally, entecavir might be a reasonable choice for rtn236t mutants. patients with rtn236t mutation are suggested to (1) switch to or add entecavir; or (2) add lamivudine or telbivudine; or (3) switch to tenofovir. compared with that on hbv wild strain, lamivudine becomes less effective on a181v adefovir resistant strain. in vitro studies show that tenofovir has reduced sensitivity to the rta181t mutation. clinically, entecavir and tenofovir can effectively inhibit the replication of a181t adefovir resistant mutants [40] . patients with rta181t mutation are suggested to (1) switch to or add entecavir; or (2) switch to tenofovir. under this circumstance, lamivudine should not be suggested in case it increases the risk of cross-resistance [41, 42] . currently, there has been no data from large sample clinical trials that can guide the treatment for entecavir resistance. in-vitro studies and case reports suggested that adefovir and tenofovir are effective for entecavir-resistant patients. based on expert opinions, patients with entecavir resistance are recommended to add tenofovir or adefovir [43] [44] [45] . in summary, the virus mutation in above regions may be associated with the pathogenesis of severe hepatitis. however, the severity of hepatitis depends on key factors of virus and host. the same mutant may lead to different clinical outcomes in different hosts. thus except for virus mutation, factors including host immune status, cytokines and hla might also account for the development of severe hepatitis. tests for hbv infection hbv surface antigen/pre-s1 protein/pre-s2 protein hbsag is the major coat protein of hbv with antigenicity but not infectivity. in a broad sense, hbsag contains the major protein, middle protein and large protein. narrowly, hbsag simply refers to the major protein, which appears earliest and has the highest titer. thus it is considered as an important marker for early diagnosis of hepatitis b. for typical acute hepatitis b, hbsag appears during the incubation period, followed by the clinical symptoms and abnormal liver function in 2-6 weeks. hbsag stays in the blood for 1-2 months, and disappears in the recovery period. persistence of hbsag more than 6 months indicates the development of chronic hepatitis. hbsag can also be detected in hbv carriers and patients with hbv-related liver cirrhosis or liver cancer. a rapid reduction of quantitative hbsag within 3 months can predict the efficacy of antiviral drugs, but no changes in the quantitative hbsag level after 6 months is not considered as a good predictor [46, 47] . pre-s1 protein, which appears early and is significantly associated with hbeag and hbv dna, can be used as a marker of acute hepatitis b early in infection. the pre-s1 protein has a strong immunogenicity, and includes the important site where hbv attaches to and invade the hepatocytes, the sodium taurocholate co-transporting peptide (ntcp). it is also a reliable reflection of hbv replication level. the synergy from pre-s2 protein is also important for hbv invasion. most patients with acute exacerbation of chronic hepatitis, or chronic active hepatitis, or acute hepatitis developing to chronic hepatitis have persistent pre-s2 protein expression in serum. therefore, serum pre-s2 protein can be used to estimate the activity and infectivity of hbv in chronic patients [48] . pre-s1 and pre-s2 protein, which can induce and regulate humoral and cellular immune response of the host, provide important immune defense for eliminating virus in blood circulation and preventing healthy liver cells being infected. hbsab, which is a protective antibody, can eliminate the virus and prevent hbv infection. hbsab appears in the late stage of acute infection, just before hbsag becomes negative, and will gradually rise to the peak levels in 6-12 months. this antibody can last for a long time, but the titers will gradually decline after 10 years. a small number of cases do not produce hbsab after hbsag becomes negative. in acute hepatitis b infection, appearance of hbsab suggests recovery of the disease. patients with severe hepatitis often present with high titers of hbsab, which forms the immune complexes with hbsag, which can induce flares of hepatitis that lead to liver cell necrosis. after the hepatitis b vaccination, hbsab (>10 iu/l) means the successful vaccination and development of immunity. in the blood, hbcag is mainly located in the core of the dane particles or virions. the only small amount of free hbcag is also combined by high titers of hbsab and presents as immune complexes. thus, it cannot be detected unless treated by detergent. hbcag on the surface of hepatocytes is considered to be the main target antigen of the host ctls. hbcag is a direct evidence of hbv infection and replication, and also a marker for evaluating the efficacy of antiviral drugs. hbcag is strongly immunogenic, so that hbcab can be detected in most patients with hbv infection. hbcab often emerges in the early stage after infection, is present in high titer in blood and can persist for a very long time. titer of hbcab above 1: 100, together with abnormal alt level, can be used for the diagnosis of hepatitis b infection. for occult hepatitis b, high titer of hbcab is also valuable for the diagnosis. hbcab consists of igm and igg antibodies. anti-hbc-igm, which suggests hbvresulted liver damage, is the main evidence for the diagnosis of acute hepatitis b. it may become positive during the active phase of chronic hepatitis b and turn negative during remission. it also appears during the flares of chronic hepatitis b, mostly in a week after infection, and disappears within 6 months. anti-hbc-igg appears late but is sustained for many years or even a lifetime. in patients with acute hepatitis b, the titer of anti-hbc-igm is higher than anti-hbc-igg, while in those with chronic hepatitis b the situation is opposite. both antibodies show high titers in fulminant hepatitis. hbeag is a soluble antigen, which appears later than hbsag. sustained expression of hbeag suggests persistence of hbv infection. in patients with chronic hepatitis b, hbeag acts as an important immune tolerance factor leading to a low immune response to hbv infection. it is a valuable marker for evaluating the efficacy of antiviral drugs. seroconversion refers to the loss of hbeag and development of anti-hbe. approximate 2-15% patients have spontaneous seroconversion every year. studies have shown that spontaneous seroconversion occurs earlier in patients with genotype a, b, d and f than those with genotype c. appearance of hbeab demonstrates the decrease or termination of viral replication and low infectivity. recent studies showed that, after 1 year since the spontaneous hbeag seroconversion, viral load more than 2000 iu/ml increased the incidence of fulminant hepatitis. for hbv chronic carriers and patients with hcc, hbeab does not mean the recovery, or elimination. in contrast, hbv dna integration is often found in these patients. x protein is capable of transactivating the expression of numerous cellular and viral genes, and is vital for virus replication. x protein can be detected in some patients with chronic hepatitis, so it is used as an auxiliary diagnostic marker of hbv infection [49] . x protein plays a central role in hbv-related hcc progression and stimulation. thus, follow-up is necessary for patients active and persistent hbv replication. serum hbv dna is the direct evidence of active hbv infection, reflecting the level of viral replication and infectivity. the quantitative detection for viral genes is a very important marker for treatment decision, efficacy prediction and observation. long-term high load of hbv dna is an independent risk factor for predicting the development of liver cirrhosis and hcc. numerous studies have shown that viral load is the most reliable marker to predict the development of hcc [50] . the differences of hbv whole genome sequence are approximately 2-4%. the diverse variants that are genetically linked through mutation are known as quasispecies. quasispecies contain a large number of mutated genes serving as a reservoir for viral selection under the pressure of immune response and antiviral treatment. when changes occur in the environmental conditions, the quasispecies structure responds by rebalancing its composition. the predominant sequence may shift by selection of a variant that is better adapted to the new environment, in the classic darwinian process of survival of the fittest [51] . this feature gives the virus strong adaptability, and makes it difficult to prevent and control. study on the relationship between quasispecies and different clinical outcomes will provide valuable information for exploring anti-hbv treatment strategies. 1. quasispecies evolution under host immune pressure. in chronic hbv infection transmitted via perinatal transmission, the different immune phases of chronic hbv infection confer different environments on hbv quasispecies. thus, the characteristics of hbv quasispecies may differ. a preliminary study on the differences of full-length hbv quasispecies between mothers and their progeny showed that, after 30 years of evolution, the dominant sequence of hbv quasispecies became different between mothers and daughters. the characteristics of hbv quasispecies in various gene regions are different in mothers and daughters with different treatment responses or disease status. among these genes, the prec/c gene had the highest substitution rate [52] . under antiviral drug selection pressure, hbv mutants are selected from the preexisting pool of quasispecies and over time become the dominant species [53] . the probability of resistant mutations depends on the effectiveness of antiviral drugs. low potent drugs have almost no selective pressure on the virus, thus lead to low probability of viral resistance; on the contrary, drugs that completely inhibit viral replication also rarely result in resistance. only medium potent drugs have the highest rates of drug resistance. a study showed that during the 4-week lamivudine therapy, distinct patterns of quasispecies evolution are found between responders and non-responders; the structures of viral quasispecies tended to be simpler in responders, but more complicated (higher diversity) in non-responders. similar phenomenon was also observed during entecavir therapy. another study detected the full length sequence of resistant virus in lamivudine and adefovir sequential therapy, and found that variation of nucleotide or amino acid sequence usually occurs in hbv hbsag or rt region. using single strand conformation polymorphism (sscp) and dna sequence analysis, researchers found that the complexity of hbv quasispecies in patients with cirrhosis was more than those in patients with chronic hepatitis b, suggesting that complexity of hbv quasispecies is associated with disease status [54] . researchers from china had also used the same methods to analyze the difference of quasispecies complexity in s region among patients with chronic severe hepatitis b, patients with chronic hepatitis b and hbv carriers. it is found that the quasispecies complexity in the s region increases along with disease progression in chronic hbv infection. analysis on quasispecies in acute hepatitis b, chronic hbv carriers, chronic hepatitis b and chronic severe hepatitis by full-length hbv genomic clone and bioinformatics methods also discovered the positive correlation between hbv quasispecies complexity and disease severity [55] . on one hand, complex evolution of quasispecies may lead to persistent infection and continuous liver damage, and increase opportunities for the emergence of new hbv variants. on the other hand, enhanced virulence of mutated virus and change of antigen epitope may cause excessive immune response and severe liver damage. however, correlation between hbv quasispecies complexity and disease severity still needs dynamic large sample research to confirm [56] . the dynamic change of quasispecies during na antiviral therapy may be related to the antiviral efficacy and drug resistance. results from a study on patients receiving lamivudine for 48 weeks suggested that the baseline quasispecies heterogeneity is not associated with antiviral efficacy, and the changes of quasispecies complexity at an early stage may predict antiviral efficacy and drug resistance more accurately than the change of hbv dna level during lamivudine therapy. another study on dynamic changes of quasispecies in patients receiving entecavir antiviral therapy suggested that, compared to the partial responders, quasispecies complexity is reduced but dispersion is increased in complete responders after 4 weeks of treatment [57] . in these two studies, the quasispecies dispersion decreased in lamivudine responders but increased in entecavir responders after 4 weeks of treatment. this might be because entecavir has stronger antiviral effect than lamivudine, thus generates more selection pressure. in addition, entecavir can still induce complete response even when some mutations occur since it has a higher drug resistance barrier. therefore, the early changes in hbv quasispecies complexity may act as a predictor of sustained antiviral effect of nucleos(t)ide analogues. the resistant strains typically already exist before antiviral therapy, and become the predominant strains under selection pressure of antiviral drugs. a study showed that adefovir treatment for 240 weeks in patients with chronic hepatitis b selected resistant virus strains [58] . study on gene heterogeneity of hbv reverse transcriptase suggested that lamivudine monotherapy is liable to induce the quasispecies that affect response rate of salvage therapy with adefovir for virologic breakthrough in lamivudine-treated patients, and reduce the sensitivity to other nucleos(t)ide analogues [59] . under different drug selection pressure, non-responders have similar quasispecies evolution patterns, suggesting that this pattern may be associated with viral resistance mechanisms. from the perspective of quasispecies, drug selection pressure changes the relative ratio of viral populations, and leads to population drift. dna sequence analysis is the most direct and reliable method to detect gene mutations. it is also the gold standard to detect nucleic acid sequence mutation. however, the high cost of this method prevents its application in large sample research. dna sequencing, which can be divided into direct sequencing and cloning sequencing, is built on the basis of high-resolution denaturing polyacrylamide gel electrophoresis. direct sequencing detects the nucleotide sequence of dominant strains; clone sequencing method can find out changes of nucleotide in other strains. rflp is a technique that applies to detect variations in homologous dna sequences. it refers to a difference between samples of homologous dna molecules from differing locations of restriction enzyme sites, which may be naturally formed or brought in by pcr mismatch. the dna sample is broken into pieces and digested by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. mismatched pcr is a modified pcr, which changes one or a few nucleotide bases in designed pcr primers to make the synthetic dna meet special requirements. here, a restriction enzyme recognition sequence is introduced into the pcr amplified fragment in order to change the target dna restriction map and distinguish the mutants and non-mutants. nucleic acid hybridisation is the pairing of complementary single-stranded nucleic acids (dna or rna) to produce dna-dna or dna-rna hybrids. when the target dna obtained by pcr amplification combines with the probe labeled by radioactive or non-radioactive labels, any mismatch between the probe and target dna can be detected. this helps to distinguish the wild and mutant strains. nucleic acid hybridization applies to high frequency point mutation and is suitable for large sample detection, but the high requirement for hybridization temperature makes it difficult to popularize. the principle of sscp is based on conformational difference of single-stranded nucleotide sequences of identical length. this property allows sequences to be distinguished by means of gel electrophoresis so as to determine whether mutations exist. it applies to the single base substitution and screening of dna fragment mutation. however, sscp is neither stable nor practical [60, 61] . primer extension starts from the 3′ end in pcr amplification, and only succeeds when the 3′ end of the primer is an exact match to the template dna. based on this phenomenon, the primer with 3′ end containing mutated bases is used to detect the mutation of target dna. the 3′ base-specific pcr technology is simple but with low sensitivity, and the results are prone to be false-negative. the incomplete block of pcr amplification also leads to false-positive results [62] . melting curve analysis is an evaluation of the dissociation-characteristics of doublestranded dna during heating, the temperature at which 50% of dna is denatured is referred to the melting point [63] . in chronic hbv infection, the peak numbers of dna melting curve in patients with moderate and severe hepatitis are significantly more than those in hbv carriers and mild hepatitis; the peak number of melting curve in patients with severe hepatitis is significantly more than that in moderate hepatitis. the melt-curve analysis is less sensitive than sscp, but is more accurate on analyzing genetic variation. the strong operability and high cost-effective make it a preferable method for genetic variation analysis. the high demand for low-cost sequencing has caused the development of highthroughput sequencing-the next-generation sequencing, which includes massively parallel signature sequencing (mpss), solexa sequencing, solid sequencing, 454 pyrosequencing, heliscope single molecule sequencing, etc. these methods apply to genome sequencing, genome resequencing, rna-sequencing, chip-sequencing and epigenome characterization [64] [65] [66] . current dna sequencing methods under development include microscopy-based techniques, macromolecule and nanotechnology that can distinguish the base signal and directly read the sequence without the use of biological or chemical reagent. the third-generation sequencing methods include: (1) non-optical microscope imaging: the dna sequence can be read out if the resolution of image is high enough to differentiate the four kinds of bases on dna when visualizing the spatial linear arrangement of nucleotides. this idea is based on non-optical microscope at the atomic level, for instance, scanning tunneling microscopy (scanning tunneling microscope, stm) [67] . (2) nanopore dna sequencing: it is based on the readout of electrical signals occurring at the single stranded dna or rna molecules passing by alpha-hemolysin pores covalently bound with cyclodextrin [68] [69] [70] . those methods still need further validation and improvement. the pathogenesis of hbv infection is determined by the interplay between both virus and host. different outcomes after infection are related to different host immune responses and viral mutations. however, the biological significance of viral mutation has not been fully elucidated because of: (1) the limited samples and lack of comparison between different groups; (2) the overlap of each hbv genomic coding regions; (3) the limitation of detection technology. with the rapid development of detection technology, the large sample, long-term, multi-level studies will help to understand more about the host-virus interaction and potential mechanisms. zhi the indications for liver transplantation are changing over time. before 1980, the major indication for liver transplantation was primary malignancy of the liver, especially hepatocellular carcinoma (hcc) which significantly influenced the therapeutic efficacy of liver transplantation and postoperative survival. currently, not only are acute or chronic liver diseases nonresponsive to other medical treatment and surgery now the major indication for liver transplantation but also liver diseases that markedly affect quality of life. the major indications for liver transplantation are shown in table 1 .7. viral hepatitis has and continues to be a major public health problem in china. it is the most common liver disease and most common cause of liver failure. the proportion of patients with hepatitis b-induced liver failure among patients receiving liver transplantation in china is significantly higher than in other countries. hepatitis b has diverse clinical manifestations and may progress to chronic hepatitis, liver failure (including acute liver failure, acute on chronic liver failure, and chronic liver failure), hepatic cirrhosis (compensated and decompensated), and even hepatitis b-related liver tumors. whether liver transplantation is necessary for patients with hbv infection and the timing of transplantation must be carefully considered. early liver transplantation may increase patient risk and be an economic burden because patients could recover and even survive for a long time in the absence of liver transplantation. however, presently it is difficult to predict patient outcome to hbv infection. in contrast, if liver transplantation is performed in the late stages of disease, numerous complications of hepatitis b-induced liver dysfunction may either increase the risk of transplant surgery resulting in a poor prognosis. thus, for patients with severe hepatitis b, the indications for and the timing of liver transplantation are crucial and should be carefully evaluated. in general, the following conditions are indications for liver transplantation in patients with chronic hepatitis b: 1. obvious manifestations of liver failure including sustained elevation of serum bilirubin to >5 mg/dl; prothrombin time > 5 s longer than the reference range; plasma albumin <2.5 g/dl; and liver failure which is nonresponsive to active and/or symptomatic therapy (such as infusion of fresh plasma and albumin) or if the patient continues to deteriorate clinically despite optimal medical therapy. 2. when there are complications related to either liver failure or portal hypertension such as the presence of severe hepatic encephalopathy, disturbances of coagulation function, refractory bleeding due to rupture of esophageal or gastric varices, refractory ascites, repeated episodes of spontaneous bacterial peritonitis, or the development of hepatorenal syndrome. 3. when hepatitis b influences the quality of life including the development of severe lethargy, uncontrollable itching, metabolic bone diseases or the development of bacterial cholangitis and 4. the development of hepatocellular carcinoma. although there are some well defined indications for liver transplantation, the ultimate determination of the timing and specific indications for liver transplantation remains a challenge in clinical practice for hepatitis b patients with liver failure. the introduction of the model of end stage liver diseases (meld) and some derivative scoring systems are now frequently used to determine whether a patient should be listed for transplantation and the urgency of liver transplantation. in 1999, the mayo clinic proposed that meld should replace the child-pugh grading system for the determination of urgency of liver transplantation. meld is calculated using the following calculation: meld = 9.6 × log e (creatinine [mg/dl]) + 3.8 × log e (bilirubin [mg/ dl]) + 11.2 × log (inr) + 6.9 × (causes: 0 for biliary and alcoholic; 1 for others). meld was originally used for the evaluation of prognosis of hepatic cirrhosis patients receiving transjugular intrahepatic portosystemic shunt. as described above, the meld score is determined on the basis of total bilirubin, international standardization of bleeding time, serum creatinine, and etiology. compared to the child-pugh grading system, meld employs objective parameters, which are helpful for the comparisons of patients from different centers. while the meld score changes with the alteration of the liver disease, the child-pugh grade has only three levels and is unable to meet the requirements of accurate and objective evaluation. saab et al. investigated the prognosis of 404 patients receiving liver transplantation and found that the 1-year survival rate was 90, 89, 90, 79, and 69% in patients with preoperative meld scores of ≤10, 11-18, 19-24, 25-35 , and ≥36, respectively. however a higher meld score negatively affected the 1-year survival rate. for example patients with a meld score ≤24 had a 1 year survival rate of 88 whereas patient survival was reduced to 65% in patients with a meld score >24. despite the benefits and the widespread application of meld in clinical practice, there remain some imperfections in the evaluation of timing of liver transplantation and the assessment of prognosis and therapeutic efficacy. one of the major imperfections is the failure to consider complications such as infection, hepatic encephalopathy, hepatorenal syndrome, and disturbances in fluid and electrolytes which may significantly influence the prognosis and timing of liver transplantation but are not included in the meld scoring system. hence inclusion of sodium (na) in the meld scoring system: meld − na [meld + 1.59 × (135 − na)], imeld [meld + (0.3 × age) − (0.7 + na) + 100]; and meso [meld/na (mmol/l)] × 10 has improved outcomes of patients requiring liver transplantation. those scoring systems, however, also fail to consider other complications. including the development of hepatic encephalopathy or ascites and therefore, their applicability is limited in china. in china, liver transplantation was initiated relatively late compared with other countries; regulations regarding liver transplantation remain imperfect; and smooth communication among transplantation centers in different regions of the country are lacking. these drawbacks markedly influence the timely and fair distribution of donor livers. determination of waiting times for transplantation is not scientific. therefore, the comprehensive evaluation of a patient's condition of hepatitis b-induced liver failure, determination of the timing of liver transplantation, and optimization of the rational and fair distribution of donor livers needs to be carefully considered by chinese clinicians in the field of hepatology going forward if improvements are to occur. there are currently additional scoring systems which have been used by chinese clinicians. one of these was developed by professor ke wm in the department of infectious diseases of the affiliated third hospital of sun yat-sen university. in that system, hepatic encephalopathy, serum creatinine, prothrombin activity, serum total bilirubin, liver size (determined by ultrasonography), amount of ascitic and pleural fluid (determined by ultrasonography), and infection (peripheral white blood cell count, proportion of neutrophils, and inflammatory findings from thoracic imaging examinations) are taken into account and objectively and conveniently scored on a scale of 0-4 according to their severities as described in tables 1.8, 1.9, and 1.10. [72] these newer scoring systems and the meld scoring system can favorably predict the mortality of acute on chronic liver failure patients with hepatitis b. the area under receiver operator curves of scores determined with a new scoring system and meld scoring system was 0.960 [95% confidence interval (ci), 0.944-0.977] and 0.886 (95% ci, 0.852-0.920), respectively. there was no overlap in 95% cis between the two, and they were significantly different (p < 0.01) as illustrated in figs. 1.7, 1.8, and 1.9 ). wenzhou medical college in china subsequently proposed a scoring system in which the total score is calculated using the following equation: x = 1.4053 + 3.6017 × hepatorenal syndrome (hrs) + 1.2069 × lc − 1.1555 × hepatitis b e antigen (hbeag) − 0.1003 × alb − 0.042 × pta. that scoring system has been used in several centers for liver diseases, and its effectiveness and accuracy have now been confirmed in these centers. in summary, to establish a new scoring system on the basis of china's national status of liver disease and to further validate this system, large multicentered studies with a large number of patients is crucial before they are adopted in china. it is important that any new system developed under these conditions be subjected to rigorous study, standardization and scientific validation. for liver transplantations in patients with hepatitis b-induced liver failure, perioperative therapy is crucial, impacting the recurrence of hepatitis b virus reinfection following liver transplantation. in fact, perioperative therapy can make a major 10 fig. 1.9 receiver operator curve of a new scoring system and meld scoring system difference in patients with hepatitis b compared to patients with other liver diseases (e.g., tumor recurrence). it is well appreciated that antiviral therapy is necessary in hepatitis b virus patients as discussed below. the guideline for the prevention and treatment of chronic hepatitis b in china (2010) recommends that (1) for patients with hepatitis b planning to receive liver transplantation, oral lamivudine be administered within 1-3 months before liver transplantation when hepatitis b virus dna is detectable (100 mg q 24 h). hepatitis b immune globulin (hbig) should be administered during the anhepatic stage in surgery and long-term use of lamivudine and low-dose hbig (800 iu q 24 h for the first week postsurgical, 800 iu q 1 week, then 800 iu q 1 month). the dose of hbig and interval between two treatments [generally, trough hepatitis b surface antibodies (anti-hbs) are >100-150 miu/ml and anti-hbs is better if >500 miu/ml within 6 months of surgery) should be determined according to the anti-hb levels. for patients nonresponsive to lamivudine, other nucleos(t)ide analogues effective for resistance mutation should be used and for patients with a low risk for recurrence of hbv infection (i.e., hbv dna negative before liver transplantation and absence of hbv infection recurrence within 2 years after liver transplantation), lamivudine plus adefovir should be considered for the prevention of hbv infection recurrence. while patients are waiting for liver transplantation, an artificial liver support system (alss) may be of use as a bridge to transplantation. liver failure may significantly compromise the detoxication, synthesis, and metabolism activities, resulting in a significant accumulation of toxins and deficiency of some important factors (such as coagulation factors). unfortunately, some patients die while waiting for either their liver function to improve or for liver transplantation. alss have been generally used to partially substitute the liver's normal activities, such as clearing some toxins and supplement some missing factors, which is helpful for life maintenance while awaiting subsequent liver transplantation or spontaneous recovery. thus, for patients with liver failure secondary to hepatitis b, alss maybe an important therapeutic strategy while patients are waiting for liver transplantation. alss can be classified as a mechanical artificial liver, biological artificial liver, or mixed artificial liver. mechanical alsss mainly utilizes nonbiological materials to clear toxins in the body and supplement some missing factors. biological alsss use biological materials to substitute the liver's activities. the core feature of the biological alss systems is to simulate hepatocyte function. however, the source and biosafety, as well as their location (bioreactor), are important factors limiting the development of biological alsss. finally, mixed alsss employ the hemodiafiltration, plasma exchange, and hemoperfusion that are able to detoxify substances (nonbiological alss) and human or porcine "hepatocytes" in the bioreactor. currently, nonbiological alsss are the most widely used in clinical practice, and biological and mixed alsss are still undergoing investigation and have not been widely applied in clinical practice. in addition to its use before liver transplantation, alss post liver transplantation may be used to correct ongoing renal failure, brain edema, severe water and electrolyte disturbances, and severe infections. another factor to consider is that liver donors are rare. as a result, the inability to perform liver transplantation in a timely fashion results in reduced survival rates. under those conditions, pre transplant complications including multi organ failure may persist. in such cases, alss therapy may prove to be beneficial. for patients with hepatitis b-related liver failure, perioperative therapies are more important than in patients receiving elective liver transplantation (such as liver tumor patients without liver failure). liver failure causes a variety of complications that directly contribute to transplantation failure and a high-risk status after surgery. clinicians need to address problems such as portal hypertension, upper gastrointestinal bleeding, severe jaundice, ascites, spontaneous peritonitis, hepatic encephalopathy, hepatopulmonary syndrome, portal pulmonary hypertension, kidney dysfunction, hepatorenal syndrome (among others) through active treatment/management in specialized liver units. preserving liver function and maintaining a normal physiological status have been shown to reduce the risk of therapeutic failure in patients receiving liver transplantation due to hepatitis b-related liver failure. diagnostic and treatment guidelines for liver failure the clinical implications of hepatitis b virus genotype: recent advances new complex recombinant genotype of hepatitis b virus identified in vietnam possible new hepatitis b virus genotype when should "i" consider a new hepatitis b virus genotype? a genetic variant of hepatitis b virus divergent from known human and ape genotypes isolated from a japanese patient and provisionally assigned to new genotype clinical outcome and virological characteristics of hepatitis b-related acute liver failure in the united states influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis b virus infection mortality secondary to fulminant hepatic failure in patients with prior resolution of hepatitis b virus infection in japan profile, spectrum and significance of hepatitis b virus genotypes in chronic hbv-infected patients in yunnan, china. hepatobiliary pancreat dis int precore/core promoter mutations and genotypes of hepatitis b virus in chronic hepatitis b patients with fulminant or subfulminant hepatitis virologic and clinical characteristics of hbv genotypes/ subgenotypes in 487 chinese pediatric patients with chb quasispecies and its impact on viral hepatitis hepatitis b virus: molecular virology and common mutants a function of the hepatitis b virus precore protein is to regulate the immune response to the core antigen cytoplasmic expression of hepatitis b core antigen in chronic hepatitis b virus infection: role of precore stop mutants different hepatitis b virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis b e antigen seroconversion hepatitis b virus e antigen variants two core promoter mutations identified in a hepatitis b virus strain associated with fulminant hepatitis result in enhanced viral replication type, prevalence, and significance of core promoter/ enhancer ii mutations in hepatitis b viruses from immunosuppressed patients with severe liver disease probable implication of mutations of the x open reading frame in the onset of fulminant hepatitis b association between the various mutations in viral core promoter region to different stages of hepatitis b, ranging of asymptomatic carrier state to hepatocellular carcinoma number of mutations within ctl-defined epitopes of the hepatitis b virus (hbv) core region is associated with hbv disease progression pre-s2 defective hepatitis b virus infection in patients with fulminant hepatitis associations between hepatitis b virus mutations and the risk of hepatocellular carcinoma: a meta-analysis potential threat of drug-resistant and vaccine-escape hbv mutants to public health early changes of hepatitis b virus quasispecies during lamivudine treatment and the correlation with antiviral efficacy hepatitis b virus variant with the a194t substitution within reverse transcriptase before and under adefovir and tenofovir therapy restoration of replication phenotype of lamivudine-resistant hepatitis b virus mutants by compensatory changes in the "fingers" subdomain of the viral polymerase selected as a consequence of mutations in the overlapping s gene lamivudine resistance and other mutations in the polymerase and surface antigen genes of hepatitis b virus associated with a fatal hepatic failure case adefovir rapidly suppresses hepatitis b in hbeag-negative patients developing genotypic resistance to lamivudine adefovir dipivoxil monotherapy and combination therapy with lamivudine for the treatment of chronic hepatitis b in an asian population comparison of adefovir and tenofovir in the treatment of lamivudine-resistant hepatitis b virus infection two-year assessment of entecavir resistance in lamivudine-refractory hepatitis b virus patients reveals different clinical outcomes depending on the resistance substitutions present telbivudine, a nucleoside analog inhibitor of hbv polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir management of treatment failure in chronic hepatitis b resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the hbv polymerase selection of a hepatitis b virus strain resistant to adefovir in a liver transplantation patient susceptibility to antivirals of a human hbv strain with mutations conferring resistance to both lamivudine and adefovir impact of hepatitis b virus rta181v/t mutants on hepatitis b treatment failure management of drug-resistant chronic hepatitis b treatment of patients with lamivudine-resistant and adefovir dipivoxil-resistant chronic hepatitis b virus infection: is tenofovir the answer? successful treatment of an entecavir-resistant hepatitis b virus variant how to diagnose and treat hepatitis b virus antiviral drug resistance in the liver transplant setting prevention and management of drug resistance for antihepatitis b treatment is there an association between the measurement of qualitative hbsag and virologic response in chronic hbv infection serum hepatitis b surface antigen quantitation can reflect hepatitis b virus in the liver and predict treatment response serological and molecular diagnosis hbv induced hcc: major risk factors from genetic to molecular level new insight in the pathobiology of hepatitis b virus infection quasispecies structure, cornerstone of hepatitis b virus infection: mass sequencing approach clinical implications of evolutionary patterns of homologous, full-length hepatitis b virus quasispecies in different hosts after perinatal infection hepatitis b virus mutations associated with antiviral therapy hepatitis b virus s gene mutants in a patient with chronic active hepatitis with circulating anti-hbs antibodies hepatitis b virus genomes of chronic hepatitis patients do not contain specific mutations related to acute exacerbation molecular virology and the development of resistant mutants: implications for therapy evolutionary patterns of hepatitis b virus quasispecies under different selective pressures: correlation with antiviral efficacy complex dynamics of hepatitis b virus resistance to adefovir emergence of hepatitis b virus quasispecies with lower susceptibility to nucleos(t)ide analogues during lamivudine treatment optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene an improved allele-specific pcr primer design method for snp marker analysis and its application product differentiation by analysis of dna melting curves during the polymerase chain reaction next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solution next-generation dna sequencing methods applications of next-generation sequencing technologies to diagnostic virology fast dna sequencing via transverse electronic transport single-nucleotide discrimination in immobilized dna oligonucleotides with a biological nanopore rapid sequencing of individual dna molecules in graphene nanogaps detecting single stranded dna with a solid state nanopore peri-operative therapies of patients receiving liver transplantation due to severe hepatitis establishment of a scoring system for evaluating the severity of hepatitis b patients with acute-on-chronic liver failure key: cord-006391-esnsa4u5 authors: nan title: abstracts 5(th) tripartite meeting salzburg/austria, september 9–11,1982 date: 1982 journal: langenbecks arch chir doi: 10.1007/bf01279099 sha: doc_id: 6391 cord_uid: esnsa4u5 nan the gastric secretion capacity increases during the first year after all types of vagotomy which is assumed to be important for the development of recurrent ulceration. however, the cause of this reestablishment of the preoperative gastric function is not adequately explained. the purpose of this study was to investigate the vagal innervation of the parietal cell mass in dogs 11/2 year after truncal vagotomy (tv), selective gastric vagotomy (sgv) and parietal cell vagotomy (pcv). material and methods: mongrel dogs supplied with a gastric fistula were used. the groups comprised 5 dogs with tv, 6 dogs with sgv and 6 dogs with pcv. the acid secretion was measured before and one month and one year after vagotomy following insulin and pentagastrin stimulations. operative technique: a gastroduodenostomy was performed in addition to the gastric fistula operation in the dogs randomised for either tv or sgv. the vagotomy operations were performed after the prevagotomy secretion experiments. tv by a 4 cm resection of the main truncs through a left side thoracotomy and sgv as described by amdrup [1] . the pcv dogs had no gastroduodenostomy added but the technique at the lower esophagus was identical with the sgv and the dissection of the lesser curvature was extended 1 cm distal of the sharp physiological and histologically determined borderline between the fundus and the antrum. about 11/2 year after the vagotomy the dogs were sacrificed by an acute experiment. the persistent or the regenerated vagal innervation of the parietal cell area was mapped out by neutral red excretion elicited by electrical stimulation of either the thoracic or the cervical vagal nerves [2] . results: one year after vagotomy no increase in insulin response could be demonstrated in the tv and sgv groups, but a gradual increase to about 50 % of prevagotomy output occured in all pcv dogs. reliable neutral red experiments were performed in 2 tv, 6 sgv and 4 pcv dogs. the stimmulated neutral red excretion was scanty to the same extent at the cardia region in all three groups, but a distinct antralfundic border was outlined in all 4 pcv dogs. the border was, however, only suggested in one of the sgv dogs. conclusion: pcv including denervation of 1 cm distal of the antral-fundic border do not prevent a functional important reinnervation of the distal part of the parietal cell area. no important reinnervation seems to occur at the cardia region after any of the three vagotomy procedures. 1 and insulin (0.2 ugkg -1) i.v. bolus; after uni-and then bilateral truncal vagotomy. each study was 120 minutes and blood was taken at 1, 3, 5, 7 and then 10 min intervals. gastric acid was measured by autobiuret titration. plasma was stored at -20°c until assayed for pp by ria using an antibody sensitive to 3 fmol ml-1. results are expressed as mean total increment _+ se. significance: p < 0.01". bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p < 0.05), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p < 0.01). bilateral and right hemi-vagotomy significantly inhibited pp release by bombesin (17 < 0.01), however; only bilateral truncal vagotomy significantly inhibited pp release by insulin (p < 0.01). these results suggest that the measurement of pp release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released pp may specifically delineate the integrity of the right vagus. since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance. glucose (g) or oleate (o) empty more slowly from the stomach than 0.15m nac1 (s). this chemoregulation may be achieved by resistance beyond the proximal stomach [1] . we sougth to define the site of this resistance. expt. 1: gastric emptying of s and 600 mm g was measured in 5 dogs with intestinal cannulas 45 cm distal to the pylorus while gastric pressure was maintained at 10, 18 or 26 cm h20 with a barostat. the dogs were studied either with an uninterrupted intestinal stream or with duodenal effluent diverted through a second barostat prior to its return downstream. since the latter ensured a constant pressure at the ligament of treitz, any observed effects on emptying could not be due to resistance further distally. expt. 2: emptying of s, g, and 40 mm oleate emulsion was measurd in 5 dogs without cannulas after control antroduodenal transection and after antrectomy while gastric pressure was controlled at 11, 17 and 23 cm h20. emptying rates expressed as the average of the volumes emptied at the 3 pressures. under both conditions in expt. 1 emptying rose with pressure yet s emptied faster than g. thus a major site of chemoselective resistance to emptying of liquids lies proximal to the ligament of treitz. after antrectomy s and o emptied faster than before, however, the differences between saline and nutrients were maintained. resistance, therefore, does not reside in the antrum or pylorus and clearly the duodenum is implicated. gastric emptying of liquids may be abnormally rapid in du. this abnormality my be due to a selective loss of inhibition in response to acid since previous studies [1] have demonstrated rapid emptying of acid solutions but normal emptying of glucose and fat. previous investigations, however, used meals of only one concentration, so differences between dus and normals (n) may have been minimized by selection of a supra-maximal inhibitory dose. furthermore intragastric ph was not maintained constant so emptying could have been slowed by higher rates of acidification in du. we therefore studied emptying of graded concentrations of citric acid, glucose and oleate in 500 ml meals. gastric volumes were measured by the george technique in dus and n at 5 rain intervals for 30 min after ingestion. gastric ph was maintained constant by intragastric titration (3.0 for acid; 7.0 for nutrients). subjects received one type of meal on separate days and the doses were randomly administered. dose dependent inhibition of emptying in response to acid and nutrients occured in n and du, but emptying was faster in du (p<0.05). with glucose and acid the difference occured within 0-5 min whereas with fat the difference occurred between 5-30 min. thus (1) dus do not have a selective loss of inhibition of emptying in response to acid, and (2) the mechanisms which control emptying of fat and which are disturbed in du differ from those which control emptying of glucose and acid. 1 gross ra, isenberg ji, hogan d, samloff im (1978) the effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load and serum gastrin in duodenal ulcer and normal subjects. a means of reducing the requirement for postoperative nasogastric intubation would be advantageous. we report the effect of cimetidine on postoperative nasogastric aspirates. thirty patients undergoing elective abdominal aortic surgery or colonic resection were entered into a double blind randomised study, receiving cimetidine 200 mg 4 hourly or placebo (saline) by continuous intravenous infusion from the morning of the first postoperative day. volumes of 4 hourly aspirates were recorded and ph and (h ÷) of samples measured. nasogastric tube removal accorded to standard clinical practice. it has been shown, that the duodenum has a better ability to resist acid than jejunum or ileum. this resistance was attributed to alkaline secretion (a.s.) of the mucosa, because pancreatic and bile secretion were excluded. recent in vitro studies demonstrated an energy dependent hco3-transport (flemstroem 1980 am j physiol g 239: 198). we were interested in studying the role of blood flow (bf) in the production of alkali in an in vivo preparation. min. three groups of animals were studied: i) controis with normovolemia for 120 min. ii) normovolemia for 60 min, thereafter 60 min of shock induced by bleeding to 40 mmhg mean arterial blood pressure. iii) 60 min normovolemia and 60 min vasopressin (0.2 i. u./kg/min) under normovolemia. in controls a small decrease in bfand a.s. as well was observed. both shock and vasopressin induced a significant reduction in a.s. and bf. the relation ofbf to a.s. was found to be exponential (y = 2.71 + 0.21x-0.003x 2 r= 0.71; p<0.05). the reduction in a.s. during shock was much less, when shock induced acidosis was compensated by i.v.-infusion of hco 3 (0.3 ,uequ/kg/min). conclusion: the alkaline secretion of the proximal duodenum is dependent on blood flow and the arterial [hco~. there has been considerable controversy as to the nature of the offending agent in the etiology in reflux oesophagitis in man. in rat studies, surgically induced reflux oesophagitis was shown to be correlated with the presence of active trypsin in the reflucting juice. reflux of bile and gastric juice with a ph of 4 did not induce oesophagitis. even short periods of oesophagitis (1-6 weeks) showed an increasing amount ofpanmural fibrosis which even further increased after a reflux abolishing roux-y operation. the pattern of the collagen content of the full thikkness oesophageal wall was similar to that found in human reflux oesophagitis. the hypothesis that active trypsin is also in man responsible for reflux oesophagitis prompted to the following investigation. 24 patients with typical gastrooesophageal reflux symptoms and 17 control patients were endoscopically examined. on entering the stomach with the endoscope samples of gastric juice were taken for ph and active trypsin determinations. trypsin activity was determined with a kinetic method using s-2160 (kabi vitrum b.v. amsterdam) as substrate. results: in all patients some trypsin could be detected in the gastric juice. in those patients with endoscopically erosive and/or ulcerative changes (n = 14) the trypsin content was significantly elevated compared to the controls (/7<0.05 wilcoxon). conclusions: the composition of gastric juice is determined by gastric secretion and duodenogastric reflux. duodenogastric reflux is increased in patients with symptoms of reflux oesophagitis [1] . trypsin in gastric juice (ph 3.5-7) will stable and partly active over prolonged periods [2] . so from our study in man one may conclude that the high concentration of trypsin in the gastric juice in patients with reflux oesophagitis, may well be the etiological factor of the oesophagitis. disinfection, using artificial contamination of hands with escherichia coil and 'surgical' disinfection directed against the resident microflora of the hands. the authors who developed the procedure have reported unexpectedly high potency for n-propanol compared to iso-propanol, and, in turn, for iso-propanol compared to povidone-iodine and chlorhexidine preparations [1] . however, using the same procedure we have been unable to find any significant differences in activity between these agents. we have identified several potential sources of error including the method of applying e. coliand its spontaneous loss of viability, the use of neutralizers before disinfection, the differing surfactant effects of the agents, and the absence both of a control untreated area and of a cross-over of disinfectants studied sequentially. in our parallel tests using an excision-sample technique [2] which is considerably more sensitive than the dghm procedure, we have observed the following mean reductions in the counts of accessible bacteria: iodine in ethanol, 96%; povidone-iodine, 89%; chlorhexidine in ethanol, 88%; iso-propanol, the purpose of this study was to compare radiation injury in guinea pig small bowel (1) devoid of contents (2) containing bile (3) containing pancreatic juice. one group was intact control animals not radiated. another was intact animals radiated. in group 3 a roux y cholecystojejunostomy was constructed and the bile duct ligated. thus one limb contained bile, the other pancreatic juice. in group 4 a blind roux y was constructed such that one limb was empty of contents and the other contained both bile and pancreatic juice. each animal was subjected to a single dose of 1600 rads via an abdominal port and sacrificed four days later. the severity of injury was judged by counting the number of surviving crypts per circumference. damage was further evaluated by histologic criteria -mucosal loss, edema, inflammation, hypervascularity and loss of mucus. the maximum histologlc grading score on this scale was 20. the microscopist was ,,blinded" as to the origin of each tissue section. for histologic grading all radiated groups were significantly different from control (p<0.02) and from one another (/r<0.05) except pancreatic juice vs. empty. intestine devoid of contents sustains a mild, but significant radiation injury. the presence of pancreatic juice enhances the damage. pure bile makes for yet more severe injury but still significantly less than normal whole intestinal contents which contain both bile and pancreatic juice. the main problem facing patients with ulcerative colitis after mucosal proctectomy and ileo-anal anastomosis is severe frequency of bowel action. our hypothesis was that either an artificial valve [1] or reversed ileal loop might improve intestinal absorption and slow transit. we tested it in dogs after colectomy and low ileo-rectal anastomosis (ira). body weight, xylose absorption, serum albumin, folate, vitamin b12, calcium, urea and electrolytes, full blood count, faecal weight and mouth to anus transit time [2] were measured before operation. the dogs then randomly underwent either ira alone (control, c, n = 7), ira with a 10 cm reversed loop (ira + rl, n = 6) or ira with an artifical valve (ira + v, n = 6). body weight, haematological estimations and faecal chemistry were measured weekly for 3 months post operatively. xylose absorption and transit time were measured a minimum of 2 months after operation. body weight decreased significantly after each type of operation. there was no difference however, between (ira + rl) and c or (ira + v) and c. likewise, haematological and faecal measurements after both test operations did not differ significantly from c or from pre-op, measurements. the grosfeld valve prevented the reduction in transit time that occurred after the control operation, whereas the reversed loop did not. use of such a valve in combination with mucosal proctectomy might slow transit, but is unlikely to improve absorption. it seems worthy of further study. histologic grading___ sem crypt count-+-sem we have examined the effects of changes in intestinal blood flow induced by hypovolaemia, the mesenteric vasoconstrictors somatostatin and vasopressin and the mesenteric vasodilator prostaglandin e1 (pge1), on intestinal absorption, electrical activity and volume. in each of 7 dogs a 75 cm jejunal segment was isolated and serosal electrodes attached. in absorption experiments the segments were perfused at 2.9 ml/ rain with a solution of 90 retool/1 sodium and 100 retool/1 glucose. to measure segment volume, a solution of 50.7 g/1 mannitol was perfused. this solution shows zero net absorption but does not alter electrical activity. intestinal motility and absolute volume were monitored by gamma camera. experiments were performed after a steady state was reached. absorption and transit time were measured using non-absorbable markers. electrical fast wave activity was reduced by (a) intravenous somatostatin 2.5 mcg/kg/h (28+1.4 to 11_+3.4; mean_+sem/5 rain) and abolished by (b) intravenous vasopressin 1.2 u/kg/h (p<0.001). motility was inhibited and mean transit time was prolonged (a) 7.4 to 15.3 rain and (b) 4.4 to 19.9 rain (/r<0.001). intestinal volume rose by (a) 12.3___ 3.7 ml and (b) 15.4-+4.7 ml (p<0,001). absorption was unchanged by somatostatin and fell with vasopressin (5.58---0.7 to 4.35-+0.68 ml/5 min). acute blood loss sufficient to lower the central venous pressure by 5 cm of water produced identical changes to somatostatin. pge1 produced no alteration in electrical activity or absorption. intestinal absorption is resistant to changes in intestinal blood flow whilst electrical activity is depressed and intestinal volume increased by mesentric vasoconstriction. the accepted views on the pathogenesis of amoebic lesions in complicated amoebic colitis are that amoebae produce a toxin resulting in cytolysis and necrosis. this concept may adversely affect the management of patients with complicated amoebic colitis by implying that once the amoebae are killed, the disease process is arrested and colonic perforation is unlikely. we present an alternative hypothesis that 'complicated amoebic colitis is the result of vascular compromise'. transmural disease is caused by amoebic invasion of vessels supplying a segment of the colon with subsequent thrombosis and ischaemic necrosis of the affected area. the ischaemic nature of the necrosis is suggested by its shape, and the demonstration of vascular thrombosis on dissection ofresected colons which have perforated. amoebic invasion of blood vessels can be demonstrated histologically. the ischaemic nature of the lesions can be confirmed by angiographic examination of the resected colon. vascular occlusion can be demonstrated pre-operatively with the use of selective mesenteric angiography, which clearly delineates the ischaemic segment of the colon. selective rnesenteric angiography in 27 patients with fulminating amoebic colitis aided the preoperative diagnosis of colonic infarction before signs of visceral perforation had developed and permitted life saving surgical intervention. there is accurate correlation between angiographic and operative findings. in postdysenteric colitits associated with amoebic strictures of the colon, mesenteric angiography will demonstrate the ischaemic nature of the stricture and accurately define the extent of resection. after ca. the mean age at operation was 33 and 61 years respectively. anal canal length was 3.5___0.1 cm (mean + s.e.m.) after ia and 3.7___0.1 cm after ca. a resting tone of 66___ 3 cm water after ia and 53 -----10 cm water after cawas recorded. squeeze pressure was 123___9 cm water after ia and 79-----3 cm water after ca. the recto-anal reflex was present in 54% of the ia patients and in 75 % of the ca patients. a mean daily bowel frequency of 3.5___0.2 followed ia and 4+_1 after ca. these findings show that after ia anal pressures are within the normal reference range for our laboratory [2] . lower pressures were found after ca, probably due to the older age of the patients in this group [3] . normal anal canal length, the integritiy of the striated sphincter and in most cases preservation of the recto-anal reflex contribute to satisfactory continence following peranal anastomoses. perineal descent is found in patients with idiopathic faecal incontinence (ifi) and patients presenting with symptoms of the descending perineum syndrome (dps), who have no incontinence. manometric, radiological and neurophysiological studies were performed in 17 patients with ifi, all of whom leaked during rectal infusion of 1500 ml saline, 18 patients with dps, who were continent of rectally infused saline, and 14 matched controls. both patient groups exhibited similar degrees of perineal descent below the pubococcygeal line of straining (ifi, -5.1___2.1 cm; dps, -5.9 + 1.7 cm) compared with controis (-2.4__+ 1.5 cm;p<0.005 in both cases), and similar prolongation of both the latency of the cutaneoanal reflex (ifi, 13___5 ms; dps, 16+6 ms; controls, 9+4 ms;/,<0.005 in both cases), and motor unit potential duration (ifi, 9.1___2.6 ms; dps, 8.4___2.0 ms, control, 6.7+1.5 ms; p<0.01 in both cases). moreover, both groups had an abnormal ano-rectal angle (/,<0.005), though this was more obtuse in ifi than dps (120___21 ° vs 107___ 15°;p<0.05). however, while patients with ifi had lower sphincter pressures than normal (basal; 39___17 vs 81___29 cm water; /7<0.005; squeeze, 96___18 vs 208___84 cm water; /7<0.001), and required a lower rectal volume to inhibit sphincter tone for more than one minute (32 + 18 vs 76___29 ml; p<0.00l), these values were normal in patients with dps (basal; 87___ 30 cm water; squeeze, 198_+90 cm water; rectal volume, 52___46 ml). these findings suggest that perineal descent and neuropathy are not necessarily associated with incontinence if sphincter pressures remain normal. aminoacids administered either enterally or intravenously stimulate gastric secretion in both dog and man. it has been suggested that absorption of amino acids from the gut into the circulation might contribute to the intestinal phase of gastric secretion. the mechanism of this stimulation by amino acids remains uncertain but in an earlier communication we reported the direct action on the parietal cell of phenylalanine, glutamine and alanine [1] . in the present study using (14c) aminopyrine uptake as an index of dispersed parietal cell secretory function, the interaction between histamine and individual amino acids and the effect of the histamine h2-receptor blocker ranitidine (10-4 molar) on these interactions have been examined. results (percentage of maximal stimulation produced by 10-4 molar histamine) results (percentage of maximal stimulation produced by 10-4molar histamine) methionine glutamine alanine amino acids only (10-2molar) 26 32 29 amino acids and histamine (10-4molar) 175 -137 amino acids and ranitidine (10-4molar) 10 17 7 histamine and ranitidine -complete inhibition -25 125 1. the response to the combination of amino acids and histamine was greater than the sum of the maximal responses to each of these agents alone. 2. the histamine h2-receptor antagonist ranitidine completely inhibited the histamine stimulated response but only partially inhibited secretion stimulated by amino acids. we conclude that amino acids act directly on the parietal cell by a mechanism which is not solely dependent on histamine. 1 sex related differences in gastric acid secretion have been documented (lilja et al, 1967) . it has been also reported that male rats have a significantly higher serum gastrin concentration than females and that oophorectomy increases serum gastrin to male levels (lichtenberger et al., 1976) . estimation of serum gastrin levels after administration of androgens has not been reported. aim. the aim of this study was to investigate the serum gastrin levels before and after oophorectomy and administration of estrogens and testosterone in female guinea pigs. method. 4 groups of guinea pigs were used for this study, 8 animals as controls, 6 were given estrogens for 2 weeks, 8 were given testosterone for 2 weeks and 5 underwent oophorectomy. results. the mean serum (-+sem) gastrin value in controls was found 35+ 1.63 pg/ml, after estrogens 13.33-+1.22 pg/ml, after testosterone 55_+1.89 pg/ ml, after oophorectomy 46-+2.45 pg/ml. conclusion. it is concluded that estrogens decrease the serum gastrin, and that the increase of serum gastrin after administration of androgens and oophorectomy is statistically significant (/r<0.01). the mechanism by which the ovarian hormones influence gastrin levels may be a sex-dependent change in the synthesis or secretion of gastrin. the reported inhibitory influence of estrogen on food consumption may be also the predominant factor in serum gastrin alterations. mean caloric intake was 2336--+427 kcal/day. moderate to severe dumping occurred in one patient. faecal fat was normal in 11 while 7 patients had greater than 10 grams fat loss per day. weight loss was 14.6_+8.6% of recall weight. muscle mass measured by arm muscle circumference (25.7_+3.9 cm) and creatinine heigth index (99___26%) was normal for our patients but triceps skin fold was 30% less than normal (8.6_+2.2 mm) indicating that in these patients fat stores were reduced. in six patients haemoglobin was less than 13 grams per cent but serum iron, iron binding capacity and serum folate were normal. red cell folate was depressed in six patients whose haemoglobin values were normal. it is concluded that with this reconstruction total gastrectomy produces satisfactory digestive and "nutritional results. examination of factors affecting faecal fat loss and folate metabolism may help improve the nutritional status of these patients. in order to assess the optimal conditions for segmental pancreatic graft viability the following experiment was conducted. the duct obliterated splenic lobe of the canine pancreas was autotransplanted by end to side fashion to the femoral vessels (n = 10). the graft was lodged in a subcutaneous pocket. a month later total pancreatectomy was completed. the dogs were supplemented with pancreatic enzymes (viokase). mean survival was 42___ 12 days. deaths were due to hyperglycemia when grafts lysed from local infection and thrombosis. in the second experimental group (n= 10) the autografts were anastomosed to the iliac vessels and placed intraperitoneally followed by total pancreatectomy one month later. construction of a distal splenic arterio-venous fistula increased blood flow from 31.4 cc/min to 108 cc/min. the pancreatic duct was obliterated with neoprene (n=4) silastic (n=3) or left open (n=3). three dogs died one month to four months due to graft fibrosis and failure. seven dogs had been followed from four months up to seven months when sacrificed. all were normoglycemic. mean k values for ivgtt were 1.5. the normoglycemic dogs had mean plasma concentrations of amino acids similar to healthy controls. a two to threefold elevation was observed in pancreatectomized diabetic dogs for plasma leucine, isoleucine and valine. immunoreactivity of pancreatic polypeptide was measured by radioimmunoassay with an antibody raised against the human hormone by re. chance, lilly research laboratories. mean plasma levels rose form 256--+28 s.e. pg/ml to over 1000 pg/ml following protein meal (20g ground lean beef/kg) in all normal control dogs (n=4), and to levels in the range of 390 to over 1000 pg/ml in the same dogs following infusion ofbombesin (1 pg/kg/ h) for 15 min. mean basal levels were lower (42___ 2.2 s.e. pg/ml) in dogs with autotransplants and did not increase significantly during any stimulatory test. the levels of pancreatic polypeptide did not distinguish between viable and failing grafts. in successful grafts histology showed fibrosis of the exocrine component. transmission electron microscopy revealed the presence of clusters of functional islet cells in the six-month implants. a and b cells were common. d cells were much less frequent. release of secretion granules into the perivascular connective tissue space was observed. the results indicate that vascularized segmental pancreatic graft comprising 25 % to 30 % of the pancreatic mass maintains normal carbohydrate and amino acid metabolism. graft survival was extended by intraperitoneal location and creation od distal splenic a-v fistula, but chronic graft fibrosis occurred regardless of the method of pancreatic duct treatment. the results further suggest the necessity of an intact vagal innervation for the physiological or pharmacological stimulation of pancreatic polypeptide release from thendocrine pancreas which is otherwise apparently functional. besides, these considerations rule out pancreatic polypeptide levels as a useful marker for evaluation of pancreas graft. polyisoprene ductal occlusion allows segmental pancreatic transplantation in man with satisfactory early islet cell function. however acute rejection remains a problem as the diagnosis, based on hyperglycaemia and glycosuria, represents a late manifestation of rejection. although llqndiumlabelled platelets have been used in the diagnosis of renal rejection [1] , their use in pancreas transplants has not been studied. ~ in 6 patients, autologous llqn-labelled platelets were injected on the second, sixth and tenth days following combined renal and pancreatic transplantation. graft radioactivity was expressed as the ratio of counts from the pancreas over a reference area on daily gamma images for 14 days. one patient developed hyperglycaemia coinciding with renal rejection on day 5. over the previous 24 h pancreas radioactivity had increased by 94 %. in a further patient whose pancreas continued to function, a perigraft haematoma was recognised on the gamma image. the remaining 4 patients had good pancreatic function and no 11xin-platelet accumulation: mean (_s.c. mean) pancreatic radioactivity was 1.76+0.190 on the third postoperative day and 1.75_+0.135 at the end of study. these results demonstrate that ductal occlusion with polyisoprene does not cause significant platelet accumulation. hence min-platelets may potentially be used for the earlier diagnosis of pancreatic rejection. isograft models of pancreatic transplantation, methods involving closure of the duct system result in severe inflammatory changes and eventual fibrosis [1] . these changes can be avoided by formal drainage of the duct into the bowel of urinary tract. inflammatory changes initiated by duct closure may contribute to and enhance allograft rejection. pancreas transplants were performed in rats using lewis (rt11) donors and streptozotocin-induced diabetic da(rt1 a) recipients, using duct-ligation, open duct and ureteric duct drainage. cyclophosphamide produced significant prolongation ofnormoglycaemia in all groups and although there was a tendency towards longer function in the unligated groups there was not statistical difference (wilcoxon's unpaired test) between the methods of duct management. management of the pancreatic duct did not seem to have any immunological consequences for graft survival but septic complications, associated with the normoglycaemic deaths, were more common in immunosuppressed animals with draining ducts. between july 1, 1980 and march 30, 1982 combined pancreatic and kidney transplantation was performed in 9 patients with type i diabetes who had all been on insulin therapy for at least 16 years. age at the time of transplantation was 28 to 45 years. the pancreatic segment used for transplantation consisted of body and tail of the organ based on a vascular pedicle of the celiac axis and the splenic vein. imediately prior to intraperitoneal transplantation to the iliac vessels the ductal system was filled with 4 to 6 ml of prolamine, a rapidly solidifying alcoholic protein solution. simultaneous kidney transplantation was performed through a separate incision. five pancreas transplants were lost for non-immunological reasons. four of them never had any useful endocrine function, one graft was lost of venous thrombosis after 48 hours of excellent function. in none of these patients did failure of the graft lead to any substantial complication. in the 4 remaining patients initial graft function was excellent with plasma glucose normalizing within 12 hours and subsequent normoglycemia on a regular diet without exogenous insulin administration. plasma glucose did not rise spontaneously during rejection episodes of the kidney and eleva-tions due antirejection treatment were promptly; reversed as high dose prednisolone was discontinued. in oral glucose tolerance test (ogtt) performed at 4 to 6 weeks in 3 patients median glucose rose from a basal 5.1 mmol/l to 8.6 mmol/1 and was back in normal range (4.7 mmol/1) at 3 h. basal insulin was 43-237 pmol/1 and also returned to the normal range after 3 h after a peak of 201-531 pmol/1 at 50-210 rain. c-peptide showed a significant 2 peaked rise over basal values (230, 233 pmol/1) in 2 patients while all values were above 4500 pmol/l in the third. two of these patients have since rejected both organs. two tranplants still function very well after 12 and 21 months, respectively. in one of these patients the ogtt after 13 months is even slightly better than the above -mentioned first test, and he shows a norreal insulin and c-peptide response. in the fourth patient an ivgtt performed at 7 months and an ogtt at sixteen months were normal with insulin peaking at 251 pmol/1 and a c-peptide peak of 1267 pmol/1 at 60 min in the latter. -results at 26 and 17 months, respectively, will be presented. three problems persist in clinical organ preservation. these are failure of current systems to replenish the ischemically injured organ, to reliably extend the period of organ preservation and to definitely determine organ viability. previous studies have documented the value of electrochemical redox control in rejuvenation of the ischemically injured kidney during perfusion preservation. this study was undertaken to develop the cost effective technique applicable to current organ preservation systems, to test the reliability of redox measurement in prediction of organ viability and to determine the ability ofredox maintenance to safely extend the period of organ preservation. a disposable cell has been developed using reticulated vitreous carbon as an electrode which is driven by a potentiostat powered by a nine volt transistor battery. short term preservation studies using ischemically injured dog kidneys (60 rain in-situ warm ischemia) were auto transplanted after 24 h of pulsatile preservation to determine the optimum redox level of the hypothermic kidney. this was determined to be a -20 mv vs the standard calomel electrode. twenty-one adult mongrel dogs were equally divided into three groups. pulsatile perfusion preservation was extended to four days in group a with redox level monitored only. group b was treated with electrochemical reduction for four days and group c for six days. three of seven dogs survived in group a following auto transplantation and immediate contralateral nephrectomy. the kidneys of all survivors were able to bring perfusate redox potential under control and maintain this level throughout the preservation period. six of seven dogs in group b survived with a mean posttransplant peak serum creatinine of 4.5 rag/100 ml. in group c four of seven kidneys supported life immediately. all redox controlled kidneys made copious amounts of urine. our data indicate that perfusate potential may be either monitored as a reliable index of organ viability or controlled to allow extended safe preservation. antithymocyte globulin (atg) has been shown to be an effective agent in combination with increased doses of steroids in reversing renal allograft rejection. since it is frequently undesirable to employ raised doses of steroids, the following study evaluated the effect of 15-21 i.v. daily doses of horse atg alone without additional steroids, on 2nd-5th rejection episodes in 10 recipients of cadaveric renal allografts (28 rejections: 25 biopsy-proven) detected within 3-18 mos. of transplantation. of 28 rejection episodes, 25 were successfully reversed, with return of serum creatinine to pre-rejection levels in 18 episodes (7 patients). three patients had primarily humoral rejections and returned to dialysis 2-4 mos. after treatment of the last rejection. levels of circulating horse immunoglobulins were obtained in all 10 patients during and follwing administration of atg. recurrent rejections (3rd-5th) following last treatment with atg (3-11 mos.) were seen in 5/10 patients. all 5 patients had rapid immune eliminiation of atg (1/2 life 2-4 days) as compared to the 5 patients who had no more than 2 rejection episodes (1/2 life 6-14 days). atg without additional steroids is an effective agent for reversal of multiple renal allograft rejections which by biopsy are primarily cell-mediated. to be effective, heterologous atg must be given in adequate total doses and/or from appropriate heterologous source, to prevent rapid immune elimination by the recipient. the use of atg alone for treatment of recurrent allograft rejections is particularly recommended for its steroidsparing effect in treatment of multiple rejections and for those patients at high risk from steroid side effects. the significance of the monocyte crossmatch in lrd recipients of hla identical kidney grafts j. cerilli, l. brasile and s. rogers ohio state university hospitals, columbus, ohio, usa in a preliminary study from our transplant center, the presence of pre-formed antibody in recipient sera directed against monocytes from their respective living-related donors correlated with a poor clinical course. a poor prognosis for graft survival was found regardless of the hla match grade. to minimize the role of the hla system, only those living-related recipient/donor pairs who were hla identical at the a, b, c, d and dr antigen loci, and who exhibited severe immunological types of rejection were evaluated. due to the small numbers found in this category at any one center, this abstract represents an international study from 12 different transplant centers. 26 patients who met the criteria were studied for the presence of antibody directed against their respective donor's monocytes both pre-and posttransplant. in eighteen of these patients, cytotoxic antibody against their donor's monocytes was found in their pre-transplant sera. there was no detectable cytotoxic activity against their donor's t or b lymphocytes. two additional transplant recipients exhibited this antibody in post-transplant sera. again, no t or b lymphocyte cytotoxicity was detected. a control group of hla identically matched siblings who incurred no or minimal rejection demonstrated no anti-donor monocyte antibody. the results of this international study points towards a correlation between a high incidence of graft rejection and the presence of antibody directed against their respective donor's monocytes. therefore, in our view, the presence of anti-monocyte antibody to the prospective donor pre-transplant is a contraindication to transplantation. in patients with different liver diseases the reduced concentration of the peripheral blood t-cells and altered immune response were observed. the purpose of our studies was to investigate the mechanism of the immunological modification of the lymphoid system in the hepatic injury. studies were carried out in 3 groups: 1) lew rates were treated with dimethylnitrosamine (dmna), 35 mg/kg b.w., i.v. for acute liver necrosis induction, 2) cc14 (0.1 ml/100g b.w., i.v.) twice weekly, over 4 weeks for chronic liver damage, 3) ccl 4 over 10 weeks for liver cirrhosis (histologically examined). serum and thymus, spleen, mesenteric lymph nodes (ln) were removed 2 and 32 days after dmna treatment and 5 and 35 days after last cc14 injection. the total number of thymocytes and spleen cells was counted and the reactivity to pha and cona was measured. normal liver perfusate (lp) was prepared 6 h after removing (20°c) by 5 times perfusion in 30 rain intervals (flow rate 1 ml/g/min with 2 ml/g of ringer). the effect oflp, sera, dmna and cc14 on the viability and pha response of normal thymocytes was tested in vitro. liver enzymes were evaluated, we found the decreased total number of thymocytes immediately after the stopping of medication (group 1: 9,2___ 1,8 %, group 2: 4,6___ 1%, group 3: less than 1% of control). proliferative response of the remaining cells in thymus to pha was much higher than normal thymocytes (group 1:325 ± 30 % group 2: 608___164%, group 3: nd). the total number of spleen cells was not changed but their response to cona was altered in the all groups and to pha only in group 3. pha response of ln-cells was decreased in the all groups. liver perfusate was cytotoxic (53___3 % of viable cells) and suppressive for pha response of thymocytes (6_+5% of control). the sera of rats with the hepatic damage showed an enhanced suppressive activity. cc14 and dmna did not have any effect on thymocyte in vitro. one month after last medication we observed the recovery of thymus involution (total i in the acute and partial in the chronic hepatic damage and cirrhosis). our results suggest that the liver origin cytotoxic and immunosuppressiv e factor(s) can be released from the damaged liver into the circulation (like to lp) and can destroy the thymus leading to the secondary changes in the other lymphoid organs. the grade of thymus alteration is dependent on the degree and the duration of hepatic damage and is reversable. the hepatic factor (s) showed the similar effect on the cortical population of thymocytes like the steroid immunosuppressants. liver grafts in the rat are in certain strain combinations not rejected and in this situation there is evidence for spontaneous donor specific tolerance [1] . we have developed a model of auxiliary liver transplantation which would allow us to study the immunosuppressive properties apparently produced by a liver allograft. the portal vein is anastomosed to the left renal artery, the i.v.c. to the renal vein and the bile duct to the ureter. simultaneous kidney or heart allografts were performed. conclusions:auxiliary liver grafts are rejected. survival of heart or kidney grafts is not influenced by a simultaneous kidney or heart allograft. heart and kidney grafts are prolonged by simultaneous auxiliary liver grafts. [1] . the role of suppressor cells in the initiation and propagation of malignant tumours in man, is less clearly defined. the present study, using in vitro mitogen assays (pha, cona, pwm) and various rosetting assays [2] with specific monoclonal antibodies to lymphocytic helper (okt4) and suppressor (okt8) cells, has revealed the presence of such suppressor lymphocytes in women with clinically localised (breast and axilla) mammary carcinoma. lymplaocyte hyporeactivity to mitogens was found in 30% of lymphocyte preparations from blood and axillary lymph nodes of patients with breast cancer. in 10% of patients nodal lymphocytes were totally anergic. the most profound hyporeactivity, however, was detected in the lymphocyte subsets (<75 %) of specimens isolated from the breast carcinomas by collagenase digestion and sephadex g-10 column passage [3] . the lymphocyte preparative techniques were not responsible for the low levels of responses detected. also, in situ prostaglandin synthesis and release did not appear to be involved in depressing lymphocyte reactivity [4] . comparable percentages of suppressor cells (okt8+) were detected within these different lymphocyte preparations. suppressor cells were not found in the lymphocyte preparations from the blood and lymph nodes of appropriate controls. from clinical and experimental studies it is known that blood transfusions may have immunosupressive as well as immunostimulating consequences. the effect of transfusions on graft survival has been extensively studied by our group in the bn to wag rat model. in this donor-host combination it was found that a donor specific pre-transplant blood transfusion could lead to a marked prolongation of heart and kidney graft survival, whereas the similar pretreatment resulted in accelerated rejection of bn skin allografts. this specific model was used to investigate the influence of a single bn transfusion on the growth of 2 different syngeneic transplantable tumors in wag rats. the first tumor was a radiation induced basal cell carcinoma of the skin (t 1), the second tumor was a chemically induced adenocarcinoma of the duodenum (t 2). the antigenicity of both tumors was assessed in vivo, using classical challenge-protection experiments. it was observed that t i exhibited strong immunogenetic properties, whereas t 2 was only weakly immunogenic. the doubling time oft i was 2.5 days, the doubling-time of the adenocarcinoma was 14 days. intravenous inoculation of isolated t 1 cells led to development of lung nodules which could be counted after 14 days. wag rats were injected i.e. with 1 ml of bn blood or syngeneic blood (controls) at 7-14 days before tumor challenge. t l was given in two different ways: a) sc. implantation of+2x2 mm pieces, b) i.e. injection of 106 isolated tumor cells. t 2 was implanted sc. only. each experimental group consisted of 8 animals. for t 1 it was found that allogeneic blood transfusions caused a slight (but not significant) inhibition of subcutaneous tumor growth. however, in the t 1 lung-metastasis model it was observed that a single bn blood transfusion led to a 50 % reduction of nodules, counted at 3 weeks after inoculation. this reduction in number and size of nodules was highly significant (p<0.01). for tumor t 2, the bn blood transfusions evoked a strong inhibitory effect on the growth of the sc. implanted tumor. at 8 weeks after implantation all tumors in the control group had grown to a diameter of 10-16 rnm (average diameter 12.2 ram). in the group pretreated with bn blood, only 3 of 8 tumors were palpable at that time (average diamter 4 mm). for t 1 it was further investigated whether a single bn transfusion, given one week after i.e. tumor cell inoculation, would have any influence on tumor growth. no significant effect on number or size of the lung nodules could be noticed, if anything, the transfusion appeared to have a stimulatory effect. the results indicate that allogeneic transfusions can lead to a substantial modification of tumor growth, depending on tumor type and site of implantation. this observation may have important clinical implications. we report here the serial study of circulating immune complexes (cic) in two human tumor systems, colorectal cancer and gestational trophoblastic neoplasia (gtn). cic were assayed by antigen nonspecific insolubilization induced by 3.75 % polyethylene glycol (peg) and monitored as a od45o changes by spectrophotometry. all 11 of the serially studied colorectal cancer patients presented with elevated cic levels (mean = 610 + 396 zt od450) as compared to our standard cic level for pooled normal human sera (202--+_4 aod450, p<0.05). initial values in these patients range from 304 to 1407 a od450 with no correlation to tumor load, site of presentation, or subsequent clinical course. in 6/7 patients who underwent ,,curative" resection of primary or metastatic colorectal cancers, serial cic elevations occured only when antigen excess (measured by simultaneous carci-noembryonic antigen [cea] assay) decreased. immunoglobulin components of fractionated cic showed predominantly iga subclass. in 30 gtn patients followed with serial cic and simultaneous human chorionic gonadotropin (hcg) assay, only those patients documented to enter hcg remission after molar evacuation showed significant elevation of cic. chromatographic fractionation of peak cic in one such patient defined three irnmunoglobulin containing fractions showing immunoreactivity to one of four paternal hla haplotypes (aw32). one ,,antigen" fraction (<25.000 mw) from this complex completely inhibited reference anti-aw32 binding. as in the colorectal cancer patients, these data show that cic rise only when antigen excess decreases (reflected in the gtn patients by hcg normalization). in addition, some gtn patients may react to immunogenic paternal hla haplotypes as part of their response to molar pregnancy. dfmo, an enzyme-activated irreversible inhibitor of ornithine decarboxylase (odc), reduces tumor polyamine levels, inhibits growth of emt6 sarcomas and hepatomas in experimental animals, and induces remission in human leukemia. a renal adenocarcinoma (ra) cell suspension, or a 1 mm segment ofwilms' (wm) tumor was transplanted intrarenally into balb/c mice (n--105) or subcutaneously into wistar-furth rats (n=80) respectively. dfmo (2 %) in drinking water was administered to half the animals in each group throughout the experiment. at 28 days ra tumors in dfmo-fed mice weighed 72 % less than tumors in control animals (p<0.001); wm tumor weight at 28 days was not affected by dfmo feeding. the mean number of lung metastases in dfmo-fed r.a-bearing mice was 0.1 and in ra-bearing control mice was 1.4 (p<0.001). dfmo caused 72-75 % inactivation of tumor odc, reduced ra putrescine levels by 710/o (p<0.001), reduced wm putrescine levels by 65% (d7<0.01), reduced wm spermidine levels by 58% (p<0.001) and increased wm spermine levels by 37% (p<0.01). dfmo feeding did not alter dna content of ra or wm tumors. although final carcass weight was similar in all animals, dfmo feeding progressively reduced total body weight (tbw) of mice, but not rats, until at day 20 the tbw of dfmo-fed mice was 10.5% less than tumor-bearing control mice (p<0.01). dfmo-fed mice bearing ra tumors survived 3.0-t-0.8 days longer than control mice (p<0.05). reduction of polyamine levels in wilms' tumors does not affect tumor growth. lowering of renal ade-nocarcinoma putrescine levels by continuous feeding of dfmo to tumor-bearing animals decreases tumor growth, reduces lung metastases, and increases host survival. we have previously demonstrated that vagal nerve stimulation releases 5-ht into the lumen of the feline gut. this study was initiated to: 1. determine if substance p (sp) and motilin (mt), other enterochromaffin cell products, are released simultaneously, 2. to evaluate if this release is under cholinergic or adrenergic control. in 6 cats, 15cm isolated in situ segments of proximal jejunum were perfused with saline at 1 ml/min (37 ~c). perfusate samples were evaluated at 5 rain intervals and concentrations of 5-ht, sp, motilin were measured by ria's developed in our laboratory. after two 5-min basal periods, the supradiaphragmatic sectioned vagus nerves were stimulated electrically (10v, 5m s, 10hz). the output from the loop in ng/5 min (5-ht) and pg/5 rain (sp and mt) was: introduced into the jejunum of conscious dogs through an external small bowel fistula. the gut was perfused at 5 ml min-1 with a physiological electrolyte solution containing the non-absorbable marker polyethylene glycol (mol. wt. 4000; 5 g 1-1); water and electrolyte absorption and transit time (tt) were measured during intravenous (i.v.) administration of each peptide, and during preceding and succeeding i.v. control infusions of 0.15m naci. separate studies showed jejunal absorption and tt to be constant over prolonged periods during i.v. nac1 administration. bombesin (10 pmol kg-lmin-1) and neurotensin (1 pmol kg-~min 1) significantly reduced jejunal water absorption; bombesin and enkephalin (0.5 nmol kag-1rain-1) significantly prolonged tt; and enkephalin encreased water absorption (/7<0.05 in all cases). measurement of plasma neurotensin during i.v. infusion indicated that physiological blood levels were not exceeded during these studies. conclusion: a number of peptides may be involved in the regulation of small bowel function. the effect of neurotensin on jejunal water transport provides a possible mechanism linking raised blood neurotensin levels with intestinal intraluminal fluid accumulation in the dumping syndrome. in order to establish whether this release was under adrenergic control, 4 cats had cervical ganglionectomies. using the same electrical paramenters, stimulation of the cut cervical vagus nerves resulted in identical 5-ht responses as above. in 4 additional cats atropine administration (lmg/kg iv) totally abolished the 5-ht responses to vagal nerve stimulation. the paralled release of 5-ht, sp, and mt following vagal nerve stimulation, strongly suggest that the ec cell is the source of these luminal hormones. this release appears to be under cholinergic control. since diabetes mellitus is markedly improved immediately after jejunoileal bypass before significant weight loss, but only gradually and often incompletely changed after gastric bypass, it seemed appropriate to investigate the effects of intestinal exclusion on experimental diabetes. studies were performed on alloxan diabetic sprague-dawley rats. two days after jejunal exclusion (je) in 8 rats (resection of proximal 1/3 of small intestine), fasting blood sugars (fbs) decreased 580, from 344 to 146mg/dl, and averaged 164 mg/dl at 3 weeks. after ileal exclusion (ie) in 7 rats (resection of distal 1/3 of small intestine), fbs fell 39% in 2 days, from 250 to 153 mg/dl, but increased 40% above preoperative levels to 350 mg/ dl at 3 weeks. sham operated rats responded similarly to ie rats. after alloxan and operations, all groups lost weight, but only je rats began to increase weight at a normal rate. increased water intake, polyuria (140 ml/24 h), and glycosuria (2756 rag/all), were present in ie rats; je rats were normal (urinary output <20 ml/24 h, and urinary glucose 36 mg/dl). oral glucose tolerance tests (gtt) were extremely abnormal in ie rats, similar to those in non-operated alloxan rats, while gtt curves in je rats were similar to normal animals, with some elevation at 30, 60 and 120 min. serum insulin levels remained low in all alloxan treated rats after jejunal exclusion. possible mechanisms, currently under study, relate to carbohydrate malabsorption and changes in enteric chemical mediators. the purpose of the present study was to investigate the changes in somatostatin release and somatostatin-containing cells of the pancreas and stomach of the streptozotocin (stz) -induced diabetic rat after the amelioration of diabetes by whole pancreatic transplantation. highly inbred lewis rats were divided into three groups: (1) normal rats, (2) stzinduced diabetic rats and (3) transplanted rats. diabetes was induced by the administration of stz (60 mg/kg). on the seventh day after stz treatment, pancreatic transplantation was performed. four weeks after the transplantation, in vivo and in vitro, studies were performed. pancreatic d cells and gastric somatostatin-containing cells were stained with antibody enzyme method. studies in vivo showed marked improvement of the impaired arginine-induced insulin release by the transplantation. studies in vitro employing isolated perfused rat pancreas and stomach revealed following results: mean basal pancreatic somatostatin release in normal, diabetic and transplanted rats were 12___3, 24-t-7, and 17__+4 pg/ml, respectively. total amount of pancreatic somatostatin release in each group during arginine stimulation (19.2. mm) were 946--+200, 2321___266, and 1013-+126 pg/15 min, respectively. significantly higher somatostatin release was obtained from the diabetic pancreas, which was, however, reduced to normal after the whole pancreatic transplantation. on the other hand, insulin release from the diabetic pancreas was severly impaired and pancreatic transplantation had not effect on insulin release from the host pancreas in the transplanted rats. as to the glucagon release, there was not significant difference among them. mean basal gastric somatostatin release in normal, diabetic and transplanted rats were 179-t-5, 173___5, and 135 + 5 pg/ml, respectively. there was no significant difference between normal and diabetic rats, though the significant decreased value was obtained in the transplanted rats. (vs. normal;/7<0.01, and diabetes; /7<0.01). on the other hand, glucagon-stimulated peak values in these groups were 498_ 36, 662___47, and 412+25pg/ml, respectively. glucagon-stimulated gastric somatostatin release in diabetic rats was significantly increased, but reduced to normal value by pancreatic transplantation. also, a number of pancreatic d cells and gastric somatostatin-containing cells were markedly increased on the diabetic rats. on the other hand, a number of these cells in the transplanted rats were descreased to normal levels. in summary, enhanced pancreatic and gastric somatostatin release and cells in the diabetic rats were both normalized after the amelioration of diabetes by the whole pancreatic transplantation. from these results, it is suggested that pancreatic and gastric somatostation are regulated by circulation and/or metablic of nutrients. doppler velocity recordings are widely used for the non-invasive diagnosis of carotid arterial disease. although detailed analysis of carotid doppler spectral information has been suggested as a method for improving diagnostic sensitivity, the accuracy of the relationship between the doppler recording and the true instantaneous velocity profile has not been established. the purpose of this study is to determine if a cw doppler velocitymeter can accurately transduce the true instantaneous blood flow velocity information. methods: a pulsatile flow model has been constructed in which it is possible to record the instantaneous doppler spectrum and simultaneously photograph and measure the true velocity profile. a computer controlled pump generates a carotid waveform in tubes without stenoses and with, symmetrical stenoses. flow is visualized using the photochromic dye tracer technique. a short burst of uv light from a laser is passed across the tube. a narrow band of the fluid turns blue, its movement is photographed, and the instantaneous velocity profile determined every 10 msec throughout the pulse cycle. at the same time, the instantaneous doppler spectral information is recorded by a frequency analyzer. the results follow. for pulsatile laminar flow, the doppler spectrum correctly recorded the true velocity spectrum, including the instant/~neous maximum velocity and mean velocity. for disturbed flow, it was not possible to show the same direct relationship between the doppler spectral recordings and the blood flow velocity. in conclusion doppler velocitymeters accurately transduce velocity information when flow is laminar but when flow is disturbed there is not a direct relationship between doppler recordings and the true velocity profile. consequently, one should be cautious in attempting to relate doppler measurements of disturbed flow directly to the true changes in the velocity pattern. early failure of arterial reconstruction may originate in poor patient selection. in aorto-iliac stenosis (ais) selection for operation relies upon clinical examination of the femoral pulse and radiology. since single plane arteriography is inadequate for accurate definition ofiliac stenosis [1, 2] , this paper compares clinical examination, doppler ankle systolic pressure (aspi) and femoral signal analysis (laplace transform damping, ltd, and pulsatility index, pi) [3] with biplanar contrast studies. i at biplanar angiography 66 of 102 ischaemic lower limbs had ais with diameter reduction from 25-84 %, of the remainder, 25 were normal (<25 % stenosis) and 11 were ,,occluded" ('_--850/0). nearly two thirds (61%) of limbs with a clinically normal femoral pulse had identifiable arteriographic stenosis (~-__.250/0), upstream abnormality was predicted incorrectly in 15 % and the overall accuracy of clinical examination was 67 %, both for detecting stenosis and predicting its severtiy. aspi (median 0.61; 95% confidence ,limits 0.3-1.0) and pi (5.4; 2.3-0.05) although correlated with stenosis (aspir = 0.65; p = 0.05, pir = 0.62; p=0.05 variance analysis for linear regression), did not aid the clinician further (accuracy 62 %). however ltd (0.72; 0.37-1.0) was well correlated (r=0.76, p=0.001) and did improve assessment ofiliac stenosis (accuracy 84 %). the need for biplanar arteriography is reiterated and its use with doppler signal analysis should improve the evaluation of aorto-iliac disease. in the absence ofa non-invasive method for estimating volume flow in an individual artery, local blood pressure measurement has proved, with certain limitations, useful in assessing the cardiovascular system. now ultrasound technology has progressed to enable blood flow in an artery to be measured noninvasively. we report the results o four evaluation ofa 5 mhz, computerized, 30 channel, pulsed doppler vessel imaging and flow measuring instrument in in-vivo experiments. computed blood flow was compared to actual blood flow (calculated by timed collection) in 17 anaesthetised dogs. correlation between computed and actual blood flow was stronger in the larger abdominal aorta than in the smaller common carotid arteries. from the regression plot, the coefficients of determination, p, were: 0.927 (exposed aorta scans); 0.857 (transcutaneous carotid scans); and 0.784 (exposed carotid scans). stepwise regression analysis showed the computed flow values to be independent of probevessel angle, depth and lumen diameter for vessels greater than 2.5 mm in diameter. these results suggest that this pulsed doppler instrument has the versatility and accuracy essential for diagnostic flow measurements in the main conducting arteries of the neck and limbs and in vascular bypass grafts. in the assessment of patients undergoing carotid artery surgery, many laboratory methods are available in addition to angiography. in a series of 210 patients experience has been gained with the use of eeg, tc 99m isotope scanning, opg, ct scanning and doppler. in a 10 year follow up over 85% of patients had a satisfactory outcome. an early mortality of 2% in the beginning of the series has been eliminated due to improved selection. in this report the application of multi-gated pulsed doppler techniques is reported. this allows a non invasive measurement of mean volume flow in the common carotid artery with a method reproducibility of + 5 %. mean volume flow in 50 undiseased arteries (25 subjects mean age 46 years) was found to be 536± 104 (s.d.) ml/min. from this a lower range for normal flow of 300 ml per minute (2 x s.d.) was selected. 30 patients were investigated before surgery and a follow up examination was performed at a mean interval of 4v2 months post operatively. 3 groups were defined. group a >300 ml/min; group b 200-300 ml/min; group c <200 ml/min, of 14 arteries in group a before surgery, 11 remained in the group and 3 dropped to group b. in group b, of 8 arteries, 5 go to group a, 2 remain and 1 dropped to group c. in group c, 7 out of 8 arteries moved to group a and 1 to group b. thus of 16 arteries with below normal volume flow before surgery, 12 were returned to normal range and further 1 improved. 2 remain unchanged and 1 disimproved. of the 30 arteries examined 4;/2 months after surgery, 23 are in the normal range and a further 1 improved. 2 remain unchanged and 1 disimproved. of the 30 arteries examined 41/2 months after surgery, 23 are in the normal range in flow values and a further 2 remain unchanged. the non-invasive and isotope techniques have a valuable and practical application in assessment of carotid artery surgery. timing is influenced by the finding fo infarction on ct or isotope scanning. doppler techniques are useful not only in defining severity of diseas and sub-radiological plaques, but valuable flow information can be obtained by pulsed doppler pre and post operatively. this may help in identifying patients who need further medical or surgical treatment. stepwise logistic regression-amodel for predicing success of femorai-popliteal bypass grafts the objectives of this study were to identify the preoperative factors that influenced postoperative patency of femoral-popliteal grafts and to develop a model that could be used prospectively to determine the probability of successful outcome. data base material consisting of history, physical examination, laboratory data, angiography, and operative findings in 199 patients undergoing femoral-popliteal bypass grafting was entered into a computer programmed for stepwise logistic regression analysis. the computer identified and ranked 24 factors that influenced outcome. the top five factors (other than technical problems) included quantity of runoff, previous ipsilateral femoral-popliteal bypass, preoperative prediction of potential amputation level, concurrent proximal vascular reconstruction, and the location for distal graft anastomosis. having established the computer data base, it is now possible to enter information from new patients into the computer which will weigh all factors and indicate the likelihood of surgical success. in addition, tables can be generated which will look at simple combinations of variables to predict patency. for example, in a patient about to undergo a primary femoral-popliteal bypass with no anticipated technical problems, the likelihood of success as a function of runoff and preoperative amputation level is as follows: irreversibility of shock and ischemic injury is generally considered a consequence of extensive cellular injury. to study the role of intravascular coagulation in shock, 56 rats were bled to a mean arterial pressure of 50 mm hg for 3 hrs or 25% uptake of shed blood, whichever occured first. return of shed blood with these data provide the patient and the surgeon with a quantitative prediction for success and permit an informed decision when considering therapeutic alternatives. potential cytoprotection by heparin was studied by similarly bleeding 52 additional rats; controls were only cannulated. twenty pairs were heparinized (h); 32 were not (nh). paired-bled and control rats were sacrificed following hemorrhage, liver (l) and kidney (k) mitochondria were isolated, and the isolates were studied by the polarographic technic with glutamate and succinate to determine the respiratory control index (rci) as a measure of cellular injury. results were: an equal volume of isotonic saline resulted in a 43 % survival; % uptake of blood during shock allowed prediction of survival. an additional 55 rats were then randomized (coin toss) to heparinization versus no heparin prior to shock, and were similarly bled and resuscitated. significantly improved survival (p<0.025) was seen in heparinized (17/23; 740/0) versus nonheparinized rats (13/32; 41%). uncoupling and inhibition of mitochondrail function were noted in both h and nh rats with rci being significantly reduced from control. however, there was no difference between h and nh compared to each other. heparin does not provide cytoprotection during shock; improved survival with heparin may rather be a consequence of improved reperfusion of tissues following the shock episode. fl-endorphin (b-end) has been postulated to play a role in the pathogenesis of shock because the opiate "antagonist naloxone improves the macrohemodynamics in various shock models [1] . however, plasma levels ofopioid peptides have not been determined as yet. the aime of our study was to measure the plasma concentrations of various peptides and to evaluate the influence of naloxone particularly on the plasma concentration of r-end. in 16 anesthetized foxhounds, the adrenolumbar vein was cannulated and hemorrhagic shock (map = 4 mm hg for 3 h) was induced according to wiggerstechnique. the plasma levels offl-end, methioninenkephalin (m-enk), and leucine-enkephaline (l-enk) were simultaneously determined in c.v. and/ or adrenal venous blood by a specific ria. crossreactivity of r-end withfl-lipotropin was about 4 %. the enk-antibodies crossreacted with less than 5 %. five dogs received an i.v. bolus of naloxone (2 mg/ kg) and a subsequent naloxone infusion of 2 mg/kg/ h after 2 h of hypovolemia. eleven dogs served as control and received equivalent volumes (1 mg/kg per h) of ringer solution. hemorrhage resulted in a sharp rise of central venous plasma levels particularly of m-enk and l-enic this effect was even more pronounced in the adrenal's effluent system.fl-endorphin levels remain elevated whereas the enk secretion began to decrease 1 h after hemorrhage. naloxone treatment inhibited any spontaneous fall of adrenal enkephalin release during the shock phase and the values remained elevated 10-15 fold. volume substitution with autologous blood resulted in a normalization of all peptide levels. these data demonstrate that hemorrhagic shock will cause stimulation of endogenous opioid peptides. the high levels of enkephalins in the adrenolumbar vein indicate that the adrenal gland is the main source of these peptides in the circulation. in addition toil-end, the enk seem to play a role in the pathogenesis of shock as well. at our present state of knowledge, however, it is difficult to design a coherent concept of mechanisms involved. this shows that cp treated cells bound nearly as much [125t]-acth analog as control cells but there was very little specific binding to sp treated cells. low concentrations of acth effectively displaced the acth analog whereas exposure of adrenocortical cells to sp resulted in a significant decrease in acth receptors. this suggests that sp has a factor(s) that binds to acth receptors of adrenocortical ceils which may adversely affect the stress response of shock. f-43 has been proposed as superior to other asanguinous fluids due to increased oxygen carrying capacity. evaluation to date has been largely uncontrolled and at extremes of hemodilution (hct. <2 %) rarely seen clinically. near infrared spectrophotometric monitoring of brain cytochrome a, a3 redox state, a sensitive indicator ofintramitochondrial oxygen availability, offers a unique opportunity to contrast f-43 with balanced salt-albumin (bsa) and whole blood saline (wbs) as a resuscitative regimen in a clinically relevant model. fifteen rats were subjected to 30 minutes of hypoxia (fio2 = 7,5%) and hemorrhagic hypotension (map = 30 mmhc), then randomly allocated to one of three groups and resuscitated by fio2 = 100% and infusion of either f-43, bsa, or wbs. cytochrome a, a3 redox state was monitored continuously at 813 run. thirty additional rats were sacrificed at baseline, end shock and 20 and 120 minutes post resuscitation for cerebral cortical atp and lactate assay. despite hematocrits as low as 15 % in the bsa and f-43 groups, there were no significant differences /7<0.05 between groups in the parameters of oxygen sufficiency; atp, lactate, and cytochrome a, a3 redox state. we assume differences in cardiac output compensated for differences in arterial oxygen content. on this basis we suggest perfluorochemical utilization should be limited to situations in which hematocrits are <15 % and when cardiac reserve is limited. metabolites of the prostaglandin endoperoxide h2 (pgh2) affect both vascular tone and platelet aggregation and thereby may influence blood flow. we, therefore, determined the metabolites formed from pgh2 by microsomes isolated from human saphenous vein used for aortocoronary bypass surgery. in the absence of reduced glutathione (gsh), the enzymatic metabolism of 14c-pgh2 produced only prostacyclin (pgi2) as measured by the formation of its stable breakdown product 6-keto-pgfla. the amount of pgi2 formed varied from 10-50% of the substrate depending upon the microsomal protein and pgh2 concentrations. in addition, the nonenzymatic breakdown of pgh2 resulted in the formation of pgf2a, pge2, pgd2 and heptadecatrienoic acid (hht). there was no formation of thromboxane a2 (txa2) as measured by the absence of its stable breakdown product txb2. in the presence ofgsh, a required cofactor for microsomal pge2 isomerase activity, the formation of pge2 was augmented 2 fold (up to 35 % of the substrate) indicating enzymatic for-mation of pge2. the gsh either did not alter or augmented (less than 1 fold) the formation of pgi2. the increased formation of pge2 in the presence of gsh was at the expense of decreased nonenzymatic breakdown of pgh2 to pgf2a, pgd2 and hht. these data suggest that prostacyclin synthetase activity may serve to protect the vessel graft from platelet aggregation and/or vessel spasms and may possibly serve as an indicator of graft viability. thrombosis is a frequent cause of early arterial bypass graft failure and platelets are known to be major determinants ofthrombus formation on arterial surfaces. pgi2 and flbriolytic activators from the vascular wall counteract intravascular thrombosis. the aim of this work was to study the effect of arterial grafting on the aforementioned mechanisms. 6 cm lengths of tanned human umbilical vein grafts (huvg) with an internal diameter of 5 mm were interposed end-to-end in the carotid arteries (c.a.) and jugular veins (j.v.) of sheep. placed in the c.a.'s, 12 grafts with restricted flow (50 cc/min) were removed on the 10th postoperative day (group i); 18 grafts with unrestricted flow (140___20 cc/min after placement) were taken out 90 days later (group ii) and 4 grafts placed in the j.v.'s were removed 10 days after surgery (group iii). upon removal, the grafts were checked for patency and sections from the proximal and distal anastomoses and midgraft were obtained for determination of pgi2 production (ria) fibrinolysis activators activity (histochemical method) and for light and scanning electron microscopy. sections from the femoral arteries were also obtained. the results of pgi2 generation are expressed in ng/ml/cm% all grafts showed fibrinolytic activity in the adventitia but 4 grafts in group ii also showed fibrinolytic activity in the intima. early neointimal fibrous hyperplasia (nfh) characterized by proliferation of smooth muscle cells was present in group ii. the, occluded grafts showed organizing thrombus material and inflammatory cells and the patent ones showed fibrin and scattered inflammatory cells. in groups i and iii, sem revealed numerous platelets and rbc's incorporated into a proteinaceous material overlying the anastomoses and in some specimens obvious thrombus material was present. in group ii, the anastomotic areas were covered with large endothelial cells, nonetheless, some areas were denuded and small thrombi were occasionally noticed. in conclusion: 1. anastomotic sites create a strong stimulus for thrombus formation despite a high production of pgi2. this suggests that antithrombotic therapy may be necessary to prevent early failures. 2. huvg develop the capacity to produce pgi2 and fibrinolytic activators and 3. although the etiology of nfh remains obscure, the decreased levels of pgi2 in group ii suggest that exhaustion of pgi2 generation from the endothelium might occur leading to proliferation of smooth muscle cells (nfh). these cells will in turn supply pgi2 if a persistent stimulus exists. permeability of intestinal capillaries to fibrinolytic products d. manwaring and p. william curreri department of surgery, university of south alabama college of medicine, usa fibrin/fibrinogen degradation product d (fdp-d) is significantly elevated in the serum of patients after trauma or sepsis. purified fdp-d infused into nontraumatized rabbits precipitates thrombocytopenia, complement depletion, pulmonary dysfunction and increased permeability of lung capillaries to i12s-albu -composite fibrin plate assay size oflytic zones (mmx) after 17 h incubation at 37 °c rain. in order to determin of products of fibrinolysis alter fluid filtration or permeability, either purified fdp-d or fdp-e were tested in an isolated, autoperfused cat ileum preparation. steady-state lymphatic: plasma protein concentration ratio (cl/cp) and lymph flows (ql) were measured at a venous outflow pressure of 5 mmhg. data was analyzed for each animal group by the paired student t test for ql, cl/cp and protein clearance (ql x cl/cp). in cats which received fdp-d (n=7), ql and clearance increased five-fold (p<0.002), but cl/cpwas not altered, which suggests a permeability change. ileal mucosal biopsies prepared for histology had villi that were de-epithelialized and platelet clots in blood vessels. fdp-e (n=7) provoked a slight increase in ql (p<0.01), but not in cl/cp or clearance. histology was normal. (fdp-e causes no pathological lung change in awake rabbits). fdp-d may contribute to various organ pathologies after trauma. the effect of aspirin on the fibrinolytic activity of viable granulocytes r.c. franz, w.j.c. goetzee, b. rotunno and r. anderson department of surgery, university of pretoria, rsa although several influential authors have suggested that low dose (150 rag) acetyl salicylic acid (asa) represents a balanced daily antithrombotic regimen probably by both inactivating thromboxane a2 production and enhancing prostacyclin synthesis little is known about the effect of aspirin on the fibrinolytic activity of live granulocytes [1] . the present study was designed to evaluate this effect in vivo. methods: granulocytes from fasting samples of heparinized venous blood taken from male volunteers were separated from monomuclear cells and platelets by density gradient centrifugation (ficoll: sodium metrizoate). viable granulocyte suspensions and plasma samples were placed as drops on a coinbefore aspirin after aspirin p = [2] . the experiment was repeated 3 h after each subject had ingested 1.8g of aspirin. the results are summarized in table 1 . 1. there appears to be a significant increase in granulocyte fibrinolytic activity 3 h after the ingestion of 1.sg of aspirin. 2. this increment is insufficient to overcome the resting inhibitor potential of plasma on granulocyte fibrinolysis. 3. aspirin does not evoke a significant increase in plasminogen activator-(urokinase) induced flbrinolysis in platelet free plasma or in the combined system of granulocyte-plasma mictures. 4. the optimal dosage of aspirin as a fibrinolytic agent requires further study. the terminal vascular bed of malignant tumors is characterized by a lack of organization, differentiation and sufficient developement of nutritional capillaries. as a result, malignant tumors reveal consistently small regions of low or even no perfusion. pre-vious data in a melanoma indicate that due to the rarefication of capillaries, the full impact of tumor treatment ±st diminished by an elevated microvascu lar resistance, which could significantly affect the impact of tumor therapy. since the improvement of the blood's fluidity has been shown as one therapeutic modality to increase significantly the capillary blood flow, it was assumed that this measure might enhance the accessibility of tumor tissue for bloodborne drugs. this study was aimed to investigate the effects of the improvement,of microcirculatory flow on tumor growth and tissue oxygenation. moreover, the response of the melanoma to chemotherapy was evaluated when isovolemic hemodilution was employed in conjunction with chemotherapy. a transparent chamber technique, intravital microscopy, a platinum multiwire electrode (local po2 measurement) and quantitative television image analysis (capillary blood cell velocity and diameter) were employed to study the microvasculature in the amelanotic melanoma a-mel-3 of 18 hamsters in the event of hemodilution without and in conjunction with chemotherapy (200 mg/m 2 dtic, dimethyl-triazeno-imidazol-carboxamid). permanent indwelling catheters in carotid artery and jugular vein served for measuring systemicpressures, heart rate, for withdrawing blood and the infusion of dtic and/or dextran 60. after inoculation of 4 x 104 cells of the amelanotic hamster melanoma a-mel-3 into s.c. tissue in the preparation, this tumor re~ched a diameter of approx 3 mm within five days. the reduction of systemic hematocrit from 0.45 to 0.31 (1.3 ±0.2 ml blood vs dextran 60, 7 animals) at a tumor diameter of 3 mm increased the growth rate of the melanoma by about 30 % while enhancing significantly the volume flow through capillaries and the mean local po2. table 1 control hemodilution capillary velocity (ram/s) 0.39 _ 0.12 0.50 ± 0.12 capillary blood flow (ml/min x 10 -5) 5.4 ± 1.7 8.9 ± 1.2 mean local po2 (mmhg) 9.4 (0 -28) 15.7 (0 -60) the frequency distribution of local po2 on the tumor's surface showed a distinct shift toward higher po2 values with still some hypoxic regions remaining. intravital microscopy, however, revealed petechial bleeding and localized, interstitial edema which compressed a small number of capillaries. by contrast, the tumor's diameter remained at app. 3 mm for a period of ten days with chemotherapy alone (3 animals). in one of the animals, a complete stop in the melanoma microcirculation was seen within four hours after infusion of dtic followed by a significant decrease of tumor diameter. when chemotherapy was initiated in hemodiluted animals, neither retardation of tumor developement nor vascular obstruction was observed (5 animals). conclusion: capillary blood flow of the melanoma can be enhanced by hemodilution thus diminishing tissue hypoxia. this measure, however, was associat-ed with an increase in melanoma diameter of 30%. at the present, we investigate whether, in hemodiluted animals, a reduction of tumor size can be obtained with a higher dose of dtic. ). however, the etiology of stress hyperglucagonemia in the immobilized rat is only poorly defined. since during restraint stress, catecholamines (ca) are elevated and stimulation ofglucagon by ca is accepted (woods sc d jr [1974] physiol rev. 54: 596), we decided to study by surgical means the the relative contribution to glucagonemia of different sources of ca, i.e. peripheral sympathetic nervous system and adrenal medulla. methods: male sprague-dawley gastric fistula rats (n=74), weight approx. 250 g, were subjected to either sham-op or various sympathectomies [microsurgical splanchnicotomy = s-sx; chemical sympathectomy = c-sx (150 mg/kg 6-oh-dopamine ip two days prior to the experiment); adrenal demedullation = amx; combinations: s-sx + amx; c-sx + amx]. gastric acid secretory trials (duration 8 h), preceded by a 24 h fasting period were carried out 6-7 days following the operation. at the start of the experiment an intraperitoneal polyethylene tube, was inserted into the abdominal cavity of the rats, allowing a constant infusion of physiological saline (4 ml/ kg/h). in addition, stress was performed by pairwise restraint of the extremities and small electric shock waves applied by a tail electrode. at the end of the experiment, blood was drawn from the vena portae and the abdominal aorta for plasma and serum. hormones (glucagon, insulin) were measured by radioimmunoassay, glucose enzymatically, volume was read to the next 0.1 ml, acidity by microtitration. results (see table) : volume and acidity are not changed by the various sympathectomies, when 179 compared to the sham group. the same is true for acid secretion, except in s-sx + amx, where it is elevated. glucagon in peripheral plasma is elevated in c-sx, amx and c-sx + amx. in the portal vein, glucagon is dccreased in s-sx + amx (408___99 pg/ ml) and elevated in c-sx + amx (2625___405 pg/ml) when compared with sham rats (930--.195 pg/ml). the portal/aortal glucagon ratio is significantly decreased only in c-sx and amx (1.7--+0.9, 1.7--.1.0, resp.) when compared with sham (3.8--_ 2.1). insulin is increased only in amx, glucose decreased in amx, s-sx + amx and c-sx + amx, insulin and glucose are unchanged in the other groups. conclusion: 1. stress hyperglucagonemia in the rat is confirmed (levels during zero stress 30-180 pg/ml) and also the rise in insulin following removal ofadrenomedullary ca (but not other sympathectomy); 2. the blood glucose fall (amx; s-sx + amx; c-sx + amx) is not uniformly paralleled by hyperglucagonemia, but in the case of amx it may be secondary to relative stress hypoglycemia owing to removal of adrenal medullary ca or reactive insulin release; 3. mechanism underlying the increased systemic glucagon despite partial (c-sx; amx) or total (c-sx + amx) sympathectomy are yet unknown. 4. during stress the enterogastrone component of hyperglucagonemia may be of minor importance. evlw showed good agreement with gravimetric lung water determinations. significant lung water accumulation was produced by pressure elevations over 20 mmhg. reductions in plasma oncotic pressure significantly increased transvascular fluxes at each level of pressure elevation. however, fluid accumulation was not significantly greater during hypoproteinemia. we conclude that a 40-50% reduction in plasma oncotic pressure does not contribute to increased high pressure edema because the lymphatic safety factor is augmented. this phenomenon may explain the well tolerated state of hypoproteinemia in patients after hemorrhagic shock. computerized gamma scintigraphy is a new technique for the analysis of albumin flux in the acute respiratory distress syndrome (ards). the objectives of this study were to obtain normal control values and to determine the method's validity in patients with cardiogenic vs. permeability pulmonary edema. methods: following 10 mci99mtechnetium-human serum albumin, lung :heart radioactivity ratios were determined. this ratio remains constant unless there is a leak o falbumin, when a rising ratio is seen, called the ,,slope index" (si). si's were determined in 5 control individuals who had :> 50 % left ventricular ej ection fraction and < 7s pulmonary circulation. thirtythree studies were obtained in 27 patients using a portable gamma camera. fourteen patients had clinical evidence of ards. results: studies were considered positive if the si was 2 s.d. > control mean ( -0.4___0.5 x 10-3 units/ min). among 15 positive studies, all had diffuse air space disease on chest radiographs. their average pulmonary capillary wedge pressure (pcwp) was 14.6_+4.6 mmhg. the average artertial: alveolar oxygen tension ratio (a/a)o2 was 0.31+--0.15 on 10.8 cm h20 peep, which were both significantly (p<0.01) different from patients with normal si's. positive si's were present from 24 hours to 6 days following the apparent onset of ards in 6 patients. recovery of gas exchange was associated with normal si's on repeat studies in 4 patients. of 6 patients with cardiogenic pulmonary edema, 5 had negative studies (18-29 mmhg pcwp) and i a positive study (40 mmhg pcwp). conclusion: gamma scintigraphy was a sensitive, non-invasive tool for the detection of a pathological increase in pulmonary protein flux, which was usually normal in cardiogenic pulmonary edema. positive scintigraphy was associated with significantly impaired gas exchange. the method documented that the leak of albumin in ards may last for days but resolves with recovery. cancellous bone thermocoagulation ph. dumontier, r. benichoux and a. vidrequin institut de recheres chirurgicales -c.h.u. de brabois 54511 vandoeuvre les nancy cedex, france electrocoagulation can not stop bleeding from the cancellous part of a sectioned bone. therefore we tested the efficiency ofthermocoagulation by hot air. the hot air generator delivers a flow, of non illtercd air, at 25-30 1/min at a fixed temperature of 300°c measured at the exit of a 3 mm diameter pipe and 60 °c at the site of bleeding. the generator sustains usual steam sterilization. 14 dogs were operated on both patellae, femoral short segments and iliac crests giving 53 different site of cancellous bone. in vitro sterility studies: the conduit of hot air was applied at various distances, from 10 cm to 2 meters, above a petri plate containing culture material. thus the turbulence in the atmosphere around the zone of thermocoagulation has been bacteriologically controlled and a particle counter used. in vivo thermocoagulation: three sites of cancellous bone were used in 14 anesthetized dogs, using a sterile procedure: the iliac crest, a small segment of the femoral bone and the divided patella. 1. five iliac crests were divided and bleeding measured after thermocoagulation. 2. the 2 segmental femoral resections were thermocoagulated. 3. the patellae were vertically divided and each section submitted separately either to thermo or electrocoagulation. the pipe of thermo-181 coagulation was directed to the bleeding surface at a 2 cm distane, sweeping it during 5 to 6 seconds. the bleeding was compared by photography and the two fragments were approximated by a wire synthesis to provide a bone fusion. in few cases both sides were thermocoagulated. results: in vitro: no contamination was found in the atmosphere, up to a distance of 2 meters. there was a significant decrease of particles around the operating site (60 % less, at 80 cm and 30 % less, at 2 meters). in vivo: the bleeding was weighted around 50% less than with conventional coagulation. thermocoagulation did not delay or disturb the healing of the patella after wire synthesis. the in vitro nucleation time of cholesterol crystals from gallbladder bile of patients with gallstones is more rapid than that from normal persons, (holan rt [1979] gastroenterology 77: 611). this study determined whether this was due to a gallbladder or liver defect and wether the defect was the addition of a nucleating factor or the deletion of an antinucleating factor. hepatic and gallbladder bile were gathered at surgery in stone patients and gallbladder bile in patients with a normal biliary tract. after ultracentrifugation, the isotropic phase was observed daily by polarizing microscopy until cholesterol crystals appeared. in gallstone patients, the nucleation time of gallbladder bile was significantly more rapid, 2.5 days+0.9 sem, than that of hepatic bile 7.9+2.1 days, although hepatic bile was significantly more saturated with cholesterol [cholesterol saturation index (csi), 2.12_+0.36], than gallbladder bile, (csi, 1.31_+ 0.12). thus the characteristic short nucleation time of stone formers is due to an alteration in bile after it enters the gallbladder. to determine whether the gallbladder defect was due to addition of a nucleating factor or the deletion of an antinucleating factor, isotropic phases of normal gallbladder bile and that from stone formers were mixed and nucleation time determined. mixtures of up to 95 % normal bile had pathological nucleation times demonstrating that the defect is the addition of a nucleating factor by the gallbladder, and that this factor is potent. the rate of formation ofgaustone precursor crystals in bile, although faster in gallstone patients than in controls, is unrelated to the degree of cholesterol supersaturation [1] , implying that other factors are involved. two competing factors seem likely; (a) secondary seed crystals in bile may trigger and accelerate gallstone crystal formation from supersaturated solution; (b) "poisons" in bile may retard or inhibit crystal growth. because of the complexity of bile itself, experiments were performed in highly purified mixtures of bile salt, lecithin and cholesterol, in concentrations closely resembling those of gallbladder bile. (a) lipid solutions were seeded with calcium carbonate, hydroxy-apatite, calcium bilirubinate and biliary mucus, all of which are found in gallstones [2] . cholesterol crystal formation was significantly faster in_ the presence of all of the seed compounds tested (x= 221.7/~g ml-lh-1) than in unseeded controls (128.0/ag ml-~h-1) (/7<0.05). (b) substances with "crystal-poisoning" properties included heparin, chondroitin sulphate and bile salt. these and changes in ph altered the quantitiy (20-80 % decrease) and rate of calcium carbonate and calcium phosphate crystal formation. we suggest that gallstone precursor crystal formation may be affected by a subtle balance between crystal seeding and crystal growth-inhibition, both due to the presence of other compounds in bile. at the last tripartite meeting we reported experimental data on a new method to destroy concrements of the kidney in situ by shockwaves allowing for spontaneous excretion via the urinary tract [1] . the shockwaves are generated externally by underwater discharge of a condensor with sparking electrodes which are localized in a focus o fan elliptic cavity. for treatment the renal concrement must be positioned exactly in the second, virtual focus opposite to the elliptic cavity. since then, altogether 126 patients were subjected to this form of treatment in our institute by the colleagues of the department of urology at the university of munich. 90% of the patients got rid of their concrements within a few days, in 8.5 % small remnants remained in the renal pelvis, and in 1.5% (2 cases) additional surgery became necessary. meanwhile this technique is employed on a routine basis in the department of urology of the university of munich [2] . the experimental as well as clinical results were considered encouraging enough to extend the technique for the treatment of biliary concrements. for this purpose, human gallbladder concrements of different composition (bilirubin, cholesterol) were implanted into the gallbladder of dogs for exposure to shockwave treatment after wound healing. under in vitro-conditions the biliary concrements could be crushed into any size desired, irrespective of their composition, while only in 8 out of 10 experiments this was accomplished under in vivo-conditions. blockage of the biliaiy duct after shockwave exposure was never observed. concrements which were experimentally implanted into the bile duct could be visualized without difficulties by contrast medium. here, destruction by shockwaves was accomplished as well. currently experiments are conducted to dissolve remnants of biliary concrements after treatment by administration of desoxycholic acid. precise positioning of the gallbladder concrements in the second virtual focus is a problem which has not been satisfactorily solved so far, because the concrements cannot be visualized by conventional x-ray techniques. alternatively it is attempted to employ ultrasound, or visualization by retrograde injection of xray contrast medium through a catheter. in experimental animals, we leave a t-formed drain in the gallbladder for injection of contrast medium. in animal experiments conducted so far, histological or clinical evidence for tissue damage has not been obtained, as is the case with shockwave treatment of kidney stones. we are convinced that treatment of biliary concrements by shockwave exposure can be employed under clinical conditions in the near future. exploration of the common bile duct (cbd) for calculi, particularly in the presence of obstructive jaundice, is a procedure with considerable mortality and 183 morbidity. to avoid the problem of retained stones, choledochoduodenostomy and transduodenal sphincteroplasty have been recommended, but have their own complications. this morbidity might be reduced by removal of cbd calculi prior to surgery. endoscopic sphincterotomy (es) allows this. a review of 123 cases of es performed for calculi indicated that this was a safe (complications 8% no deaths) and reliable procedure (85 % success rate). a study was conducted of patients with known cbd stones who had either preliminary es followed by operation at a later date (group i) or operation alone (group ii). this study showed a lower morbidity in group i. a prospective randomised controlled study has begun on the basis of these findings and the data from both studies are shown in the table. these results suggest that pre-operative endoscopic sphincterotomy my reduce the morbidity of cbd stones. group ii n = 28 two controversies regarding the physiology of the biliary sphincter (bs) concern its functional independence from the duodenum [1] and those aspects of its acitivity which control bile flow [2] . the rabbit was chosen as the experimental animal as it has an easily identifiable sphincter. during anaesthesia induced by intravenous pentobarbital sodium, recordings of the electrical and mechanical activity of the bs and duodenum were made from (a) starved, (b) fed and (c) starved animals during administration of cholecystokinin, pentagastrin, secretin and glucagon. spike complexes (sc) were ordinarily associated with mechanical acitivity of the sphincter and duode-num. of 2.172 sphincter sc, recorded in 18 animals, 775 (36%) were not associated with duodenal acitivity, whereas 1.397 of 1400 (99.8 %) duodenal sc were accompanied by synchronous bs activity. this supports the hypothesis that the rabbit's bs can contract independently of the duodenum but that duodenal contraction is usually accompanied by simultaneous contraction of the bs. sphincter scs correspond to its phasic acitivity. food and cholecystokinin increased the number of sc without altering the baseline pressure of the perfused common bile duct. pentagastrin produced a transitory increase in sphincter activity whereas :secretin and glucagon were without effect. phasic activity of the spincter may influence bile flow through the choledochoduodenal junction. natural blood coagulation finally results in the formation of fibrin, which is one of the most important components of hemostasis in the human organism and thus provides the basis of all reparative procedures that are part of wound healing. it stands to reason to utilize the properties of fibrin for hemostasis during surgery and for joining severed tissue. first attempts of this kind were made at the beginning of this century. but only after greater insight had been gained into the coagulation proc-185 ess and the manufacturing techniques of blood derivatives had become more sophisticated, the essential breakthrough was made. a biological adhesive system has been developed, which consists of highly concentrated fibrinogen, thrombin and clotting factor xiii. this tissue sealant is completely resorbable and of high adhesive property. further advantages are elasticity of consistence and excellent tissue compatibility. after extensive animal experimentation, first clinical experience was made in 1973. in the meantime the fibrin-adhesive-system (fas) has been introduced into numerous surgical disciplines with excellent results. the outstanding properties are: atraumatic tissue synthesis; enhancement of fibroblast proliferation and promotion of rapid wound healing; obtaining of local hemostasis by sealing bleeding surfaces, which is of special importance in the treatment of patients suffering from hemophilia or during operations under heparinization. the authors experience in using the fas within the last 10 years is reported and a review over indications, techniques and advantages of this method is given. bile salts have been shown to enhance the stability and prolong the activity of intraluminal pancreatic enzymes and may therefore influence the effects of impaired exocrine secretion in patients with pancreatitis [1] . individual bile salts in the peak 15 min collection of duodenal fluid following cck/secretin administration have been quantitated by high performance liquid chromatography in 7 patients without pancreatic or hepatic impairment (group c), 6 patients with acute pancreatitis (group ap) and 8 patients with chronic pancreatitis (group cp) all with functioning gallbladders, and 4 patients with gallstone related acute pancreatitis (group gs). the peak total bile salt output in moles and the trihydroxy: dihydroxy (tri:di), primary:secondary (p:s) and glycine:taurine (g:t) bile salt ratios are shown below (mean___ sem). the duodenal aspirates contained detectable amounts of taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate but not lithocholate or free bile salts. the low total bile salt output in groups cp and gs were due to decreased levels of all the individual bile salts. although the bile salt pattern in groups c and ap were similar, the relative proportions of trihydfoxy and secondary bile salts were higher in groups cp and gs respectively. these results indicate that patients with chronic pancreatitis without obvious large bile duct obstruction have an impaired bile salt output into the duodenum and this may exacerbate the effects of pancreatic exocrine insufficiency. an elevated amylase creatinine clearance ratio (accr) was considered a specific test for the diagnosis of acute pancreatitis (ap). however, it has been found elevated in other diseases as well as after surgery. the aim of this study was to evaluate prospectively the accr levels in patients with ap and in several groups of surgical patients. we studied 197 subjects divided into 6 groups: group a: acute pancreatitis (n=67). group b: patients undergoing biliary tract surgery (n=38). group c: peptid ulcer patients undergoing gastric surgery (n=25). group e: patients undergoing cardiac surgery under extracorporeal circulation (n = 16). group f: control group of healthy subjects (n = 40). the accr was determinated using the levitt method. in the surgical groups the accr was measured before and after the operation. amylase was determinated by the phadebas amylase test. ap was diagnosed on the basis of both clinical and radiological findings and the presence of high serum amylase levels. this diagnosis was confirmed through laparotomy in 18 cases (26%). the accr was 1.96 + 0.8 % (mean___ sd) in group f, control group, and 7.5+5.7% in group a, acute pancreatitits p<0.01. accr values below 4 % were found in 21 cases of acute pancreatitis (31%). among those patients whose ap diagnosis was confirmed through surgery the accr was 10.6+7.9% (mean_+sd), higher than in the rest of the ap patients, 6.4±4.2% (p<0,05). in groups b,c,d and e (surgical groups) the accr before the operation was 1.83±0.8%, 2.34±0.9%, 2.28+ 1.6% and 1.54+ 1.06% (mean___ sd), respectively. after the operation it was: 3.85+2.2%, 3.18+2%, 2.83± 1.5% and 2.05+ 1.6%, respectively. on the average, we found an increase in the accr levels after the operation in the biliary tract group (p<0.01), but not in the other surgical groups. in 22 patients (24 %), the accr after operation was above the upper limit of normal. none of these patients had symptoms compatible with clinical pancreatitis. in conclusion: 1. the accr increase in acute pancreatitis (sensitivity: 69o/0) 2. the average accr increase after biliary tract surgery, but not after either gastric or thyroid surgery or after cardiac surgery under extracorporeal circulation. however, it is possible to find isolated cases with high accr after any type of surgery without any symptoms of pancreatitis, suggesting that an increase of accr may be an unspecific finding in postoperative patients which require further investigations. out of 217 patients with acute pancreatitis 25 developed a fulminant type. 18 were males and 7 females, mean age 45 years (range 23-83 years). all 25 patients were primarily treated by peritoneal lavage applied at laparotomy. indication for laparotomy was sudden deterioration with (20) or without (5) organ failure. a necrotic or hemorrhagic pancreas was found in every patient. the pancreas was exposed and 2 soft and large catheters were placed close to the pancreas. mean duration of lavage (ll/h) was 9 days. due to secondary deterioration a pancreas resection was performed 2-10 days later in nine patients. 7 patients with acute fulminant pancreatitis died, a mortality of 27.7%. all 192 patients with the mild type of acute pancreatitis survived, thus the overall mortality was 3.2%. none treated by peritoneal lavage only developed diabetes mellitus, whereas 4 out of 5 surviving patients with an additional pancreas resection had this complication. 19 patients with acute fulminant pancreatitis displayed 3 or more ranson criteria [1] and six died. however, no less than 32 of those with a mild type of acute pancreatitis fulfilled 3 or more of these criteria and they all survived. a laparotomy -not a laboratory test -is necessary to confirm the diagnosis of acute fulminant pancreatitis. the indication for laparotomy is mainly clinical and therefore such a patient should preferably be handled by surgeons or physicians experienced in this disease. after confirmation of a correct diagnosis peritoneal lavage is one of the methods by which the mortality of acute fulminant pancreatits -which by conservative means is 80-100% -can be reduced. coincidences ofhyperparathyroidism and pancreatitis have been given up by different author varying between 1% and 19%. frequently a causal relationship has been defended. recently, however, any causality has been queried [1] . we analysed our own series of patients with surgically and histopathologically confirmed primary hyperparathyroidism (phpt) (n=686) and found a coincidence with a coexisting or prior pancreatitis of 1.5 % (n = 10). from this a causal relationship cannot be concluded. however, 3 out of these 10 patients (i.e. 0.4%) had an acute onset or exacerbation of a pancreatitis immediately following the parathyroidectomy, which is strikingly more than one would expect after an operation without any anatomical relation to the pancreas (<0.01%) [2] . none of these 3 patients had another cause of the pancreatitis such as cholelithiasis or alcohol abuse. hence a causal relationship cannot be excluded. it is imaginable that excessive amounts of parathyroid hormone are released during the surgical manipulation of the pathologically altered parathyroids. the postoperative pancreatitis may be caused by the acute elevation of parathyroid hormone levels in the presence of hypercalcemia. in our series the parathyroidectomy was combined with a partial or total thyroidectomy in 27 patients. in another 7 patients a thyroid operation was performed prior to the parathyroidectomy. although calcitonin has been employed as a possible therapy in patients with acute pancreatitis (ap), the normal levels of this substance in ap are not well documented and have not been correlated with pth. furthermore no definitive role in human calcium homeostasis is accepted for calcitonin, whereas high pth levels have previously been correlated with hypocalcaemia [1] . in 21 patients with ap the mean serum calcitonin on admission was 121 ng/1, and the peak level mean 173 lag/1 (upper limit of normal 75 ng/1). calcitonin levels were higher in severe than mild ap, and 5 of 6 patients with levels >200 ng/1, were objectively graded as severe ap [1] . in the 3 hypocalcaemic patients, with corrected serum calcium <2.0 mmol/1 all recorded calcitonin levels >100 ng/1 and had elevated pth. nine of the 21 patients had significantly elevated pth ('>700 rig/l) and of these only 1 did not have an associated elevation in calcitonin. the high calcitonin levels in patients with ap suggest that supplementary calcitonin intended to inhibit pancreatic secretion is unnecessary. at present it is merely speculative to suggest a role for calcitonin in ap but intriguing to report a tendency to parallel the elevations of pth. nuclide labelled microspheres were used to measure pancreatic and other visceral blood flow in two groups of conscious dogs before and after intravenous alcohol infusion. blood alcohol concentrations at the time of blood flow measurements were similar to those encountered in intoxicated humans. thus 8 dogs (groups a) were given a somewhat low dose of alcohol to produce a mean blood alcohol level of 0.16 gm d1-1, while 6 dogs (group b), received substantially greater doses of alcohol, and reached a mean blood alcohol of 0.32 gm dl-1. wilcoxon matched pairs signed rank test confirmed a biphasic, concentration-related, response of pancreatic blood flow after alcohol infusion; no such response was found in blood flow to other viscera. moderate alcohol levels (group a) were associated with a decrease in pancreatic blood flow (p<0.03), while high blood alcohol concentrations resulted in increased pancreatic blood flow (p'<0.02). colonic blood flow increased in both groups a and b, but blood flow to the gallbladder, small intestine and the parotid gland increased in group b only. gastric, duodenal, renal, hepatic (arterial) and cerebral perfusion did not change. in addition, direct observations of the surface of the pancreas showed occasional haemorrhagic areas and mottling. these findlings however could not be confirmed by objective attempts to measure blood flow in such discrete areas in conclusion pancreatic blood flow shows a biphasic, concentration-related, response shortly after intoxication. this response appears to be peculiar to the pancreas and does not occur in other viscera. we recently showed that acute ethanol (e) and/or aspirin (a) ingestion increased the permeability of the pancreatic duct to large molecules, this suggested that pancreatic enzymes might leak from the duct into the parenchyma, causing pancreatic disease. this is a new concept in the study ot he pathophysiology of this organ (to be presented at aga, may, 1982, plenary session). in the present experiments wie studied the effects of chronic e/a ingestion on pancreatic function in dogs. methods: 6 dogs were fitted with duodenal and gastric cannulas. after recovery, baseline secretory sutdies were performed by cannulating the pancreatic duct and collecting pancreatic juice during secretin infusion (0.1 (submax) or 2.0 u/kg-hr. (maximal) iv). at least 2 studies were performed in each dog at each dose. after baseline studies, 3 dogs (group e) were given daily intragastric ethanol (2 gm/kg-day). 3 dogs (group a/e) were given e and a (100 mg/kgday). after 36-48 weeks, secretory studies were repeated. results: all dogs gained weight (x = 1.28 kg). the pancreases appeared normal by light microscopy. drug treatment increased volume and hco3 output by 47 and 67%, respectively, in group e, submax secretin, but decreased them by 29 and 34 % in group a/e animals. (p=0.0001 vs. predrug values and group e vs. a/e values). drug treatment decreased volume and hco3 output in both groups by 5-10°/c (p=0.0001) after maximal secretin stimulus. (manova test for all statistical analyses). conclusions: consumption of e alone increased volume and hco 3 output after submaximal and decreased them after a maximal secretin stimulus. this confirms the work of sarles. consumption of e and a reduced volume an i-ico3 output after secretin at both doses. thus chronic a ingestion further impaired pancreatic function in these animals. since only a small proportion of chronic alcoholics develop clinically significant pancreatic disease, an aggravating "cofactor" may be operating in this group. chronic asa ingestion, not uncommon in alcoholics, may represent such a cofactor. the development of diabetes mellitus in pancreatic cancer is well known but the standard oral glucose tolerance test is not recognised as a useful diagnostic indicator. glucose homeostasis, insulin and c-peptide secretion in response to intravenous glucagon were studied prospectively in patients with suspected pancreatic cancer and assessed as to their diagnostic value. 34 fasting patients were given glucagon, 1 m.g., i.v., and serial measurements of blood glucose, plasma insulin and c-peptide concentrations made for 60 min. subsequently it was shown that 12 patients had pancreatic cancer and the remainder constituted a control group. there was not significant difference in the rise in blood glucose between the groups after glucagon. the mean plasma insulin concentrations rose rapidly in both groups peaking between 2 and 5 rain but the values were sginificantly lower in the pancreatic cancer group (p<0.01 at 5 min: p<0.001 at 15 min: p<0.01 at 60 min). a similar pattern was observed with c-peptide. in patients with obstructive jaundice the plasma insulin response was a better discriminator of pancreatic cancer. we conclude that abnormal pancreatic beta cell funktion exists in patients with pancreatic carcinoma, detectable before any change in glucose homeostasis, particularly in patients with obstructive jaundice. the glucagon stimulation test may have a useful role in the diagnosis of pancreatic cancer. t-suppressor cells (t~) have previously been implicated as mediators of graft surival in baboons tolerant to their renal grafts [1] . in addition, it seems that t-helper cells (th) are also affected by totallymphoid irradiation in that they are unable to provide help in mitogen-induced t-cell responses or pokeweed mitogen induced immunoglobulin synthesis in bcells. in this study the evolution of t~ and th is followed in baboons undergoing graft rejection at different rates. peripheral blood was collected from the animals at weekly intervals after renal transplantation, defibrinated and the lymphoid cells isolated by flotation on ficol hypaque. the t-lymphocyte fraction was purified by filtration through a column packed with nylon wool. th, ts and total t-cells were enumerated using monoclonal antibodies okt4, 8 and 11 respectively (ortho). the th/ts rations reported were taken immediately before a rapid rise in serum creatinine occurred. in longlived baboons who maintained normal creatinine values, a mean of the ratios over the last month was used. b rats are produced by sublethal (750 rad) x-irradiation of thymectomized animals, reconstituted with bone marrow from syngeneic, thymectomized, thoracic duct drained donors. in these lew rats, (lew x bn)f1 cardiac allografts survice indefinitely (>100 days); unmodified lew rats acutely reject such allografts (8-+ 1 days). in this study, we have tried to restore the processes of acute rejection in b recipients. graft survival appeared independent of blocking factors or suppressor cells, as transfer of serum or lymphocytes from b recipients into syngeneic normal animals failed to increase survival of test allografts, placed subsequently. similarly, immunogenicity of long surviving grafts was unchanged; such grafts functioning >100 days and retransplanted into normal animals were rejected acutely. adoptive transfer of 108 unseparated spleen cells (sl) from nonimmune syngeneic animals produced slow rejection (28--+5 days) in b rats; 108 sensitized slwas somewhat more effective (22_+ 1 day). transfer of 2 x 107 syngeneic peritoneal exudate (pe) cells plus 108 sensitized sl caused acute rejection in 50 % orb recipients (11 _ 1 days), the remainder experiencing rejection at c 3 weeks. pe cells harvested from rats injected ip with thioglycollate 3 days previously, were primarily macrophages/adherent cells, as the cells used were that fraction sticking to plastic dishes and removed with lidocaine (purity >90 %). in vitro, b rat macrophages were abnormal, having only 50 % of capacity of normal macrophages to promote production of interleukin 2 (il2) when co-cultured with purified t lymphocytes. however, b recipients experienced acute graft rejection (10-+ 1 days) after transfer of 108 sensitized sl plus semipurified il2, thus bypassing the above defect. addition ofll2 to the t cell (purity >95°/0) equivalent of 108 sl (purified over degalan bead columns coated with rabbit antirat lgg, nonadherent fraction) failed to reestablish acute rejection (17-+7 days), while further addition of b lymphocytes (degalan bead adherent fraction, purity >90%) or macrophages was uninfluential (16___2 days and 16___3 days, respectively). transfer of il-2 alone never produced rejection. acute rejection can be re-established, however, by increasing the number oft lymphocytes (108, transferred concomitantly with il2). thus, the state of unresponsiveness in b rats can be reversed in vivo by adoptive transfer of particular cellular elements in the presence of growth factors; increased graft survival seems dependent ultimately upon il-2 production by sensitized t cells, presumably t helper cells. the relative inability of b rat macrophages to promote production of il-2 by t cells may be primarily responsible for the immunological deficit of the b rat. one of the most intriguing findings ofcyclosporin a (cya) immunosuppression is that in some species a short course of treatment will produce very prolonged allograft survival. we have tested the ability of cya to prolong the survival ofvascularized heart, kidney and pancreas allografts by direct comparison in a da (rt1 a) to lew (rt1 ~) rat allograft model. accessory abdominal heart and orthotopic left kidney transplantation were performed using standard microsurgical techniques. in renal transplantation the left kidney was removed at the time of transplantation, the remaining right kidney 5 days thereafter. streptozotocin-diabetic animals received ductligated pancreas whole organ grafts isolated on the portal vein and a segment of the aorta giving offthe coeliac axis and the superior mesenteric artery. rejection was taken as complete stop of palpable pulsations in heart transplantation, the day of death in renal transplantation and recurrance of hyperglycemia above 14 mmol/1 in pancreas transplantation, respectively. cyclosporin a 15 mg/kg body weight, dissolved in olive oil, was administered intramuscularly for 14 days starting with the day of transplantation. in all instances functional demonstration of rejection was confirmed by histological examination. cyclosporin a is effective to prolong the survival of vascularized heart, kidney and pancreas allografts. while cya is administered none of the grafts has been rejected. however, following withdrawal of the drug pancreas grafts are rejected within 14 days and heart grafts within 33 days. none of the kidney grafts has been rejected so far. the differential susceptibility of vascularized heart, kidney and pancreas allografts to cya immunosuppression may be caused by differences in immunogenicity due to organ specific alloantigens or a differential representation of spezialized antigen presenting cells. it may also reflect different patterns of rejection of the various organs. during cya administration all rejection processes are effectively suppressed. in the maintaince phase after withdrawal of cya such immune responses may prevail and ultimately lead to rejection of pancreas and to a lesser degree of heart allografts. the venous allograft still remains an attractive alternative for the reconstruction of small caliber vessels. however, when the venous graft is introduced to a non-histocompatible host, rejection and early occlusion is the rule. this study evaluates the use of cyclosporin a (cya) as a graft pretreatment, or systemic immunosuppressant for venous allografts. in addi-tion, cryopreservation techniques for pretreated venous allografts was investigated. 45 adult mongrel dogs, weighing between 12 and 24 kg, were used as recipients for donor jugular vein segments (5-6 cm) which had been excised and flushed with 100 cc of plasma protein fraction (ppf) at 4°c. these venous allografts were anastamosed end-to-end into a carotid artery of the recipients. the animals were divided into five groups as follows: group i (n=6) received untreated venous allografts without subsequent immunosuppression, group ii (n = 5) was the same as group i with minimal immunosuppression (azathioprine 2.5 mg/kg/day). in group iii (n= 10) the animals were transplanted with venous grafts stored in 25 cc of plasma protein fraction (ppf) containing cy a (50 mg/1) at 4 ° c for 24 hours, immunosuppression was as in group ii. in group 1v (n=9) the animals received allografts that had been cryopreserved in a 15% dmso solution at -196 ° c for 25-35 days and then the animals had azathioprine as in group ii. in group v (n= 15) venous allografts recipients were treated with systemic cya (20 mg/kg/day x 2 weeks, followed by 15 mg/kg/day x 2 weeks) as the only immunosuppression. the patency of the allografts was evaluated at 1, 2, 3, 4 and 8 weeks post transplantation. patency results at one month showed that azathioprine alone failed to improve the patency rate (gr. i and ii = 0% patency). cya graft pretreatment, however, significantly improved the one month patency (gr. iii = 57%). in addition, cryopreservation appeared to enhance the graft pretreatment effect of cya (gr. iv; one month patency = 85.7%) of the allografts. finally, systemic cya proved very effective in preventing rejection and occlusion (gr. v; one month patency = 100 %). grafts that remained patent for a initial critical period of 4-6 weeks, all showed long term patency. the effect of cya in preventing graft rejection was further documented by histiological studies of the allografts which showed a marked cellular infiltration and degenerative changes in all the grafts of the control group as compared to minimal or no cell infiltration in the patent grafts of the treatment groups. in summary, it appears that cya used as a graft pretreatment with minimal immunosuppression of the recipient, in conjunction with cryopreservation or given systemically as the sole immunosuppressant can significantly improve the survival of venous allografts. in our previous reports, it was shown that isolated hepatocytes transplanted into the splenic parenchyma of syngeneic rats, proliferated markedly and recomposed the hepatic tissue. this experimental system provided a new model to elucidate the mechanism of hepatic regeneration which could not be obtained in in vitro cell culture experiments. in the present paper, fetal hepatic tissue instead of isolated adult rat hepatocytes were transplanted into the rat spleen. we document briefly long-term morphological observations on the transplanted fetal hepatic tissue with special reference to proliferation of the hepatocytes and bile ducts. materials andmethods:wistar rats were mated in our laboratory for a fetal liver source. gestation day 0 was when a plug or sperm were observed in the vaginal smear. fetuses used were of 18 to 20 days gestation. about ten fetal livers which were obtained from one maternal rat, were minced with scissors. the liver fragments were washed three times with saline solution. transplantation was carried out by direct injection into the spleens of syngeneic adult rats using a 15 gauge needle. half of the liver fragments obtained from one maternal rat were innoculated into the spleen of one animal. a total of approximately 30 rats with transplanted liver fragments were killed 1, 3, 7, 14 and 30 days and then every two to three months until one year after transplantation. the spleens removed were stained by h.e., pas and silver nitrate for histological examination. results: fetal livers exhibited no lobular architecture or hepatic cord structure. the very sparse cytoplasm of the hepatocytes and many hemopoetic cells among the hepatocytes were characteristically found only in the fetuses. one week after transplantation, the survived hepatocytes revealed almost the same morphological features as in fetal liver except for the presence of several proliferated bile ducts around the hepatocytes. two weeks later, the hepatocytes formed apparent hepatic cord structures and the extoplasm of each hepatocyte increased abunduntly and became acidophilic as seen in normal neonatal hepatocytes. hemopoetic cells disappeared. four weeks later, hepatocytes began to proliferate sporadically among the markedly proliferated bile ducts, groups of survived hepatocytes with cord structure were very similar to a neonatal liver except for the lack of the glisson's area. two or three months later, proliferation of the hepatocytes became prominent. there seemed to be no interrelationship between proliferated hepatocytes and bile ducts. one year after transplantation, a white nodule was observed on the spleen macroscopically and it consisted of numerous bile ducts and hepatocytes with or without cord structure on histology. summary: 1. fetal hepatocytes transplanted into the spleen, differentiated to almost normal neonatal hepatocytes two weeks after transplantation. 2. hepatocytes began to proliferate about 4 weeks after transplantation. 3. three days after transplantation, proliferation of bile ducts was already observed independent of the transplanted hepatic tissue. 4. when comparing the difference in proliferation between fetal hepatic tissue and isolated hepatocyte transplantation, marked proliferation of the bile ducts in fetal hepatic tissue was observed and fetal hepatocytes proliferated more rapidly, while there were no proliferated bile ducts in isolated hepatocyte transplantation. pretransplant splenectomy (sx) has been of disputed benefit since its introduction two decades ago. 220 of 315 patients with first cadaver transplants treated at our institution between dec. 1968 and dec. 1981 have had pretransplant sx. at six monts, sx patients had 10% better kidney survival, but this benefit was lost shortly after 1 year and by 5 and 10 years was 10% and 15% worse in sx patients. patient survival for sx and no sx was identical for the first year but was 10% and 20% worse by 5 and 10 years respectively in sx patients. thus, the early improvement in kidney survival was more than offset by a late high mortality. a rational basis for selecting patients who might benefit most from pretansplant splenectomy is urgently needed. since july, 1978, 34 patients ages 16-45 have received first cadaver transplants after having been tested for reactivity to dncb. nine of 20 dncb negative patients had splenectomy as did 6 of 14 dncb positive patients. kidney survival at 1 year for dncb negative patients without sx was 79%; for dncb negative with sx, 29%; for dncb positive without sx, 21%; for dncb positive with sx, 62%. rejection was the sole cause for kidneyloss in dncb positive patients without sx. however, 5 of 9 dncb negative patients with splenectomy died, primarily of septic complications. since 1978 survival of sx patients has been 60% compared to 95% in non sx patients (p<0.04). sx appears to be beneficial in dncb positive patients but has an adverse effect in dncb negative patients because of an increased susceptibility to fatal infections. prior blood transfusion improves renal graft survival [1] . plasma from uraemic patients suppresses the in vitro responses of normal lymphocytes to antigen (plasma suppressive activity, psa) and this effect is mainly attributable to the plasma protein macroglobulin (a2m) [2] . the aims of the present study were: a) to identify changes in psa and a2m concentration in uraemic subjects following primary blood transfusion. b) to correlate the psa of transfused renal transplant recipients with subsequent graft survival. a) ten potential transplant recipients were studied before and after their first blood transfusion. following blood transfusions the psa increased significantly (p<0.01) reaching a maximum at two months. there was no significant change in the plasma a2m concentration over the same period. b) the plasma of 137 consecutive chronic renal failure patients was tested for psa prior to renal transplantation and before institution of immunosuppressive therapy. all but two patients had received previous blood transfusions. after transplantation patients were followed for a minimum of 3 months and a maximum of 42 months. 16 grafts failed for non-immunological reasons and were excluded from the study group. patients were divided into two groups according to the degree of suppressive activity of their plasma. a volume of5/t 1, producing a 50% inhibition of normal lymphocytes was used as a treshold to differentiate those with a high or low suppressive activity. graft survival in the first three months was significantly better, 92% (/7<0.05) for those recipients with a high psa as compared to 74% for those with a low psa. we conclude that blood transfusion causes a significant increase in psa although not a2m concentration and that patients with high psa have a better graft survival. the effect of in vitro steroid on antibody dependent cellular cytotoxicity (adcc) was studied in patients awaiting renal allotransplantation and the results were correlated with transplant outcome. 85 recipients of primary cadaveric allografts were classified as steroidsensitive or steroid-resistant from the degree of adcc suppression induced in vitro by methylprednisolone, 36 patients being steroid-sensitive and 49 steroid-resistant. following transplantation patients received azathioprine and prednisone, and rejection crises were treated with bolus doses of methyl-prednisolone. graft failure occured in 5 of the 36 steroid-sensitive patients, and in 34 of the 49 steroid-resistant patients. the observed one year graft survival rate was 57.6% for the whole group, 83.1% for the patients with steroid-sensitive adcc and 36.9 % for those with steroid-resistant adcc, the difference between the two groups being highly significant (xz=23.6). a high incidence of early graft failure was seen in steroid-resistant adcc patients, 49.7% of grafts being lost in the three months after transplantation, as compared with only 2 of 36 graft failures in the steroid-sensitive adcc group in the same period. analysis of hla-a, hla-b and hla-dr incompatibilities showed no significant difference between the groups, and since all patients had received deliberate pregraft blood transfusion, the difference in survival rates between the two groups appears to be independent of these two variables. these findings confirm our preliminary observation that pregraft assay of adcc response to in vitro steroids identifies those patients who are unlikely to respond to steroid therapy in the treatment of rejection, and in whom alternative forms of therapy may be appropriate. post-operative dxt, whilst not influencing survival, protected patients from loco-regional recurrence 07<0.001, hazard ratio (hr) = 3.1). interestingly it was found to be most effective against axiallary node recurrence (p<0.001, hr = 4.0), reasonably effective against chest wall recurrence (/7<0.001, hr=2.1) but conferred no protection against supraclavicular node recurrence (hr = 1.2) in spite of a supraclavicular field being routinely employed in the radiotherapy technique. with such large numbers involved, this trial has facilitated the study of the prognostic significance of sub-groups of patients with different patterns oflocoregional recurrence as first evidence of treatment failure (see table) . of those patients developing loco-regional recurrence who have since died (222 out of 356 in wp group; 73 out of 128 in dxt group) 63% in the wp group and 69% in the dxt group did so with evidence of persistent loco-reglonal disease. however, the incidence of uncontrolled local disease at death was higher in the wp group overall. stress as well as dietary fatty acids have been shown to prolong allograft survival in rats [1] . poly unsaturated fatty acids (linoleic acid, arachnoidic acid) have been reported to depress immune response [2] . depressed immune response was suggested to correlate with a higher incidence of spontaneous tumor [3] as well as with an increased growth rate of inoculated tumors [4] . the objective of this study was to elucidate the effect of two environmental factors i.e. chronic stress (change in light/dark pattern) and diets low and high in linoleic acid on immune response and growth of transplantable tumors in bn rats. immune response: four experimental groups (n >10) were used in immune response studies. group i: high linoleic acid dietl; group i1: low linoleic diet 2, group iii: l/d shift weekly, normal diet and group iv: controls on normal diet, normal lighting. seven weeks after the start of the experiment the immune response was measured. the results showed that corticosterone levels were slightly increased in all experimental groups, although only the high linoleic group showed statistic significant difference with the control group. cellular immune response (con a stimulation and popliteal node assay) was decreased in all experimental groups compaired to controls. transplantable tumors: 1 x 106 leukemia cells were injected i.v. and pieces of 1 mm 3 of an spontaneous adrenal cortical carcinoma, a urethral squamous cell carcinoma and a round cell cervix sarcoma were implanted subcutaneously. all tumors were inoculated in groups of 10 animals each. spleen weight as a measure of leukemia growth was high in the control group and low in the experimental group. the same pattern was seen in the growth of the subcutaneously implanted adrenal cortical carcinomas. both the urethral squamous cell carcinoma and the round cell cervix sarcoma, being non-immunogenic, did not show any difference in growth. so far, it can be concluded, that the immunosuppression as induced by mild chronic stress or dietary fatty acids does not lead to enhanced tumor growth. in contrary, the results of both leukemia and adrenal cortical carcinoma show a possible reserve effect. little is known of the derivation or content of human breast cysts. recent reports have shown wide variations in the content of steroid hormones, particularly dehydroepiandrosterone sulphate (dhas) [1, 2] . no explanation for this is apparent. to confirm the large variation in dhas concentrations and to further define the contents of cyst fluids, 100 cysts from 85 patients have been analysed for dhas, sodium and potassium. dhas concentrations ranged from 1.5-1155 pmol/1. both sodium and potassium content also varied widely (sodium 30-200 pmol/1 and potassium 3-158 ~umol/1). there was a significant direct correlation between the content of potassium and dhas in cyst fluid (p<0.001) and a significant negative correlation with sodium content (/7<0.001). three separate subpopulations of cysts could be identified according to the sodium and potassium content and these were, predominantly potassium cysts (47), predominantly sodium cysts (44) and mixed cationic cysts (9). the median dhas concentration of the potassium cysts was 215 pmol/1 similar to the levels found in human breast secretions [3]. in contrast the median concentration of dhas in the predominantly sodium cysts was 14 pmol/1 and significantly different (p<0.0005), with many of these cysts having dhas concentrations in the same range as those found in plasma. the remaining mixed cysts had a median dhas concentration intermediate between the two main groups. it may be that the variation in cationic content and dhas concentration in these two major subpopula-tions of human breast cysts represents either, derivation from two different sources, namely breast secretions and plasma or marked differences in the secretory activity of the epithelium lining these two groups of cysts. there is no uniform agreement on the correct management of patients with invasive lobular carcinoma (ilc). it is widely considered that in ilc there is an increased risk of developing a contra-lateral carcinoma and the major controversy surrounds the management of the second breast. the survival of patients with ilc was significantly better than that of idc fp<:0.025). six patients had bilateral carcinomata at diagnosis and a further 14 developed a contra-lateral carcinoma during the period of follow-up (10 to 21 years). survival data showed poor survival for patients with simultaneous bilateral disease, but no difference in survival for patients with metachronous bilateral or unilateral disease. this suggests that the later development of a second carcinoma does not necessarily reduce the probability of survival for patients with ilc. the major factor predicting patients at risk of developing a contralateral carcinoma was histologi-195 cal type. of 22 patients with a particular histological pattern of ilc [4] with a classical pattern of spread but showing nuclear pleomorphism and cellular cohesion, 10 developed a contralateral carcinome, compared with a further 10 in the remaining 81 patients (p<0.001). if bilateral mastectomy is justified it ought to be restricted to patients with this histological type of ilc. both the anti-oestrogen tamoxifen and cyclical combined chemotherapy will provide significant palliation in advanced breast cancer. the optimal use of these agents requires further evaluation and thus this trial was designed to compare a combination ofcytotoxic therapy and tamoxifen, against cytotoxics alone in patients with advanced breast cancer. post-menopausal patients presenting with metastatic breast cancer, locally advanced cancer extending beyond the breast and regional nodes, or with tumor recurrence following primary local treatment were allocated to the 3 treatment arms via sequential manner. doxorubicin, cyclophosphamide, 5-fluouracil, and vincristine were given intravenously once every 3 weeks. tamoxifen was prescribed in a dose of 20 mg. b.d. on failure or relapse from one of the single modality arms, a crossover of those arms occurred. the combination consisted of both the above therapies. assessment of therapies was made in terms of objective response (uicc criteria), duration of response, and survival. we have previously reported that the combination results in a significantly greater response rate [1] . as a result of stenosis reducing flow or by platelet embolisation [1] . as neither aniography nor ultrasound can identify thrombotic activity we have evaluated gamma camera neck imaging using n 1indium platelets. labelled platelets on endarterectomy specimens were also measured and the activities found were then examined in a theoretical model. twentyfive patients with tia received rain platelets and sequential gamma images were interpreted by two observers, carotid endarterectomy in 11 patients allowed measurement of specimen radioactivities. angiography and doppler spectral analysis [2] were also performed. all endarterectomy specimens contained labelled platelet deposits with the most active equivalent to platelets from 1.8 ml of blood. this activity level was at the threshold of resolution in the theoretical model. both observers agreed that 22 of the 50 carotid bifurcations showed platelet accumulation on imaging. of the 12 atheromatous ulcers demonstrated by angiography 11 were visualised, but only 5 of 10 stenoses greater than 80 per cent were detectable~ since ultrasound identified all stenoses only one angiographically diseased carotid was not detected by combining doppler and platelet imaging. diseased carotids accumulate rain platelets with the more thrombogenic ulcerated plaques identified more frequently than stenoses. long term follow-up is required to establish the clinical relevance of platelet deposition. major problem in vascular endoscopy is the existence of blood which prevents clear visualization. we devised a new technique using a combination of balloon catheter and slender fiberoptic endoscope, by which clear visualization was obtained experimentally and clinically. three to four pairs of orifices of intercostal arteries were also visualized in one visual field. in some dogs, acute aortic dissection was experimentally created by means of blanton's method. the entry, which was located at the descending aorta just distal to the left subclavian artery, was clearly identified. complete occlusion of blood flow and clear visualization could be obtained when balloon pressure exceeded systemic blood pressure. clinical study: in six patients requiring major vascular reconstruction of the aorta (abdominal aneurysm 4, leriche's syndrome 1, dissecting aneurysm 1), vascular endoscopy was performed intraoperatively. in five patients, balloon catheter was introduced through the one of the limbs of y graft after proximal anastomosis. in each case, orifices of the major abdominal aortic branches were clearly observed. irregular orifices and atheromatous plaque of the aortic intima which were not expected from aortogram, were also identified in all patients. intimal tears by vascular claps were more extensive than expected and anastomotic suture lines were able to be checked from inside. in a case of dissecting aneurysm, balloon catheter was advanced through the 11 mm graft which was sutured to the common femoral artery with finding the entry just above the left renal artery. using fiberoptic endoscope and balloon catheter was useful to observe orifices of the major aortic branches, unexpected intimal tears by vascular clamps and atheromatus plaques. it was particularly usbful to check the anastomotic suture line from inside of the aorta and to identify the exact location of the entry in dissecting aneurysm. vascular endoscopy could be one of the invaluable methods to examine, diagnose and treat the patients requiring aortic, caval and other major vascular surgery. (3) produced endothelial injury and a local increase in shear stress in 14 cynomolgus monkeys by suture plicating and constricting the aorta and then feeding an atherogenic diet for 6 months. our findings reveal that carotid plaques localize on the outer wall of the internal carotid (plaque thickness 0.7--+0.1 mm) which is an area of low flow velocity ( -4___ 1 cm/s at re 800) and shear stress (0-+2 dynes/cm 2) and not at the flow divider (thickness 0.1___0.1 mm, p<0.01) which is an area of high flow velocity (81---3 cm/s) and shear (161-+48 dynes/cm2). distal to the carotid bulb, velocity and shear increased on the outer wall and little or no plaque was observed. in experimental coarctations, no endothelial damage was observed (sem and tem) within the high-shear coarct channel and the channel was noted to be free ofatherosclerotic plaque despite the development of extensive diet-induced lesions proximally and distally. thus, high flow velocity and shear stress do not appear to produce endothelial damage in vivo. in addition, plaques were minimal in high shear areas in the human carotid bifurcation and high shear appears to have an inhibitory effect on experimental plaque formation. these data contradict previous investigations implicating high shear stress in plaque pathogenesis. in contrast, host aortic endothelium (ae) fails to cover large vp by pannus ingrowth even over much longer times. to see if iaes succeeds because of inherent differences in growth potential between ae and ve, we used ae to seed 8 cm x 6 mm diameter dacron velour infrarenal vp in dogs. an average of 4 x 105 cells obtained by trypsin/collagenase digestion of the bypassed aortic segment was used to seed each vp by a 4 step preclotting method. the identity of ae was confirmed by stains for factor viii antigen. viability of seeded ae was verified by growth of subaliquots in tissue culture. six weeks after surgery central segments of aeseeded (n = 9) and control unseeded (n = 9) vp were compared by light and scanning electron microscopy using an endothelial coverage score range of -1 (for fibrin/platelet thrombi) to +1 (for confluent endothelial coverage). ae-seeded vp had a score of+0.89 ___ 0.33 (mean___ sd) versus. -0.67___ 0.70 for controls (p<0.001). in addition to endothelial coverage, the subluminal smooth muscle and intramural vasa vasorum previously reported in ve-seeded vp were also seen in ae-seeded vp. since ae and ve seeding give identical results, the success of iaes with ve cannot be due to inherent biological differences in mitotic potential between ae and ve. iaes must instead achieve additional endothelial growth either through a) the action of the proteolytic enzymes used for cell harvest or b) mitogenic stimuli to nonconfluent cells at the edges of seeded cell clusters on the vp. further improvement of the efficiency of iaes to allow use of less harvested vein per cm 2 of vp should come from enhancing one or both of these effects. pyrolytic carbon is a crystalline form of carbon that has been extensively used in the construction of cardiac and bone prostheses. since it has also been suggested that pyrolytic carbon will prevent thrombosis from occuring in vascular prostheses, the aim of the present study performed in dogs was to test the immediate blood compatibility of this material and to evaluate its biocompatibility when inserted as vascular substitute. after pryolysis of a gazeous hydrocarbon, the carbone crystalite was deposited on a knitted textile surface or tube. its surface examined by scanning electron microscopy (sem) was rough and porous to a depth of 40p. this material was tested 1 °) for immediate hemocompatibility as inserts within the vascular lumen (aorta and inferior vena cava). the specimens were examined sequentially by sem and histology at 10, 20, 30, 180 s and 10 min after reestablishment of the blood flow, 2 ° ) for long term biocompatibility as vascular cylinders (7 mm id) inserted either in the aorta or inferior vena cava or as intraatrial (left or right) implants. patency of vascular cylinders was tested during 2 postoperative month by doppler ultrasound investigations, specimens were examined by histology, electron microscopy (scanning transmission) at 15, 30 and 60 days following implantation. satellite lymph nodes were examined by histology. already 10 s after establishement of the blood flow, platelet adhesion and limited fibrin mesh with few erythrocytes developed on the material. platelet aggregates of limited extent were only observed on intravenous implants. plasmatic protein deposition, an early event on polymeric vascular material was not observed. after 30 s a fibrino-erythrocytic membrane recovers completely the material. except in the case of intravenous insert, no thrombosis developed at the contact of intraarterial or intracardiac implant. after 15 days it was completely recovered by a 5-7 fibrocellular layer consisting of large myofibroblasts with microfilaments, newly synthetized collagen and elastin. the blood interface was of fibrous nature. at one month by sem, endotheliallike cells developed in a mosaic-like pattern, characterized by transmission e.m., by microvillous projections, numerous pinocytic vesicles and intercellular tight junctions. this endothelial-like cell lining was complete 2 months after implantation. their immunocytochemical properties are now under investigation using specific anti-dog factor viii-rag sera. although preliminary, the present results suggest that among the numerous vascular biomaterials tested, pyrolytic carbon may represent a unique feature of rapid cell development and differentiation of endothelial lining at the blood material interface. department of connective tissue biology, institute of anatomy, university of aarhus, aarhus, denmark in the surgical clinic a significant number of patients report that their incision wound has burst, even though the scar appears to be intact. by mechanical testing of strips from skin wounds we have noticed a breaking pattern, in which the deepest layer of the wound ruptures earlier than the superficial part. therefore, we have investigated the strength and extensibility of rat skin wounds at different levels (superficial-deep) of the epidermis (0.03 mm), dermis (1.8-2.2 mm) and m. panniculus carneous (0.6-1.5 mm), average thickness is indicated. 10, 20 and 60 day old standardized skin wounds from the dorsal region of rats have been used. strips were punched out at right angle to the wound line and mounted in a materials testing machine. the strips were stretched until rupture and load-strain curves registered continuously. simultaneously, the strips were transluminated and the breaking pattern was studied by taking photographs of the wound specimens during the mechanical testing (30-35 photographs of each specimen). the photographs were marked on the load-strain curve by means of a connection between camera and x-y-recorder. from the load-strain curves the maximum load and the failure energy were calculated. the breaking patterns of 10, 20 and 60 day old wounds were found to be similar. the deep part of the wounds ruptured first. the force required to break the deep part was less than that required to break the superficial part of the wounds. the musculus panniculus carneous was very extensible and did not break. however, it possessed only minimal strength. quantitative measurements of the strength of the combined superficial-deep layers were performed on 2 mm wound strips. specimens contained the superficial 0.5, 1.0 and 1.5 mm of the wound area and were produced by cutting off the deep layer parallel to the skin surface. specimens containing the total wound area down to the musculus panniculus carneous were produced by cutting off the muscular tissue. these specimens were mechanically tested as described above. the present studies demonstrate the mechanical inhomogeneity of incision wounds. a new method for testing the mechanical properties of the tissue of incision wounds at various levels (superficial-deep) is presented. the superficial layer of an incision wound contributes a major part of the strength of the wound and is more extensible than the deep layers. these results may explain the clinical observations. the effect on wound healing of different kinds of vitamins is worth investigating, since the efficiency of vitamin c has been dearly demonstrated. the possible action of vitamin bs-whose trophic effect on skin is well known -has been experimentally studied on skin and aponeurosis healing after a standard laparotomy. materials and methods: experiments were carried out on 42 five months old rabbits which were randomly divided into three groups: in group i, 18 animals served as controls (3 animals in sequence of 5 days from the 5th to the 30th post-operative day), group ii, 12 animals injected with vit b5 (25 mg/kg of body weight/24 h) and group iii, 12 animals injected with a placebo (2 animals in sequence of 5 days for each group). in each case four samples were tested of skin and aponeurosis for determinating tensile strength, directly recorded with an original technic [1] : this new apparatus allowed us to obtain simultaneously two dynamic parameters, the healing tensile strength and stretching of the scar. results: 1. no significant difference was found between controls (group i) and the placebo group (group iii) both for resistance of skin and the aponeurosis. 2. as far as vitamin b5 treated animals were concerned (group ii) there was no significant difference regarding skin resistance when compared with the other two groups. 3. inversely aponeurosis resistance become significantly greater when measured on the 20th (p<0.050), 25th (/r<0.025) and 30th (p<0.050) post-operative day. in 17 mongrel dogs (7x 15 cm cranial based rectus abdominis) mc and corresponding rp flaps were raised. in group i (9 dogs) skin bf was determined from the clearance curve for ~33xenon injected intradermally and measured with a computer-linked gamma camera. in group ii (8 dogs) subcutaneous pro2 was determined by a recently developed method using a silastic tonometer, subcutaneously implanted. the pro 2 inside the tonometer was measured in infused saline, by a platinum oxygen needle electrode and a silver/silver chloride reference electrode. b f and pto2 were measured before and after the flaps were raised and on postoperative days (pod) 1, 3, 6 and 15. pto2 were taken at 6 various inspiratoric oxygen levels (f~o2) ranging from 21% (air) to 100 % oxygen. intact areas lateral to the flaps and in flap regions prior to surgery served as controls. immediately after surgery bf in the mc increased while in the rp flaps was 24 %, 23 % and 31% of the flow in the mc flaps, in lateral intact area and in the preoperative areas (p<0.001). during pod 1-2 bf in the rp flaps increased to the preoperative level, but not to the increased levels found in the mc flaps and the lateral intact areas. by pod 6 there were no differences in bf between the two types of flaps and the lateral areas, but all were higher than corresponding preoperative values (/'<0.005). tissue oxygen tension showed a dramatic fall pod 1, 3 and 6 in the rp flaps for all fio2, and for all days the values were lower than the preoperative level (p<0.001). one rp developed pod 3 distal necrosis and the pro 2 was then 0 even with a fio2 of 100%. the mc flap showed an increased pro2 on the operative day but at pod 3 the values were slightly lower than the preoperative level, but pod 1, 3 and 6 the values for all fio2 were higher than for rp flaps (p<0,005). at pod 15 the pro2 reached preoperative level for rp as well as mc flaps. lateral intact areas showed comparable changes to that observed in the mc flaps. it is concluded that the mc flap demonstrates superior bf as well as pro2 when compared to the rp flap. early postoperative pro 2 in the distal part of the rp flaps is critically low despite of increasing f~o2 to 100 % and increasing bf. differences in bf and pto2 may be the biologic factors responsible for the superior healing characteristics of the mc flap. (1) atp~adp + pi (inorganic phosphate) (2) pcr + adp~atp the net result ofreaotjoxas 1 and 2 is a fall in pcr and a rise in pi while atp ~'emains relatively constant. all of the phosphorus metabolites are easily measured in gastrocnemiaas muscle using 31pnmr spectroscopy. normal volunteers and patients with angiographically documented arterial occlusions were studied in a 101/4" bore oxford research systems tmr-32 spectrometer at rest and after exercising each limb separately. normal resting values ofpcr/p iwere >10 and the nmr index = p/(p~+pcr) was 0.07_+0.03 (s.d.). limbs with femoral arterial occlusions whose ankle systolic pressure index was <0.5 had nmr index which was significantly elevated above norreals (0.18 + 0.09p<0.01) indicating a failure of metabolic compensation for reduced bloodflow and oxygen delivery, although atp concentration was norreal. exercise produced a five-fold rise in nmr index in both normal and diseased legs. spectra were taken over one minute intervals during the recovery period and in normal limbs returned to resting values within 2 rain. the recovery period was considerably slower in the diseased limbs indicating abnormal mitochondrial oxygen delivery and impaired mitochondrial formation of atp. these data demonstrate the feasibility of using 31pnmr to non-invasively probe the biochemical abnormalities of energy metabolism in patients with peripheral vascular disease. the incidence of urinary calculous disease (ucd) in the south african black population is very low in comparison with the white population group. no biochemical differences in serum nor urine account for this discrepancy and no other measurable parameters have demonstrated any difference between the two groups. urinary particulate activity measurements have demonstrated differences between normal persons and those with ucd who are otherwise biochemically similar, and it would therefore seem rational to expect such measurements to demonstrate differences between the two population groups. urinary particulate activity was measured in the urin of 100 normal whites and 100 normal blacks, the two groups being matched for age, height and weight, and monitored under normal dietary hydrational and environmental conditions. the three parameters of particulate nucleation, growth and aggregation were measured and the two groups compared. particulate nucleation demonstrated the most significant contrast between the two groups with the production of new particles through nucleation being far greater in the white group than that which occurred in the black group (p<0.001). particulate growth occurred at similar rates in the two groups although at slightly higher rates in the white group. particulate aggregation occurred at a greater rate in the white group but the difference between the two groups was not statistically significant. the differences between the two groups are shown to occur as a consequence of differing rates of particulate nucleation although the rates of particulate growth and aggregation are parallel. whilst the factors responsible for the low nucleation rate in black person remain unknown their effect can now be measured quantitatively through the parameters of urinary particulate activity. blood levels of ketone bodies appear to determine skeletal muscle amino acid release; high levels conserve protein and attenuate gluconeogenesis. starvation indt/ced ketosis is suppressed by infection [1] . to determine if the relative hypoketonaemia following sepsi s in turn contributes to increased glucogenesis, arterial substrates and glucose production (constant infusion 6-3h(n)-glucose) were measured before and after infusion of na-dl-./3-hydroxybutyrate (/3oh) to raise levels three-to fourfold in fed (n= 11), in fasted (n = 8) and in fasted-infected (n= 9) animals. in fasted-infected animals before infusion ketosis did not occur (/3oh 0.81±0.1 mm/1 fasted; 0.45 ±0.55 fasted-infected) and basal glucose turnover was increased (5.05±0.18 /tm/kg/min fasted; 9.5±0.83 fastedinfected). with infusion of glucose and alanine concentrations decreased as expected in fed and fasted animals but not in fasted-infected (glucose 2.33±0.17 ram/1 befor; 2.44+0.11 mm/1 after). glucose production also fell significantly in the fed (10.11±1.33 /~m/kg/min before; 8.44±1.05 after) and fasted (5.05±0.28 v. 4.11±0.33) groups but was unaffected by infusion in the fasted-infected group (9.5+-1.83 v. 9.11+1.44). the accelerated rate of gluconeogenesis in infection is thus not a consequence of hypoketonaemia. the usual reciprocal relationship between glucose and ketone utilisation during feeding and fasting has not been demonstrated~in sepsis. preliminary experiments in a hindlimb model support the hypothesis 201 that during infection amino acid release from muscle is not affected by ketone levels. we have developed a technique for measuring the total body carbon of the living subject which is suitable for measuring the critically-ill as well as the ambulatory patient. by combining this measurement with that of total body nitrogen and calcium [1] an estimate of total body fat is derived. measurement at the beginning and end of a given period enables the changes in total body protein and fat to be obtained, as well as the patient's energy expenditure if energy intake is also known. the method is a radiation technique. the supine patient is irradiated laterally with a horizontal beam of fast neutrons and the resulting gamma rays from the body are detected by a radiation detector placed unterneath the subject. the nuclear reaction employed is the inelastic scattering of fast neutrons by the carbon nuclei of the body with the emitted gamma rays having an energy of 4.43mevi in the initial application, measures of total body fat obtained using the technique were compared with those derived from skinfold thicknesses in six volunteers: there was no significant differences between the two measurements, (see table) . the method is being employed in studying the changes in total body protein and fat, and the energy requirements of surgical patients receiving nutrition. in order to investigate the mechanism of this effect, normal monocytes were incubated at 37°c for 30 min (with intralipid 40/a/ml) and their function assessed by three different techniques (chemotaxis, phagocytosis and chemiluminescence). all three methods showed impairment of function following exposure to intralipid. in order to try and prevent this potentially damaging effect, heparin was added to the various in vitro tests and found to cause marked impairment of phagocytosis. (p<0.01) to assess its effect in vivo, 11 volunteers were given 5,000 units of subcutaneous heparin 2 h prior to intravenous intralipid (as above). although the use of heparin did not affect either immunological function, it completely prevented the fall of monocyte chemotaxis following intralipid alone. these findings suggest that monocyte function may be impaired by the presence ofintracellular lipid particles. the use of s.c. heparin may help to alleviate this problem and could, therefore, be beneficial to ill and often septic patients requiring intravenous nutrition. to investigate the effect of elevated glucocorticoids of stress and trauma on peripheral glutamine metabolism, 0.44 mg/kg bw dexamethasone was injected daily intramuscularely in adult mongroel dogs over a period of 2 weeks. at least 3 weeks prior to the experiments catheters were placed into the animal's abdominal aorta (2) and caval vein (1) in order to measure a-v differences and hindquarter blood flow. during dexamethasone treatment nitrogen balances were negative, -7.1-4-1 g n per day, whereas slightly positive n-balances were observed during the control period (3.1 +__ 1.7 g n/day). muscle glutarnine concentrations declined constantly from 22.1 +2.3 mmol/1 intracellular water to 10.0-4-1.2 by 56% within two weeks. whole blood arterial and venous plasma concentrations remained constant. to test the hypothesis of increased peripheral glutamine utilisation or decreased glutamine formation, the activities of glutaminase and glutamine synthetase were measured in a muscle homogenate obtained before and 8 days after dexamethasone treatment. both enzyme activities were found to be unchanged. hindquarter glutamine efflux increased from 20.3+22.6 pmol/min in the control state to 80.0+24.1 during dexamethasone treatment indicating a 4 fold muscle glutamine output. this increased glutamine output was enirely due to increased a-v differences and despite decreased hindquaarter blood flow during dexamethasone. it is concluded that dexamethasone reproduces the metabolic response of trauma and sepsis in terms of negative nitrogen balance and muscle glutamine depletion. muscle glutamine is shifted from peripheral tissues to visceral organs with muscle compensating for visceral demands rather than skeletal muscle being the primary target of corticoid action. it has been suggested that there is abnormal glucose utilisation in malnourished patients and that this may explain the adverse clinical sequelae of high rates of glucose infusion during intravenous feeding. we have investigated the hypothesis that there is a depression of the key enzymes of glucose oxidation in the muscle of malnourished patients which is due to an alteration of muscle fibre type proportions. 14 malnourished patients (p) (9m,5f 56+12 yrs) our results demonstrate that there is a positive correlation between preoperative cp and stage of cancer 0' =34.4+-3.88x; r=0.62;/9<0.001). nevertheless before surgery there is no difference between cp values in the two groups considered (g.c.= 46.2___9.7 mg %;p.u. =44.4----9.0 mg%), but after surgical trauma cp presents a positive response in patients with p.u. (mean increase +14.9 %), whereas it acts as a negative ap protein in g.c. patients (mean decrease 5-60/0) (p<0.02). moreover malnourished g.c. patients present a reduction of cp values ( -10 %) which is greater than g.c. patients with albumin >3.5 g% ( -1.5%); this difference is not statistically significant. cancer patients undergoing palliative (n=10) or radical surgical procedure (n=31) show parallel decrease of preoperative cp ( -6%), the first group presenting higher preoperative values in relation to the tumor diffusion. in conclusion our results demonstrate that cp is not only a positive ap protein, but in some circumstances it may act as a negative pa protein depending on the underlying disease and the preoperative nutritional status. in this study the free aa concentration in liver tissue of 4 non septic patients (cholecystectomy) were compared with those of 7 septic patients (abdominal sepsis). the liver specimens were taken intraoperatively..the nature and possible risk involved in this study were explained to the patients and their consent obtained. the data presented in this abstract are part of a metabolic screening program of septic patients including the determination of aa (plasma, muscle), hormones (insulin, glucagon, cortisol), nutritional parameters (prealbumin, retinol-binding protein, transferrin), and of energy metabolism (atp, adp, glucose, free fatty acids). for the determination of the free aa the intra-and extracellular water content (chlorid method) and of fat content of the liver specimen were analysed. a membrane potential o f -45mv was assumed. the aa analysis were performed with an automatic aa analyser (kontron, svitzerland) by means of an ionexchange resin (durrum dc-4) and a lithium buffer system (durrum-pico buffers). conclusions: 1. this study reveals decreased concentrations of nearly all aa in liver tissue of septic patients (exception: phenylalanine, tyrosine, cystathionine). 2. the significantly decreased concentrations of the gluconeogenetic aa (thr, ser, ala) indicate that the gluconeogenetic capacity of the liver is not exhausted through an increased uptake of those aa as shown earlier by wilmore et al. [4] 3. an increased administration of gluconeogenetic and basic aa (lys, his) may normalise the aa pattern in the liver of septic patients. the liver is being increasingly recognized as a critical organ in postoperative multiple organ failure. the principle factors precipitating postoperative multiple organ failure were sepsis, hypotension and injury to the liver. previous studies from our laboratory have shown that hepatic failure, which has a high mortality rate, is linked to the marked decrease in energy charge. in order to evaluate the possible presence of metabolic blocks, the changes in the ratio of acetoacetate to fl-hydroxybutyrate (ketone body ratio), which reflects the hepatic mitochondrial redox potential, were analyzed in relation to energy charge in hepatectomized, jaundiced, hemorrhagic-shokked and septic animals, as well as patients with postoperative multiple organ failure. experimental: 1. in hepatectomized rabbits, mitochondrial phosphorylative activity increased to 170 % of the control and the energy charge level decreased from the normal level of 0.87 to 0.76 at 24 h after hepatectomy (/7<0.001). afterward, these values returned to preoperative levels within a week. the ketone body ratio in arterial blood was positively correlated with hepatic energy charge (r=0.696, p~0.01). 2. in jaundiced rabbits, the hepatic energy charge decreased rapidly after the bile duct ligation along with the decrease of mitochondrial pbospborylative activity. the hepatic energy charge fell from 0.87 to 0.73 at 48 h postoperatively with a maximum incidence of mortality. moreover, changes in the blood ketone body ratio were positively correlated with the hepatic energy charge (r= 0.844,/~0.01). the decrease in the blood ketone body ratio was attributed to the restricted mitochondrial reoxidation of nadh due to an inhibition of oxidative phosphorylation. 3. in hemorrhagic-shocked rabbit with a mean arterial blood pressure of 40mmhg, the changes in the blood ketone body ratio were correlated with hepatic energy charge (r=0.772, p<0.01). 4. in septic pigs subjected to the ligation and perforation of the cecum, the hepatic energy charge level decreased gradually from 0.86 to 0.80 and the mitochondrial phosphorylative activity was enhanced to 150% of controls in the hyperdynamic state. in the hypodynamic state, the hepatic energy charge level fell drastically 0.69 concomitant with the decrease in mitochondrial phosphorylative activity and blood ketone body ratio. from these results, the blood ketone body ratio may be regarded as a reliable indicator for assessing the degree of decreased energy charge. clinical: changes in the blood ketone body ratio were measured in 55 patients who underwent major surgery such as hepatectomy. these patients were classified into 4 groups according to the postoperative changes in blood ketone body ratio: group a without decrease to below 0.7, group b with transient decrease to 0.4, group c with progressive decrease to below 0.4 and group d with terminal decrease to below 0.25. all 35 group a and b patients tolerated the operation well. by contrast, the 20 group c patients showed multiple organ failure with 85 % mortality rate, which involved pulmonary failure (70%), hepatic failure (70%), gastrointestinal bleeding (30%), renal failure (600/0), cerebral failure (70%) and coagulopathy (65%). all patients who transitioned to the terminal stage of group d died of cardiogenic decompensation. in 5 patients of group c, the decreased blood ketone body ratio was restored with the amelioration of clinical symptoms after ex vivo pig or baboon liver crosshemodialysis and 3 patients of them were later discharged. evidence presented indicates that the decreased blood ketone body ratio has a direct bearing on multiple organ failure. conclusion: sepsis, hypotension or injury to the liver are a metabolic burden to the liver mitochondria which can result in mitochondrial impairment leading to a marked decrease in hepatic energy charge. such impairment ultimately leads to multiple organ failure as a result of the critically decreased energy and substrate store and the reduced protein synthesis relative to demand in the various organs. 110 in interferon-treated cells, the 2'-5'a synthetase, activated by double stranded rna, polymerizes atp into a series of oligonucleotides characterized by 2'-5' phosphodiester bonds and collectively designated 2'-5'a. these activate an endoribonuclease which cleaves rna. other regulatory functions of this enzyme may be expected because of its wide occurence in mammalian cells (untreated with interferon), where its activity appears, in vitro, to be dependent on the growth conditions, hormone responses regenerating liver after partial hepatectomy is often used as a model for the study of control growth and cell proliferation in vivo. in order to evaluate the role of the 2'-5'a synthetase in the processes leading to initiation of cell division, we measured this enzymatic activity in the rat liver during the first 40 h after partial hepatectomy. partial hepatectomy (50 rats) was performed under neuroleptanalgesia by removing the median and the left lateral lobes of the liver according to the method of higgins and anderson. control animals (3 rats) were subjected to a sham operation. after selected time intervals, the animals were sacrificed and the enzymatic activity in the regenerated liver was measured. the 2'-5'a synthetase activity present in the two first removed lobes was defined as 100%. we observe a very rapid decrease of enzymatic activity which reaches 50 % already 10 h after partial hepatectomy. the lower level of enzymatic activity (25 %) is measured between 20 and 24 h after partial hepatectomy. this minimum is followed by a slow restoration of the activity (at 40 h: 60 %). during this early phase of liver regeneration, a maximal incorporation of tritiated thymidine in dna takes place 24 h after surgery. so well differentiated liver cells have elevated levels of2'-5'a synthetase. but, after partial hepatec-tomy, the 2'-5'a synthetase activity decreases dramatically before the first wave of cell mitosis. these observations clearly illustrate the relationship between 2'-5'a synthetase activity and the growth status. moreover, this drop of enzymatic activity may be a trigger for the initiation of cell division. the primary event or events setting in motion the process of liver regeneration after partial hepatectomy (ph) remain unsettled. regarding the so called hepatotrophic factors, i.e. insulin, glucagon and recently egf, present evidence suggests that they would play mainly a promoting rather than a initiating role. early changes such as glycogen breakdown, fat infiltration and changes in adenine nucleotides and mitochondrial phosphorylative activity are usually, at least the first two, ascribed to metabolic overload of the remaining liver (bucher et al (1969) johns hopkins med, j, 125: 250). so far, however, little attention has been paid to a possible involvement of this phenomenon in the initiation of liver regeneration. attempts were therefore made to modify metabolic overload through early changes in energy metabolism in order to study their influence on the pattern of dna synthesis. fed or 16 h fffsting male wistar rats weighing 200+20 g were examined after ph at 4, 12, 18, 24, 30 and 36 h for adenine nucleotides, oxidative phosphorylation and dna synthesis, based on the rate of~h thymidine incorporation. fasting animals received continuous infusion of 20 % dextrose for 24 h at the rate of 0.45 ml/100 g/h. in addition to the above mentionned parameters hepatic glycogen and fatty acids were measured. within 12 h partial hepatectomy caused a decrease in hepatic atp which was maximal at 3 h (from 2.99___0.13 to 2.36+0.07 /~ moles/g p<0.001); in energy charge (atp + 0.5 adp/atp + adp + amp) from 0.851-+0.01 to 0.816___0.03 (p<0.01) and increase in phosphorylation potential which was maximal at 3 h (from 76+3 to 106_2p<0.001). dna synthesis began at 18 h reaching a peak by 24 h. glucose infusion to ph rats suppressed the decrease of hepatic atp, 2.97___ 0.07/1 mole/g at 3 h vs 2.99_ 0.13 in the control group, prevented glycogen depletion (histochemical estimation) and the increase in fatty acids (two folds increase in ffa and triglycerides (tg) at 24 h vs 10 folds in ffa and 8 folds in tg in the control group),with little effect on mitochondrial activity. the initiation of dna synthesis was delayed and the whole pattern was considerably modified. cessation of glucose infusion restored the usual rate of 3h thymidine incorporation after a late fall of hepatic atp. in conclusion, glucose infusion was shown by one of us to modify the hormonal response to ph, but insulin and glucagon administration to glucose treated animals failed to normalize the pattern of dna synthesis. it is suggested that metabolic overload as estimated on the basis of early changes in energy matabolism may account for one of the events involved in the initiation of dna synthesis after ph. infusion on liver cell regeneration after partial hepatectomy in the rat b. de hemptinne, j.f. ngala and l. lambotte university of louvain, laboratory of experimental surgery ucl 5570, 1200 brussels, belgium after partial hepatectomy (ph) the portal and the peripheral blood serum concentration of immunoreactive insulin suffers a drastic fall and levels ofglucagon show a rapid increase which is maximal 4 h after the liver resection. as these changes appear closely correlated to the blood glucose levels which show a 30 % decrease at 4 h and progressive restoration towards normal values up to 24 h, attempts have been made to alter the insulin/glucagon ratio by glucose infusion after ph and study its relation to liver regeneration. the purpose of this work is thus to determine after ph and hypertonic (20%) glucose infusion: 1. the effect of glucose on insulin and glucagon blood levels over 24 h; 2. the repercussion of the insulin/glucagon modified ratio on dna synthesis; 3. the possible improvement of dna synthesis by extensive glucagon infusion. male fisher rats underwent a standard 70 % ph. a 20 % glucose solution or isotonic salin was infused at a constant rate of 0.9 ml/h through a cannula placed in the iliac vein. the rats were sacrified at 0, 4, 8, 12, 22 and 24 h. (3h) thymidyne (27..3/~ci/mmol) was injected 2 h prior to sacrifice and the liver caudate lobes removed for analysis of (3h) thymidine uptake into nuclear dna. blood samples were withdrawn from the portal vein, the inferior vena cava and the aorta for glucose, insulin and glucagon assays. compared to the salin treated group, the infusion of glucose while keeping a normal steady blood glycemia was responsible of a marked increase of insulin (0.38--+0.01 ng vs 2.24_+0.4 ng,/7<0.01) and decrease of glucagon (0.615-+0.085 ng vs 0.335+0.037 ng, p<0.01), with a major switch of the insulin/glucagon ratio at 4 h after ph (from 6.68 to 0.61). dna synthesis started at 22 h in both series, but was very significantly impaired at 24 h in the glucose infused group (12800-+2280 cpm/mg dna vs 27450+2050 cpm/mg dna, p<0.01). infusion of increasing doses of glucagon (from 0.01 to 2 mg/kg/day could not restore the impaired dna synthesis. only a slight improvement was recorded at 0.5 mg/kg/day as the insulin/glucagon ratio tended to approach that of the control group. fractionation of various doses of glucagon over the 24 h perfusion time, in such a way that the changes in concentration of glucagon after partial hepatectomy was imitated, remained unsuccessful to improve thymidine incorporation. in conclusion: 1. the infusion of hypertonic glucose which impairs dna synthesis after ph, modifies markedly the insulin and glucagon secretion. 2. if a specific insulin/glucagon ratio after ph is important to sustain normal regeneration, its modification does not seem to be the major factor contributing to the blunted dna synthesis response in the hypertonic glucose infusion model. operative mortality of emergency shunt operations or esophageal transection during acute hemorrhage from ruptured esophageal varices in cirrhotic patients (child's category c) has been intolerably high. hence, the emergency operations in these patients should be avoided when the bleeding could be stopped by non-operative measures. when the emergency operation eventually becomes inevitable, the operative procedures should be simple and of short duration. in 1976, we have introduced the endoscopic balloon tamponade method for the management of esophageal variceal bleeding. the new balloon tube used in this method has essentially the similar structure as the sengstaken-blakemore tube, but is made of translucent plastic materials, and has a larger internal diameter so that a small caliber optic fiberscope (ex. bronchofiberscope) can be passed through the tube. by this method, the endoscopic observation of the esophagus is possible through the translucent balloon tube during tamponade of the ruptured varices. it is possible to know directly whether the bleeding from varices has been successfully stopped or not, which seems to be an advantage over the blind tamponade method using the sengstaken-blakemore tube. this endoscopic tamponade method has been used for emergency hemostasis of 166 acute bleeding from ruptured esophageal varices in 66 patients. the mean initial tamponade pressure by the esophageal balloon necessary to stop bleeding was 30_+12 mrnhg and the average duration of tamponade was 50 h. the location of the ruptured v~ices observed by this method was in the lower esophagus within 10 cm of the esophagocardiac junction in 86% of the cases. the bleeding has been stopped in 156 occasions (93.9 %) by this method. no significant complications other than atelectasis in 2 patients have been observed. in 4 patients, the bleeding was initially stopped by the new translucent balloon tube, but recurred within 24 h after decompression of the esophageal balloon. tamponade was repeated for several times since these patients were in the category c of the child's classification, however, the emergency operation eventually became necessary. transthoracic esophageal transection was performed using autosu-ture apparatus, eea, in these patients. one patient died of liver failure in the 6th postoperative day, but others survived the operation and recovered. the use of eea in transthoracic esophageal transection simplifies the esophageal anastomosis, and shortens the duration of operation by 40 rain. we have so far used the autosuture apparatus, eea (28 mm cartridge) in elective esophageal transection of 33 patients. neither anastomotic bleeding or insufficiency has been encountered. acute variceal bleeding in category c patients should be treated initially by endoscopic balloon tamponade, when emergency operation is inevitable, transthoracic esophageal transection using eea is the operation of choice. arterialisation of the portal vein in conjunction with an end-to-side portacaval shunt has been proposed as a method of improving survival following shunting [1] . however, there is little experimental evidence to support this suggestion and so we have examined the effects of arterialisation of the portal stump in cirrhotic rats. 24 rats with dimenthylnitrosamine-induced cirrhosis were used in this study. 8 of the rats received an end-to-side portacaval shunt, 8 had a portacaval shunt and the portal stump arterialised with the left gastric artery, eight were sham-operated. liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before and after shunting. three weeks after surgery lbf and whvp measurements were repeated, the animals bled and the liver removed and weighed. an end-to-side portacaval shunt led to an immediate fall in lbf (31.3_+5.6 to 15.5_+4.3 mls/min/ 100g-~) and whvp (7.6_+0.8 to 3.1-+0.5 mmhg). however, if the portal stump was arterialised lbf (34.6-+2.9 to 45.3+5.7) and whvp (7.9_+1.0 to 7.3_+1.3) did not change significantly. no further changes in lbf and whvp were observed three weeks after operation in any group of animals. arterialisation of the portal stump prevented the loss in body weight, loss in liver weight deterioration of liver function tests and hyperammonemia observed in animals with a portacaval shunt alone. these findings suggest that arterialisation of the portal stump may prevent some of the deleterious effects after shunting. departments of surgery and pathology, university of virginia hospital, charlottesville, virginia 22908, usa we have hadexperience with operative restoration ofhepatopedal portal blood flow in five patients intolerant of total splanchnic shunting. hepatopedal flow was reestablished by takedown of the total shunt and construction of a selective, distal splenorenal shunt, or by isolation and arterialization of the hepatic limb of the shunted portal vein. in two patients, shunt revision was undertaken electively for chronic encephalopathy, unresponsive to low protein diet, intestinal antibiosis and oral lactulose. each individual had been hospitalized more than eight times for encephalopathy or coma. nine and 34 months postoperatively, both patients have had no encephalopathy on unrestricted protein intake, and work. actively as homemakers. serial liver needle biopsies have shown bilobed nuclei and enhanced mitotic activity suggesting hepatocyte regeneration. in three patients, shunt conversion or arterialization was undertaken in desperate circumstances, characterized by liver failure (bilirubin >10 mg/dl, albumin <2.5 girl, prothrombin time >1"6 s)~ coma and respirator dependency. although two patients showed immediate, marked improvement in mentation, all three died of intraabdominal hemorrhage in the first few postoperative days in spite of prolonged attempts to achieve hemostasis and maximum blood product support. three conclusions can be drawn from this limited experience: 1. total shunt procedures which are anatomically suitable for subsequent conversion to a selective configuration or for hepatic limb arterialization should be favored over those not offering such potential; 2. at a time of election, restoration of hepatopedal portal flow can be accomplished in patients with side-to-side portacaval or hemodynamically equivalent shunts with considerable benefit; and 3. similar procedures in patients with fulminant liver failure are unlikely to succeed. peritoneal-venous (leveen) shunts are associated with a significant incidence of disseminated intravascular coagulation (d.i.c.). this study identifies a site of origin, a pathogenic mechanism, and the hemostatic pathway which accounts for the thrombogenicity of human ascites. 500 ml of peritoneal fluid were removed percutaneously from individuals with malignant and cirrhotic ascites. ficoll-hypaque column chromatography and ultracentrifugation were utilized to prepare four fractions: cellular; a low speed cell-free fluid; a high speed supernatant; and the precipitate from the high speed centrifugation. the cellular fraction from both types demonstrated an ability to shorten a one stage clotting time by 60 % relative to saline and endotoxin controls. similarly, low speed cell-free fluid shortened the clotting time of pooled normal plasma by 61%; was also effective in factor viii (required for intrinsic pathway of coagulation) deficient plasma; but had no effect on factor vii (required for extrinsic pathway of coagulation) deficient plasma or platelet aggregation and release. the high speed supernatant was demonstrably less thrombogenic. the resuspended precipitate shortened the clotting time of pooled normal plasman by 67 % and of factor viii deficient plasma from infinity to 99 s. in contrast, this material was ineffective on factor vii deficient plasma. we conclude that the thrombotic potential of human ascites derives from peritoneal cells, either leucocytes or malignant cells. consistently, the thrombotic factor exists in suspension and is thromboplastin-like in its behavior, operating through the extrinsic pathway of coagulation. thus, a site of origin and a pathway of activity for the thrombotic agent in human ascites is identified. the effect of somatostatin in hepatic haemodynamics in the cirrhotic rat s. jenkins, p. devitt and r. shields department of surgery, university of liverpool, liverpool, u.k. although somatostatin has been suggested as an alternative treatment to vasopressin in the emergency control ofbleeding oesophageal varices [1] , recent studies on its effect on wedged hepatic venous pressure in cirrhotic patients have provided conflicting results [2, 3] . therefore we have examined the effect of somatostatin on hepatic heamodynamics in cirrhotic rats. rats with dimethylnitrosamine-induced cirrhosis received a bolus injection of 2, 4 or 8 pg/kg body weight of somatostatin followed by a 30 min infusion of either 2, 4 or 8 pg/h/kg body weight. control rats received saline only. portal venous flow (pvp) portal pressure (pp), liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before, during and after somatostatin infusion. at the lowest rate of somatostatin administration (bolus injection of 2 pg/kg body weight followed by an infusion of 2 pg/h/kg body weight)there was a rapid decrease in pp (9.8+0.5 to 8.0--+0.4 mmhg), whvp (7.5___0.4 to 5.8---0.3 mmhg) and pvf (31.2 ___ 4.9 to 21.0___ 3.8 ml/min). 30 rain after the start of the infusion pp, whvp and pvf were still signaflcantly lower than preinfusion levels. at higher rates of somatostatin infusion there was no furhter decrease in pp, whvp and pvf. lbf was significantly reduced at all the doses of somatostatin. the highest rate of infusion of somatostatin (8 pg) produced a significantly greater reduction in lbf than either 2 or 4 pg. these data suggest that somatostatin may have a role in the management of portal hypertension, but that higher doses may have a detrimental effect on lbf. er positive breast cancers preferentially metastasize to bone (1) prostaglandin e2 (pgez) is synthesized by breast cancers and potentiates bone resorption in vitro (2) . this study investigates the relationship between er status and pge2 synthesis in breast cancer cells. pge2 was measured by radioimmunoassay in 75 primary breast cancers: a) after ethanol inhibition of further prostaglandin (pg) production -,,basal pg" b) after stimulating pg production with excess arachidonic acid -,,total pg". ,,total" minus ,,basal" = ,,synthesized pg". in order to relate pg production to breast cancer cells, measured pge2 values have been corrected for the epithelial cellularity of each tumour. pge2 x 100 corrected pge2 = actual (%) cellularity cellularity was evaluated by a proportional count of cancer cells expressed as a percentage against non cellular material in all fields of 3-5 histological sections at 63x magnification. we conclude that er positive tumour cells synthesize greater amonts pge2 than er negative cells. this may account for the greater tendency of er positive cancers to recur in bone. oestrogen receptor activity (er) is of prognostic value in breast cancer [1] . however, the chosen cutoff between er-negative and -positive is arbitrary and likely to affect its prognostic value. in 201 patients with invasive breast cancer treated by mastectomy, tumour er was determined [2] and the patients were followed up until first recurrence. using a computer program to perform repeated logrank analysis, the effect of varying the cut-off on prognostic value of erwas studied between 1 and 25 fmol/mg protein. er levels were higher in postmenopausal patients (0-1301, median 72, n=121) than in pre-menopausal patients (0-201, median 25, n=80). for the whole group, optimal prognostic discrimination was achieved with a cut-off 6 fmol/mg protein. in premenopausal patients, the effect of varying cut-off was complex, leading to an optimum of 17 fmol/mg protein, whilst in post-menopausal patients the optimum was 3 fmol/mg protein. these findings were unexpected and may relate to the relationship of er to tumour cellularity [3] . it is concluded that: 1. the failure of some centres to relate er to prognosis may be due to the inappropriate cut-off point chosen; 2. in our patients, the optimal cut-off was 6 fmol, which agrees with the level suggested by the british breast group [4], though it differed between pre-and post-menopausal patients; 3. optimal cut-off for prognosis may differ from that for predicting response to endocrine therapy. previous studies have reported that the use of adjuvant chemotherapy improves relapse free survival following mastectomy. the effect on overall survival is less certain. 290 patients with histologically confirmed axillary node metastases were randomised to receive postoperative radiotherapy (rt), chemoterapy (cmf) or radiotherapy plus chemotherapy (rt + cmf). 244 patients have now been followed for between 6 months and 5 years. patients initially treated with rt received chemotherapy on failure where possible. of 79 patients receiving rt alone, 28 have developed distant recurrence and 20 have died. of 78 receiving cmf alone 22 have developed distant recurrence and 15 have died (see table) . although the use of adjuvant chemotherapy significantly prolongs the relaps free interval there is no corresponding improvement in overall survival. a sample of 89 patients with histologically proven prostatic carcinoma underwent transrectal find needle aspiration at six month intervals, for a mean follow-up of 18 months, as an out-patient procedure, without significant side effects. 49 patients were followed from time of initial diagnosis. the others had been on treatment for a mean period of 36 months before the study commenced. in 25 of the new patients, cytology was positive at the time of histological diagnosis, and correlated with the histological grade. there were no false positives. repeat cytology on 15 patients after 12 months showed 6 positive and in 5 of these the disease was progressing, and in one it was stable. of the 9 who were negative, 8 were stable and one progressing. 16 of the 48 patients already on therapy had positive cytology at their initial aspiration -9 showing progressive disease and 7 being stable. 5 patients whose cytology was initially negative became positive 12 months later and all had disease progression at this time_ 19 patients had negative cytology on 3 or more occasions and in only 2 cases was there evidence of disease progression. cytology showed evidence of squamous metaplasia on follow-up in 13 of the patients whose condition was stable. the prognosis for all patients was assessed on the basis of gleeson's score and aspiration biopsy has been shown to be a safe and useful procedure for monitoring disease activity and response to treatment. the optimum method of restoring the ability to swallow in patients with unresectable carcinoma of the oesophagus remains controversial. this study evaluates the palliative potential of pulsion intubation versus retrostemal gastric bypass of the excluded oesophagus in unresectable carcinoma of the upper thoracic segment (20-32 cm from the incisor teeth). patients and methods: 71 patients were prospectively randomised for treatment by pulsion intubation (37 patients) or gastric bypass (34 patients). non-resectability was indicated by (1) tumour lengths greater than 6 cm, (2) tracheal or bronchial invasion, (3) disrant dissemination, (4) mediastinal invasion. the operative mortality, morbidity, palliation of dysphagia and postoperative nutritional status was compared in the two groups. results: mortality: intubation resulted in 2 deaths, a mortality rate of 5,4%. gastric bypass resulted in 3 deaths, a mortality rate of 8,8 %. complications: intubation was complicated by respiratory infection (8), tube migration (1), respiratory obstruction (1), oesophageal perforation (1), bleeding (1). gastric bypass was complicated by chest infection (9), pneumothorax (3), wound infection (5), subphrenic abscess (1), anastomotic leak (4), pulmonary embolism (1), purulent neck discharge (3). ability to swallow: palliation of dysphagia was achieved in 92 % of patients following intubation and 91% of patients following bypass. postoperative nutritional status: improvement in nutritional status was more rapid following intubation. conclusion: pulsion intubation is the preferred palliative procedure because of fewer complications and lesser degree of postoperative catabolism. the current technique for investigating the response of vascular prosthetic materials to infection is by challenge with a sub-lethal dose of bacteria, usually an intravenous infusion of 108 organisms in an animal model. this large bacterial innoculum, however, obscures any difference in the infectibility of prostheses that may be inherent in the material, its incorporation into host tissues, or its resistance to infection. we have developed a sensitive method for determining the susceptibility to infection of vascular prostheses based on calculation of the number of bacteria required to infect a specific prosthesis in 50% of trials (ids0). following implantation of the prosthesis to be tested in the canine infra-renal aorta, a dose-response curve was generated by the intravenous injection of known innocula of s. aureus (at log intervals from 102 to 108 bacteria). at six weeks the prosthesis was harvested and cultured to document infection with s. aureus. a characteristic sigmoid curve resulted from which the ids0 was determined. to test this method, a comparison was made between commercial human umbilical vein grafts (huvg) and huvg impregnated with silver sulfadiazine in 35 dogs. although both prostheses were infected by the standard 108 innoculum, a greater than tenfold increase in the resistance to infection by the treated huvg (<102 to 101) was demonstrated in the dose-response curves. since the number of bacteria in a postimplant bacteremia rarely exceeds 102 organisms/ml, such differences in infectibility are clinically significant. ids0 determination provides a sensitive, reproducible method for quantitating resistance to infection in vascular protheses. prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. it is accepted that infection arises from operative contamination, bacteraemic seeding and abscesses or viscus eroding into the graft. this study investigates the role played by distal sepsis on groin grafts. ten dogs had a specific strain of staphyloccocus aureus inoculated onto a surgical wound in the right foot pad. five days later interposition 6 mm dacron grafts were implanted into the ipsilateral and contralateral groin in continuity with the superficial femoral artery. ten days following this the grafts were removed for bacteriological and histological examination. blood cultures and lymphnode cultures were also taken at this time. in seven dogs the specific staph, aureus, was grown from both grafts. two dogs failed to grow the specific staph, aureus from either graft. these results are significant at the 1% level using fischer's exact test. blood cultures grew staph, aureus from only one dog. ipsilateral lymphnode cultures yielded the specific staph, aureus in seven dogs. we believe that distal sepsis plays an important role in the estabishment of graft sepsis. the mechanism of spread would appear to be via the lymphatics. the influence of tumor growth on wound healing p.w. de graaf and a. zwaveling laboratory experimental surgery, state university, leyden, the netherlands leakage of a colonic anastomosis is a complication each surgeon fears, because it usually leads to a prolonged hospital stay and is accompanied by a high mortality. carcinoma as an indication for operation and preoperative malnutrition are considered to be high risk factors. the subject of this study was to measure the influence of malignant tumor growth at a distant site on woundhealing in colon and skin, and to find a correlation between the influence of the nutritional state of the experimental animal, and the influence of malignant tumorgrowth on woundhealing. we also tried to find ways to compensate the possible unfavourable influence a malignant tumor might have on woundhealing. the study was performed in rats, the tumor used was a rhabdomyosarcoma, transplanted subcutaneously in the rats right flank. parameters for woundhealing were tensile strength of the skin-incision, and bursting pressure of a colonic anastomosis. six groups of rats were studied, each rat undergoing a standardized colonic operation after two or four weeks. the groups consisted of: control animals (20); tumorbearing animals (20), without tumor removal; malnutrltioned animals (20); tumorbearing animals receiving intravenous hyperalimentation (20), without tumor removal; animals undergoing tumor removal and colonic operation in one session (10); animals undergoing the colonic operation two weeks after tumor removal (10). woundhealing was negatively influenced in an early stage of tumorgrowth (f<0.05). four weeks of tumor growth did not impair woundhealing to a greater degree than two weeks. laboratory determinations showed no deviations. malnutrition, leading to less weight gain than in control and tumor bearing animals took much longer to influence woundhealing than tumor pearing. this is in accordance with publications by irvin and hunt (1974), devereux et al (1979) and garattini and guaitani (1981) , which led us to the conclusion that the negative effect of tumor bearing on woundhealing was not solely the result of anorexia. hyperalimentation in tumorbearing rats resulted in undisturbed woundhealing, despite the fact that hyperalimentation as practised by us (with amino-acids and carbohydrates) stimulated tumorgrowth. we concluded from these experiments that tumorgrowth caused a metabolic chaos in the animal, which in turn led to a disturbance in woundhealing. intravenous hyperalimentation could apparently compensate this. woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in two sessions (p<0.05). irvin an hunt (1974) have demonstrated however that extraabdominal trauma had no influence on colonic healing. this led to the conclusion that the diminished woundhealing found in rats operated in one session was an effect of the tumor. obviously this influence is not instantly removed with tumor excision. this study offers a theoretical foundation for the clinical observation that in operations for colonic carcinoma with a substantial operative trauma, the anastomosis needs extra protection and/or needs to be performed in a second session. the aorta of the blotchy mouse undergoes dilatation during adult life, resulting in the formation of fusiform aortic aneurysms. the mutation is x-linked, so only the males are affected. we have found a shift in the fluorescent spectrum of insoluble, hydrolyzed, dermal collagen in these mice; suggesting the presence of an abnormal crosslinkage compound. the purpose of this report is to describe additional studies carried out on the soluble collagen fraction in these animals, which lend support to the hypothesis of a crosslinkage abnormality. dermal collagen was prepared from one-monthold mutant mice and their normal male littermates by sequential extractions in salt solution, acetic acid, and urea. amino acid analyis and sds gel electrophoreses of the salt-soluble fractions revealed similar profiles of amino acids and collagen-chain types, suggesting that there is no abnormality of the basic building blocks of collagen in the mutants. fluorescent spectroscopy of the acid hydrolysates of these fractions also demonstrated similar absorption and emission peaks. however, reduction with sodium porohydride abolished fluorescence in the collagen fraction from the mutants. results (2emission m a x ± sem): normal blotchy before reduction 424.3 ± 3.0 420.7 -----2.9 after reduction 420.0 + 2.1 none sodium borohydride is used experimentally to stabilize labile collagen cross-linkages. the present studies indicate that the salt-soluble collagen from the blotchy mice is borohydride-reactive and is thus chemically ,,unstable". these studies suggest a rational for developing probes based on these fluorescent croperties of investigate collagen from human subjects affected with aneurysms. one third of testes are removed after torsion (1, 2) because of delay in presentation and diagnosis. in a retrospective study of 89 patients with torsion our salvage rate was 64%. nineteen percent were misdiagnosed as epididymitis by a junior doctor and 7 % by a surgical registrar. doctors could not distinguish between torsion of the testis and its appendages. in the literature, eight factors have been reported to be of discriminant value (table 1) . a computer program, using these parameters and bayes' theorem, was written to calculate the most likely diagnosis. data from the 89 patients with torsion and 96 patients with acute epididymitis were analysed by this program ( table 2) . the program correctly diagnosed all 96 cases of epididymitis. of 75 patients with torsion of the testis . in this study we determined the critical ischemic time for the development of an arrhythmogenic potential. egg and bipolar electrograms were recorded from the his bundle, ventricular endocardium and epicardium in twelve anesthetized dogs. short bursts ofventricular pacing (3 beats; 240-420 beats/min) were used to induce ventricular arrhythmias before and after lidocaine administration (2, 4, 6 mg/kg). previously, lidocaine has been shown to exacerbate conduction delay in ischemic myocardium leading to reentrant ventricular arrhythmias. in the control state, ventricular pacing with or without lidocaine induced either no response or single or repetitive ventricular responses. sustained ventricular tachycardia was never evoked in the control studies. in six dogs, after 20 min of left anterior descending coronary artery ligation and reperfusion, ventricular pacing with and without lidocaine produced sustained ventricular tachycardia in one animal. in another six dogs, after 30 rain ofligation and reperfusion, one dog showed sustained vertricular tachycardia in response to ventricular pacing alone. the other 5 showed sustaine~l vertricular tachycardia after ventricular pacing with lidocaine. sustained ventricular tachycardia averaged 400/min and degenerated into ventricular fibrillation within 1-2 rain. in conclusion 20-30 min of ischemia appears to be the critical period for development of malignant arrhythmogenic potential in the canine heart. administration of lidocaine during this critical period enhances the inducibility of cardiac arrhythmias, which may have clinical relevance in the management of acute myocardial infarction. untreated coronary artery air embolism in the experiments without ecc (group i) resulted in a transmural myocardial ischemia with a mortality rate of 28 % in contrast there were no death in the experiment when extracorporeal circulation was used (group ii to v). the best therapeutic effect with regard to the reversibility of temporary myocard ischemia following coronary artery air embolism was achieved by increasc of the postembolic perfusion pressure (group v), a finding being significantly different (p<0.05) to groups i through iv. also volume depletion of the left ventricle on ecc (group ii) resulted in a faster regression of the left ventricular hypothermic area compared to group i (p<:0.05). the application of dipyridamole (group iii) and st.thomas cardioplegic solution (group iv) was not able to produce a significant therapeutic effect. the increase of perfusion pressure following coronary artery air embolism has demonstrated to be the most effective procedure to achieve reversibility of myocardial ischemia. to transfer these experimental findings into clinical application is suggested because of its practicability and convenience. transmural biopsies were obtained before and 5, 15, 30, 60, 90, 120 min after acc for biochemical and structural analysis. myocardial temperature (t) ph (mph), and pco2) were measured continously with a thermistor, a new ph electrode, and a mass spectrometer respectively. in group i (n=8) acc was under normothermia. in group ii mean t was reduced to 19°c by the administration of cold potassium cardioplegia immediately after acc and consecutively every 30 min. mph at the end of acc reached 5.39-t-0.08 group i) and 6.49___0.13 (group ii) (/7<0.001); pmco2 rose from 41___4 to 234_+13 mmhg in group i and did not change in group ii. tissue content of atp decreases by 51% group i) and by 14°/0 (group ii) (f<0.001); tissue creatine phosphate fell by 98 % (group i) and by 48 % (group ii) (p<0.001). changes in atp and creatine phosphate as well as in tissue glucose and glycogen related ton concomitant changes in ph. the degree of ischemic damage assessed histologically by a mean ischemic score was 2.5+--0.06 in group i and 0.75_+ 0.19 in grup ii (p<0.01). irreversible structural demage assessed by electron microscopy occurred in group i 60-90 min after acc and was associated with a mph below 6.2. no such damage was observed in group ii. we conclude: 1. irreversible damage during normothermic arrest occurs considerably later than has been reported for regional ischemia in the beating heart; 2. during 2 h of acc cold potassium cardioplegia does not completely protect against id when mean t is 19°c; and 3. on-line oaeasurement of mph reflects the inadequacy of' mp more accurately than do either pmco2 or t and thus, provides a useful potential method for intraoperative monitoring of mp. in rabbits, infusion cardioplegia was installed at 37, 27, and 17°c respectively. the infusate contained na 27, k 5, 30, 120, 160 or 240, ca 0, 1 or 5 mmol/1, and varying amounts of glucose. measured osmolality varied from 305 to 500 mosmol/kg. the hearts were excised at the end of a 5 rain infusion period; one half was immediately frozen, the other half was incubated for varying periods of time. specimens of left ventricular myocardium were analyzed for metabolite and electrolyte contents. at termination of the cardiplegic perfusion, total adenine nucleotides (tan) and total creatine (tcr) were within control ranges under all conditions. however, atp and ecp = (atp + 0.5 adp)/tan as well as creatine phosphate (crp) and the ratio crp/tcr decreased significantly with increasing dosages of k and ca and higher temperatures. there were corresponding increases in adp, amp, and free creatine. at 17°c, there were no such changes except in hearts perfused with 240 retool/1 k and 5 mmol/1 ca. on the contrary, crp and the ratio crp/ tcr were elevated above the control values in hearts perfused with ca-free solutions containing between 30 and 120 mmol/1 k. the decrease in atp served as the parameter of the ischemia-induced metabolic alterations and the energy deficit developing during cardiac arrest. at 37°c, it averaged 1.67 and 2.55/zmol/g (p<0.02) after 10 min of arrest induced by 30 and 240 mmol/l k respectively. ca, 1 or 5 retool/1 respectively, significantly increased the atp-decay to 2.41 and more than 3.63 pmol/g/lo min respectively (/7<0.02 and p<0.05). hypothermia of 27°c reduced the rate of metabolic deterioration and suspended the influence of the k-dosage. however, the ca-effect was still visible: induction of cardiac arrest by 30 and 240 mmol/l k without/with ca decreased atp by 2.07/ 2.58 pmol/g (p<0.025) and 2.07/2.41 pmol/g (n.s.) respectively within 20 rain. at 17°c, the rate of metabolic alterations was further reduced. the effects of k-dosage and of ca were completely abolished. the atp-decreased 2.2 pmol/g during 40 min of arrest. although higher concentrated k-solutions for infusion cardioplegia do not enhance the ischemiainduced myocardial metabolic alterations in deep hypothermia, they are not recommended for practi-despite the advantages of cbk carrdioplegia, the severely impaired myocardium and/or long ischemia time continue to be a challenge. because of the association of ca ~ with cell injury and death the use of ca ~ channel blockers is logical. investigation of cbd revealied no advantages over cbk. the combination of k and d is appropriate because of their different mechanisms of action. ten dogs had one hour of myocardial ischemia (mi) with topical ice (temp 7___2°c) after coronary perfusion with 200 ml cb (5___ l___c) containing k, 30 meq/l and d, 400 pg/kg. eight dogs had two hours of mi after perfusion with 200 ml cb containing k, 30 meq/l and d, 200/zg/kg. six dogs received the same treatment as the previous group except that d was increased to 1600/zg/kg for all four perfusions. baseline studies were repeated after 60 min ofreperfusion without the use of ca ~ or inotropic agents. heart rate, peak systolic pressure, vc¢, v~,~x, peak + dp/dt, peak -dp/dt, dp/dt over common peak isovolumic pressure, left ventricular compliance, stiffness and elasticity and great water were unchanged from control. coronary vascular resistance was unchanged in groups one and two but declined in group 3. creatine phosphate returned to control but atp, adp and the adenosine pool were depressed. ultrastructure was well preserved. in 16/24 dogs defibrillation was not required whereas 48/48 dogs with cbk and 13/13 with cbd required defibrillation. these data suggest that d is a worthwhile addition to cbk. early one-stage repair of some congenital vascular anomalies might be accompleshed readily if the material used for grafts grew along with the reconstructed tissues. we have evaluated the capacity of tubular grafts constructed from anterior rectus sheath to serve as growing segmental replacements of the descending thoracic aorta of puppies (age 9 weeks). grafts measuring 3 cm in length were placed in 17 animals; 10 were implanted with attention to tissue preservation (live grafts) while 7 received grafts subjected to prior freezing and thawing in order to kill the donor tissue cells (devitalized grafts). grafts were measured initially and at sacrifice 1, 2 or 3 months later. each months 3 animals with live grafts and 2 with devitalized grafts were sacrificed. no aneurysmal dilatations or grafts ruptures were observed in any of the specimens. by 3 months, the live grafts had increased 26.9-1-1% in length and 27.3___ 3 % in diameter, while length and diameter of the devitalized grafts showed minimal growth (2.1___20/o and 5.3___5o/o respectively). during the same period the length of the thoracic aorta increased 4.5___5% in the live graft group and 41.1___ 8 % in the devitalized graft group. grafts were lined by endothelium and organized into 3 transmural zones. the thickness of each zone in the live grafts remained static from 1 to 3 months, whereas thickness of the middle and outer zones in the devitalized grafts increased 4 to 6 fold. in living grafts the layers consisted principally of newly formed fibrocellular tissue; the rectus sheath was reduced to a atrophic fibrous band. by contrast, the rectus sheath remained prominent in the devitalized graft specimens. cellularity (cells/field) of the live grafts was much greater than that of the devitalized grafts (ratio 15.9___3 : 6.0-----2). a newly formed, fibrocellular layered structure replaces the rectus sheath in the live grafts. these findings indicate that rectus sheath grafts are capable of growth in the young animal, providing that at implantation the tissue is well preserved. it is well known that durability of valvular bioprostheses (vbp) depends mainly on the structure of constituent biomaterial and long-term preservation of the collagen network. to our knowledge, no studies on ultrastructure of tissues currently used for vbp has been reported so far after a long time of chemical preservation. the presentation of a new anysotropic tissue, xenogenic cervical duramater (xcd), with a collagen tridimensional network, and a comparative study of scanning (sem) and transmission (tem) electron microscopy of bovine pericardium (bp), human duramater (hd) and procine aortic-cusp (pao) represent the purpose of the present communication. samples of the tissues were obtained in semisterile fashion, rinsed and fixed in karnovsky medium at 4°c for 24 h. materials were then minced on paraphine plates and washed with 0.2m cachodylate buffer for 1 h and dehydrated in increasing concentrations of ethanol (30 %-100 %) and intermediate exposure to 1% uranyl acetate for the in-bloc contrasts. were conducted, and results are exposed in table 1 . as a general rule, histologic evaluation was by far, more satisfactory for tissues preserved with 0.5 % glutaraldehyde (xcd, pap) than those with 98 % glycerol (hd) and 4 % formaldehyde (bp) in combination. it is concluded that the new anysotropic tissue herein presented, xcd, can be appropriately preserved with 0.5 % glutaraldehyde as collagen preservation is concerned, and it's anysotropic and ultrastructural features, maintained for as long as 12 months as assessed by sem and tem. albeit, clinical use of this new biomaterial is subjected to further experimental and animal trials. tetralogy of fallot and absent pulmonary valve (tapv) is associated with massively dilated pulmonary arteries which cause tracheobronchial compression in the newborn and heart failure and cyanosis in older patients. corrective operations have been attended by mortality rates exceeding 40% due to pulmonary insufficiency causing right heart failure (rhf) and pulmonary complications. pulmonic valve insertion (pvi) with complete repair has resulted in improved survival. the purpose of this paper is to assess the results of total correction and pvi in ten patients. during the last five years, 152 patients with tetralogy were corrected. of these, ten patients (ages 51 days to 34 years) had absent pulmonary valve. one patient (51 days) present with severe rhf and pul-monary insufficiency and nine patients presented with mild rhf and cyanosis. chest roentgenographs showed increased cardiothoracic ratio and pulmonary prominence in all. arteriography revealed massively enlarged pulmonary arteries with a mean right pulmonary artery to aorta size ratio of 2.7 to 1. associated pulmonic stenosis and insufficiency was present in all. seven patients underwent closure of ventricular septal defect (vsd) and pvi. of these, three had pvi (2 tissue and 1 prosthetic) with outflow patch and four had right ventricle to pulmonary artery (rv-pa) tissue valved conduits. two patients had repair without pvi, and one had repair with a monocusp pericardial valve patch. nine patients have done well with no episodes of thromboembolism or infection. death occurred in a 51 day old infant who had vsd closure and relief of pulmonic stenosis. pulmonary valve insertion seems indicated in these patients at it lowers peak pulmonary artery pressure and thus reduces compression effects on the trachea and bronchi. as well, when pvi was used right heart failure was not noted postoperatively. although in experimental acute myocardial ischemia intraaortic balloon pumping (iabp) appears to increase regional contractility of ischemic segments of the left ventricle by increasing the collateral flow, evidence for this effect of iabp in patients with refreactory myocardial ischemia is not available. this study was done to analyze the effect of labp on global and segmental left ventricular performance in patients with refractory, acute myocardial ischemia using gated blood pool scanning with tc-99 albium tracer. eight patients were studied on-and-off iabp prior to and following coronary bypass surgery. left ventricular (lv) wall motion analysis was undertaken and both cardiac output (co) and left ventricular filling pressure (lvfp) were determined from thermistor-tipped pulmonary artery catheters. when placed on iabp, mean preoperative global ejection fraction (gef) increased (0.264-0.05 (off) to 0.354-0.05 (on); p<0.01) abut no changes in co or lvfp were observed. when comparisons were made praend postoperatively, co increased after operation (p<0.05), due chiefly to an increase in heart rate (824-6 to 1074-8 beats/rain; p<0.01), but gef was unchanged whether or not iabp was on. only segmental wall motion analysis pre-and postoperatively revealed that iabp increased regional ejection fraction (ref) and contraction velocity by 50% (mean) in the angiographically determined regions of myocardial ischemia and these regional changes were maximal during the last one-third of the lv contraction cycle. it appears that ref is a more sensitive indicator of changes in lv performance than co and gef in these patients. further, this study indicates that iabp does increase the regional wall contracility in the ischemic areas of the myocardium in man. the use of aspirin (asa) to inhibit platelet thromboxane synthesis (tbx2) in patients undergoing coronary artery bypass grafting (cabg) has been advocated to reduce the potential for graft occlusion. however, asa in conventional doses also inhibits the venous synthesis of prostacyclin (pgi2) a potent anti-thrombotic factor, and may contribute to excessive surgical bleeding. to investigate the relationship between asa dose, blood loss and inhibition of venous synthesis of pgi2, 27 patients undergoing cabg were studied. control patients (no asa) were compared to those receiving 80 or 325 mg. asa as a single dose 8-16 hours prior to surgery. production of pgi2 by saphenous vein specimens removed at the time of surgery was measured by radioimmunoassay (ria) of 6-keto pgf~a following incubation with 25 um na arachidonate. blood loss was assessed by chest tube drainage and transfusion requirement in the perioperative period. serum tbx2 was measured by ria following asa ingestion. transfusion (units), drainage (cc), vein pgi2 (pg/mg tissue) and serum tbx2 (ng/ml) were (mean 4-sem): therefore, asa 325 mg or 80 mg did not increase blood loss during cabg. however, asa 325 mg, significantly reduced saphenous vein pgi2 synthesis (/7<0.01) while the lower dose asa 80 mg spared the production of pgi2. asa is both doses inhibited tbx2 (p<0.001) and blocked platelet aggregation. low dose asa deserves further investigation as an anti-thrombotic agent since it does not appear to increase operative bleeding or significantly inhibit venous pgi2 production. transfusion postoperatively, igg increased progressively in patients in group a, reaching preoperative levels on the 6th or 7th day, whereas in patients in group b, igg remained at lower than preoperative levels during the first week. the number of patients with abnormally low lavels of igg during the whole po period was significantly higher in group b (p"~.01). no significant differences were found in the others studied variables. one patient in group a had infection. three patients in group b had infections. conclusion: high doses of fab2igg administered during ohs and the first po days are effective in lessening the po decrease oflgg and thus may be beneficial in preventing po infection. a water insoluble but very hygroscopic polyvinyl alcohol powder, zy-15044", is capable of significantly stimulating the cicatrization of open, contaminated skin wounds in rats. when it is compared to other substances in clinical use such as powders containing antibiotics, antiseptics, amino-acids, enzymes or a dextranomer, no other agent tested was capable of producing a similar beneficial effect. these excellent experimental result justified pilot clinical trials on 30 patients: 18 varicose vein ulcers, 4 atherosclerotic leg ulcers, 4 infected traumatic wounds and 4 infected postoperative wounds. whether in rats or in patients, zy-15044 powder removed secretion, bacteria and tissue debris; it produced an early and increased proliferation of fibroblasts with the appearance of dense granulation tissue; it reduced wound size after less than three applications permitting very early grafting of the wound of eventually, cicatrization advanced to complete epithellzation. regardless of whether the treatments were applied daily in the hospital bed or every other day in outpatients, there were not complaints of unpleasant sensations such as itching, burning or pain. few studies have dealt with long term healing in stomach and none is available for duodenum. this study evaluates morphology and development of mechanical strength and collagen concentration in and adjacent to wounds after 20 to ]g0 days of healing. incisions were made in the non-glandular (rumen) and in the glandular oxyntic (corpus) part of the stomach and in duodenum of 64 male wistar rats, 23 intact rat served as controls. the wounds were dosed with 6-0 polypropylene sutures using a single sutur technique. after 20, 40 and 80 days of healing complete load deformation curves were determined on strips perpendicular to the incision line after the sutures were cut. collagen distribution was measured as hydroxyproline in strips parallel to the incision line. wound tissue continued to gain strength up to 80 days of healing (breaking energy significantly increased in all tissues from 20 to 80 days of healing). no such increase was found for wounds tested togetherwith adjacent tissue, because the "point of maximum weakness" had moved laterally from the incision line with healing time. wound collagen concentration increased in corpus and duodenum between 20 and 40 days of healing, but was unchanged between 40 and 80 days. the biochemical active zone around the incision line was unchanged 4-5 mm on each side from day 20 to 80. conclusion: while the wound tissue itself continues to gain strength during the 80 days ofheaiing studied, the increase seen after 20 days does hot,enhance the functional properties of the organ as a'whole. the "point of maximum weakness" has at that time moved to the borderline of the biochemically active zone, which lies outside the tissue area enclosed by sutures. myocutaneous flaps based on either the inferior or superior epigastric artery may be used to cover extensive soft tissue defects from the mid thigh to the clavicle. we report our experience with 33 rectus abdominis flaps, 29 based on the superior epigastric artery and 4 based on the ingerior epigastric artery. the flap is easily elevated, no skin loss has occurred and primary closure of the donor defect has been achieved in every case. in a follow-up period of between 1 month and 15 months no patient has developed an incisional hernia. the flaps have been used to cover extensive chest wall resection for recurrent carcinoma of the breast (6 patients), extensive radionecrosis of the chest wall (1 case) as part of a primary breast reconstructive procedure (22 patients) and to replace soft tissue overlying the femoral vessels after radical dissection of the groin for metastatic tumour (4 patients). of the 22 patients undergoing primary reconstruction 20 required additional subpectoral prosthesis but in 2 adequate bulk was provided by the flap itself. primary healing occurred in all cases. this easily raised and reliable flap will shorten hospital stay after major ablative surgery for recurrent disease and provides a useful addition to methods of breast reconstruction. a. watson department of surgery, royal lancester in girmary, lancaster, u.k. occult gastro-intenstinal bleeding which defies readiological and endoscopic diagnosis provides one of the greatest diagnostic challenges in surgery. lesions producing this clinical situation are often very small by definition and not infrequently mucosal angiodysplastic lesions, localisation of which may be extremely difficult pre-operatively and indeed at laparotomy. various methods have been described in an attempt to improve pre-operative localisation of such lesions. string tests an dthe detection of sicr labelled red cells in the faeces are notoriously inaccurate. arterioghraphy is invasive and usually necessitates a bleeding rate in excess of 500 ml per day. scintiscanning after red cell tagging with 99mtc gastro-intestinal blood loss, but localisation is difficult. a method oflocalisation of such lesions using slcr labelled red cells and intestinal intubation is described. after labelling of the red cells, a miller-abbot intestinal tube with the balloon inflated and rendered radio-opaque is passed and samples of gastrointestinal secretions aspirated at each 5 cm during passage down the gastro-intestinal tract. samples are counted on a well scintillator counter and when a sample of high radioactivity is obtained, localisation is assessed both by the length of tube passed and by instilling dilute barium down the miller-abbot tube underfluoroscopic control. this method has been used successfully in five patients which were subsequently treated surgically with localised resection resulting in cessation of bleeding. case histories together with photographs of the method and respected specimens will be presented in poster form. postoperative sepsis is tlae most frequent complication of surgery and is the commponest cause ofprolungation of hospital stay. purpose of the study is to prospectively evaluate incidence predisposing factors, bacteriology and costs of postoperative infections. 758 consecutive surgical patients admitted to our institute from may 79 to july 81 were studied. patients undergoing minor surgical procedures (wound less than 2 cm) were excluded from the study. patients were evaluated daily during hospital stay for onset of infection and results recorded in a data sheet. hemocultures in septic patients and free plasma, activated partial thromboplastin time, prothrombin time, and thrombin time, ristocetin test for soluble monomer complexes and fibrin degradation products (fdp, welco test) euglobulin lysis time (elt) and platelet counts. conclusion: 1. thrombocytopenia is not a typical feature of boomslang coagulopathy. 2. the demonstration of soluble monomer complexes is blood samples (positive ristocetin test) appears to be an early and consistent indication of intravascular coagulation. 3. despite unequivocal evidence of advanced consumption, platelet function seems to be maintained, thus preventing complete heamostatic failure. 4. support for this view is found in the clinical observation that bleeding is essentially mild and late in onset. 5. thrombelastographic hyperretractility the "spinning top" appearance is a constant feature and is probably mediated via the platelets. 6. although d. (vpus venom is extremely potent and often fatal, the antivenom is rapidly effective despite advanced consumption. this mechanism which may involve the platelet release reaction has not been fully elucidated and deserves detailed study. in most cases artificial ventricles are driven pneumatically with the advantage of easy handling and the disadvantages of regulation problems, high weight and volume of the driving units and the danger of air embolism in the case of membrane rupture. the driving unit developed at innsbruck universiy consists of a microprocessor-controlled electromagnet driving a pump that functions as a safety chamber (sk). force transmission to the artificial ventricle (ellipsoid heart-eh) is effected hydraulically. thus there is a fixed connection between armature stroke and membrane movement in the eh. the armature position is measured by the microprocessor by means of a position sensor with programmable switch-over points determining the change from systole to diastole so that idling and beat volumes, respectively, of the eh can be programmed. the sk is a newly designed double rolling membrane pump for pressure and suction with very low compliance. pressure and suction are determined by the armature force which is proportional to the microprocessor-controlled current in the solenoids. thus a very positive control of the pumping action is possible. in mock circulation experiments with the hydraulic safety driving unit the cardiac output (co) was between 21/min and 121/min at beat frequencies of 40 to 130 bpm. with constant piston-stroke a direct relation between frequency and co was observed. the influence of preload (starling's law) and afterload decreases with increasing armature force (decreasing periods of systole and diastole, respectively) which is due to friction in the hydraulic transmission system. improvements in the geometry of the next system should reduce these losses. in vivo experiments confirmed the hemodynamic efficiency of the system. applied as lvad the system proved to relieve the beating heart considerably. in the case of heart fibrilation the circulation could be maintained by the safety driving unit. the variable height differences between driving unit and eh as they occur in long-term experiments (animal lying or standing) affect only the ratio systole/diastole periods with the co remaining constant. mechanically assisted circulation is indicated in patients with cardiac failure after open heart operations. the clinical experiments show that left ventricular assist devices are capable only in a few cases to maintain full circulation. the limiting factor is the additional right heart failure syndrome. in patients with postoperative cardiac failure, a distinction must be made between isolated left heart failure and total heart failure. in patients with total heart failurebased on diffuse coronary sclerosis or on an increased pulmonary resistance -left ventricular assistance alone is not effective and right ventricular support is desired. therefore, a simple, quick and safe biventricular assist device is necessary. with the biventricular bypass it is possible to maintain complete circulation in cases of cardiac insufficiency. for a successful outcome in patients with low cardiac output after cardiopulmonary bypass, the e-bvad was evaluated in animal experiments (with 9 calves in 4 acute and 5 survival experiments) in cardiac failure situations. the cannulas for the inflow are put into the left and right ventricle, the outflow tracts into the descending aorta and the pulmonary artery. both parts are connected with the ellipsoid hearts. with the e-bvad it is possible to have a replacement of the heart -a total heart assistance similar to the total artificial heart. in cases of clinical emergency this device can be recommended because of the satisfactory hemodynamic effects achieved and the small degree of traumatic hemolysis (1,8 mg% free hemoglobin). it represents an easy and quick implantable system for total functional heart replacement. the realtionship between acute pancreatitis (ap) and the enzyme and hormone level in blood and gastric and duodenal juices is the factor that had driven us to do this experiment that would complete the study of the physiopathology of this illness. material and methods. we used eight 5-year-old dogs. a) production of two antiperistaltic fistula in the mann and bullman way; one gastric and the other duodenal (first part). 1. blockage of the gastric and duodenal compartments: by the gastric fstula we introduced one foley's bucket with closed point and inflatable globe to block the pylorus. by means of the duodenal fistula we introduced another foley's bucket into the duodenum. once inflated, the ball blocks the first duodenal portion. 2. juice extraction: by adjacent openings in the gastric bucket blocking the pylorum, we extracted the gastric juice separately. with another thin foley's bucket, that was introduced by the duodenal fistula near the already blocked one, to block the third part of duodenum when the globes inflates. b) production of ap: 15 days after the first surgical operation, by the morandeira method with intraparenchymal injection of a mixture of autologous bile and olive oil. results. one of the animals died on the fifth day after the first operation. + no animal died spontaneously after the second operation. they were killed on the 4th day. in each one acute necrotizing pancreatitis could be verified. + the radiograph showed that the gastric and duodenal compartments were sealed. -it was easy to separately extract blood and gastric and duodenal juices. conclusion. we believe that this method is complete, simple, with good results and very useful for the study of the physiopathology of ap. liver samples were taken form 18 patients operated for cholelithiasis and common bile duct obstruction by gallstones or pancreatic tumour. early evident histoenzymatic deficiency was found even without jaundice or liver structural lesions. in the bilio-obstructive jaundice, the histochemical methods revealed more marked liver impairment, as compared to the histological picture. the degree of the enzymatic depression could explain the occurence of the postoperative liver failure in some patients. experimentally, the effect of common bile duct ligation was studied in 80 rats. group i = 10 healthy controls. group ii = aspartate (1 ml 5% sol.) was injected i.p. in 10 rats daily, for a week. group iii = the common bile duct was ligated under other anes-thesia, in 20 rats 48 h before killing, and group iv=in another 20 rats 7 days before killing. group iv =in 20 rats aspartate was administered 3 h before an twice after choledocus ligation, and group vi = in another 20 rats aspartate was injected daily for a week after surgery. the liver slices frozen in liquid nitrogen were cut in a slee-type cryostat and the histochemical reactions were performed according to arnold, chayen-bitensky and lojda-gossrau-schiebler's methods. the biochemical reactions were performed using merckotests and merz-dade test-kits. after 48 h, and still more after 7 days, very severe structural, histochemical and enzymatic lesions developed. the liver cell glycogen and rna, as well as the "marker" enzymes of infrastructures markedly diminished: nach2-tetrazolium reductase -21.33%, glucose-6-phosphatase -48.33%, alphanaphthylacetat esterase -31.33%, atp-ase -71.0%, 5'-nucleotidase -38.67%, pas-reaction -56.0%, mgp-staining -30.67%. aspartate treatment seems to exert a protecting effect against the noxious action of retained bilirubin and conjugated bile salts upon the liver cells: nadh2-tetrazolium reductase +17.37%, gsp-ase +40.64%, esterase + 24.27%, atp-ase +67.81%, 5'n-ase +41.85%, pas + 25.05%, mgp +29.32% and lipids -33.12%. but it does not influence the biochemical signs ofcholestasis: bilirubinemia, alkaline phosphatase, leucinearylamidase, gamma-glutamyltransferase, lactate dehydrogenase. aspartate prevented partially but significantly only the increase of cytolysis enzymes: asat -40.48% and alat -52.97%. the histochemical and enzymatic results are in agreement with the severity ofmorphologic changes. therefore, aspartate treatment might be adequate for the preoperative improvement of the liver function in cases of obstructive-jaundice, in order to reduce the incidence of liver failure, without influencing cholestasis. oral glucose tolerance tests (ogtt) y. yamoaka, a. sugitani, ic kimura, ic ozawa and y. tobe department of surgery, kyoto university medical school, sakyo-ku, kyoto, 606 japan two patients with cholecystographic evidence of septate gallbladder underwent dynamic hepatobiliary radionuclide scanning with 99mtc-ehida. when a steady state of emission had been reached from both gallbladder lobes, cholecystokinin was infused incrementally in four doses (0.005, 0.01, 0.03 and 0.06 ivy-dog-units kg-lmin -1) and counts from the gallbladder analysed by computer. in each an obvious functional difference was revealed between the lobes: both distal lobes ceased emptying abruptly during the third hormone infusion whereas the proximal lobes continued to empty predictably [2] . in one case, histology revealed the septum to be a well-developed smooth muscle ring with cholecystitis glandularis proliferans confined to the distal lobe; the other case awaits surgery. the cause of the difference in response was probably contraction of the muscle in the septum acting as a sphincter. this study provides indirect confirmation that pain in septate gallbladder is due to septum contraction and raised distal intralobe pressure. it is known that the gallbladder stone passes into the common bile duct. furthermore, common bile duct stones pass to the duodenum. these gallstone movements were analysed in 334 cases during the past six years. the phenomenon in which gallbladder stones pass through the cystic duct down to the common bile duct are seen in cases where stones are small and numerous, the cystic duct is wide and connected with the common bile duct angularly or in parallel. in 13.75% of our cases common bile duct stones passed into the duodenum. there were neither inflammatory stenosis of the terminal choledouchous, high grade pappilitis nor severe obstruction of the common bile duct in these cases. it appears that the movement of gall stone is related to size and number of stone and morphological variations in the biliary system. the study was conducted in order to investigate whether intraoperative mano~etry of the bile ducts could be of additional valtie in diagnosis of afflictions of biliary tract or of the papilla. in contrast to current methods of water manometry it seemed important to the authors to use a simple and safe method which could deliver precise results. method: electromanometrics studies were carried out with a cannula placed in the common duct. three manometric parameters were obtained for evaluation. 1. resting pressure (rp); 2. pressure increase after injection of 10 ml saline (lml/s) (pi); 3. time in seconds needed for return of initial pressure (bile flow) (tr). for pressure measurements a pressure transducer with a recorder was used. results: (table) manometry results in patients with calculi in the common duct or with organic stenosis of the papillawere significantly changed as compared with normal findings in the bile ducts. false positive results (proven by cholangiography and biopsies of the papilla) were found in 3.5% of cases, false negatives in 6 % 97 % of all patients with patho-histological changes in the papilla had pathological bile pres-. sures. although false positive and negative results were mainly caused by methodical errors, the described method delivers a high percentage of correct results in agreement with the final diagnosis. it is avery sensitive method in indicating impaired and reduced bile flow and discovers early pathological changes in the papilla as proven by histological examinations. a functional anhepatic state in experimental animals for biochemical studies and as a model for treatment of acute liver failure can be achieved by different surgical procedures like portocaval shunt and complete arterial devascularization or by replacement of the liver with vascular prosthesis and protocaval shunt. we developed a simple hepatectomy procedure using t/4 inch silastic tubing, carbon-y-connector and specially designed "vascular spirales" to restore normal portal and caval blood flow. this model was used for auxiliary liver perfusion studies in heparinized animals; the operation takes 15 min, requires no surgical skill, vascular occlusion is less than 1 min, no signs ofsplanchic hypertension are present and blood loss in fully heparinized animals over 24 h is insignificant. one of the main problems in atypical and anatomical liver resections consists of achieving appropriate hemostasis. also because bile capillaries are opened when liver tissue is separated there is the danger of infection and subphrenic or parahepatic abscesses. the authors have used the human fibrinogen clue to seal the surface of the resected liver in 7 cases (3 hemihepatectomies and 4 resections). even in anatomical resections exists, in the majority of cases, a diffuse bleeding or oozing of the resected area. this can be completely controlled by sealing the surface with the fihrinogen adhesive. also one of the main advantages of the fibrinogen consists of having the possibility to avoid the insertion of numerous tubes for drainage. all our cases were drained by a single silicon tube. the postoperative courses were uneventful in all cases no major complications were observed. liver regeneration is thought to be stimulated by changes in portal blood constituents, or flow or by a substance in the hepatocytes. rice et al. have documented increases in total hepatic perfusion in rats during liver regeneration. this study has measured the portal and arterial components sequentially in large animals. young pigs (6-8 weeks) were subjected to shamoperation or 50% partial hepatectomy (ph). blood flow in the portal vein or hepatic artery were measured pre-operatively and at intervals of 24 h postoperatively using cuff probes of an s.e.m. electromagnetic flowmeter; these were positioned daily under light anaesthesia. systemic arterial and portal venous pressures were measured via indwelling catheters. previous studies have shown that the peak of regenerative response occurs in pigs 3 to 4 days after ph. the mean pre-operative total liver blood flow increased from 1.02+0.36 (s.e.m.) ml.g-lmin-1 to a peak of 226+ 33 % on day 2. thereafter flow declined towards normal by day 6. the pre-operative portal component was 71 ___3 %; this increased to 85+2.5 % on day 2 and returned to normal level within 4 to 6 days. it seems that both total hepatic flow and the portal component increase markedly just preceding the peak of regeneration. this response may stimulate regeneration, or merely accompany it, or may be proyoked by similar stimulators. the analysis of individual bile acids is relevant to several clinical investigations although established techniques have a number of disadvantages. to overcome these we have developed a simple hplc method using a waters associates rcm100 radial compression module with a radial-pak c18, 10,u, 8mm id, reverse-phase cartridge (column). bile acids were eluted from the column at a flow of 2.0 ml min-1 with a mobile phase of methanol:water (75:25 v/v) containing 2.5 % (v/v) acetic acid and adjusted to ph 5.25 with 10 m naoh. a mixture of standards of cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids and their glycine and taurine conjugates were resolved, while the 10 conjugates alone were completely separated in under 20 rain. the technique has been applied to the study of human bile from the common duct, gallbladder, tube and duodenal fluid, and serum from patients with hepatobiliary disorders. bile acids in these samples were rapidly extracted using a sep-pak c18 cartridge before being applied (10/a to 200pl) to the hplc system, although bile could be analysed directly without extraction. quantitation (~mol ml-1 sample) of separated bile acids was achieved by comparison with standards using an on-line integrating computer and less than 5 nmol could be detected using a refractive index detector. acute hepatic failure induced by total liver devascularization in pigs -amino acid uptake by combined charcoal -resin hemoperfusion support system where losses averaged 38 %. jib patients had progressively greater losses with increasing preoperative weight (/250 lbs-30 %, 250-299 lbs-35%, 300-349 lbs-38 %, 350-399 lbs-43 %, 1400 lbs-50 %). weight loss in patients dbs was statistically and clinically greater with jib. 2. diabetics, especially insulin-dependent, were rapidly cured after jib. normal plasma glucoses, serum insulins, and oral glucose tolerance curves were usually seen within l postoperative mouth, unrelated to weight loss. all 13 patients requiring insulin or oral medication preoperatively could discontinue mediation usuallywithin a weekpostoperatively. improvement after gb was gradual, appeared related to weight loss, and was often incomplete. 3. hypercholesterolemic patients had 51% decreases, from 275 to 134 mg/dl, after jib, but only 19 % decreases after gb. all serum cholesterols were normal after jib, but 23 % remained elevated after gb. because of complications after jib, some needed reoperation and conversion to gb. fortunately, most benefits were retained after conversion to gb. we suggest considering jib for "superobese", diabetic and hypercholesterolemic patients. in the past 9 years 140jenunoileal bypass procedures were performed. the most common complications and side effects were frequent abdominal pain, or discomfort and flatulence in almost half of the cases. in addition kidney stones, blind loop invagination, electrolyte and liver dysfunction problems could be observed. the over all complication rate was 20%, the postoperative mortality rate 2 %. to eliminate the blind loop of the small intestine, to maintain enterohepatic circulation of bile, and to diminish undesirable side effects in the conventional jejunoileal bypass, biliointestinal bypass was introduced in 1980. twenty patients with a mean weight of 128 kg were subjected to primary biliointestinal bypass within the last two and a half years. three additional patients had secondary biliointestinal bypass due to side effects, especially diarrhea and flatulence, of jejunoileal bypass, performed two to four years previously. the surgical procedure entailed establishment of an end-to-side jejunoileostomy. 25 cm of the jejunum and 25 cm of the ileum were left in the continuity. the blind loop of the jejunum was anastomosed to a functioning gallbladder. so far the above mentioned complications of conventional jejunoileal bypass could be remarkebly diminished. there have been no deaths in this material and no metabolic side effects. frequent diarrhea is avoided by reduced bile spill-over to the colon. the weight reduction has been satisfactory. in summary: due to a lesser complication rate biliointestinal bypass seems to be superior to the simple jejunoileostomy in the treatment of morbid obesity. hyperplasia or neoplasia g.w. geelhoed, c.j. schaeffer and p. daudu department of surgery, george washington university medical center, washington, usa patients with various thyroid disorders who were undergoing thyroid operation were studied for the presence of absence of estradiol and progesteronebinding proteins in thyroid tissue. steroid receptor assays were carried out using similar techniques and standards as those routinely employed for study of breast cancer specimens. quantitative data were collected by coded specimen number by an observer unaware of the patients' clinical diagnoses, and diagnostic correltions were drawn following de-coding. there was no correlation with age or sex and the presence or quantitative value of steroid-binding site. specimens with the histologic diagnosis of follicular adenoma had estradiol binding sites, and had them at high levels (14.19 to 143.83) femtomoles/mg cystosol protein). patients with the histologic diagnosis of thyroid hyperplasia had marginally positive estradiol-binding and lacked progesterone binding. patients with adenocarcinoma of the thyroid exhibited neither estradiol nor progesterone-binding in significant quantity. presence or absence of steroid binding sites in thyroid tissue appears independent of sex or age, but correlates with benign neoplasia of the thyroid, suggesting a possible etiologic association for thyroid adenomas. 77 patients with esophageal cancer underwent resection and primary reconstruction of the esophagus by posterior invagination esophagogastrostomy which we devised. the anastomosis was made in the cervical level in 58 cases and in the left thoracic cavity in 19 cases. functions of stomach placed in the posterior mediastinum were examined in 7 patients surviving more than 5 years and in 13 patients surviving less than 3 years. within one year postoperatively, the absorption ofvitamine b12 decreased markedly. one and a half year after operation, however, the secretion of castle's instrinsic factor recovered and it showed normal values in patients surviving over 5 years. in secretion of gastric acid by tetragastrin, both total acidity and free hydrochloric acid increased in the progress of the postoperative period and showed normal values in all 4 patients surviving over 9 years. this fact was also confirmed by endoscopic observation of color development by congo-red in the gastric mucosa; its coloring area was scattered in 2 patients surviving 5 years, but extended to the whole surface in patients surviving over 9 years. roentgenographycally in head-down position, the surgically created fornix with a sharp angle of his was effective in preventing gastroesophageal reflux completely. the strain combination of donor and recipients. the nature of the mechanisms determing the different fate of allografts remains obscure and all interpretations of the above findings must be speculative. however, the long-term acceptance of hepatic allografts in the rat appears to be similar to that in other species and emphasizes the role of the liver as an immunologically favoured organ. footnote: part of the work (intrahepatic bile duct proliferation!) contained in this abstract will be presented at the xvii essr meeting 1982. peoples' friendship university, tamojenii pr. 4, department of operative surgery, moscow, ussr a transplanted kidney, besides tissue incompatibility reaction, is influenced by non-specific injuring factors, including decentralisation. for normal functioning of a kidney transplant over a long period of time we carried out an experimental study. the purpose of the study was the investigation of operative reinnervation possibilities and its influence upon the functional state of a transplanted kidney. for the kidney reinnervation kirpatovski's method for suturing perivascular fascial tissue flaps of anastomosed vessels with branches of vegetative plexus nervosus on them was used. experiments were carried out on 175 mongrel dogs, both male and female. in the first group of experiments a kidney was transplanted into the pelvis while the opposite kidney was preserved or ablated. in the second group the kidney autotransplanted into pelvis was reirmerved by the described method with preserving the contralateral kidney in part of the animals and ablating the kidney in the rest of them. the third, fourth and fifth were control groups. in the third group the kidney was denerved, in the fourth it was denerved and reinnerved by the mentioned method, in the fifth group unilateral nephrectomy was performed. after the operation the animals, in different periods of time (from 3 days to 2 years), were studied by isotopic renography and by scanning kidney's by jbl-hippuran and neohydrin-hg 2°3 intravenous injections. results of the isotopic renography were estimated qualitatively and quantitatively by universal renographic index (hirakawa-corcoran, 1963). results of the experiments indicate that autotransplantation and denervation of a kidney without its reinnervation were followed by lowering of a kidney vascularisation and its secretory and excretory functions. during the first 2 or 3 months after the operation a tendency to improvement of the kidney function took place; later those functions gradually oppressed and the renographic index lowered down to 13.9+__2.1, 16.6+--.5.5 (p<0.001) respectively according to the groups. after operative reirmervation of a kidney autotransplant and denervation of a kidney without its transplantation gradual improvement of separate functions of the kidney took place: in 1 or 2 months the kidney circulation was restored; in 2 or 4, and in 4 or 6 months respectively, secretory and excretory functions of the kidney restored; renographic index reached its norm after 4 or 6 months and remained stable during 2 years (according to the groups 54.9+___5.5, 52.3-1-4.3, p>0.05 respectively). thus, the operative reirmervation of a kidney prevented depression of its functions and provided its normal functioning during a long period of time. introduction of fine needle aspiration cytology in renal transplantation gives the possibility not only to distinguish acute tabular necrosis, arterial and venous obstruction and viral infection from acute rejection, fnc also seems to allow judgement of the effectiveness of various types ofimmunosuppressive therapy during rejection episodes. method: after sterile fine needle biopsy the cytological evaluation of the aspirate was performed on cytocentrifuged smears after staining the cells by the method of may-grtinwald-giemsa. thus normal and "activated" mononuclear cell populations as well as parenchymal cells such as tabular and endothelial cells and their pathological changes could easily be recognized. the white blood cell types in fnc were compared with those of the peripheral blood. the difference in number due to the graft invading cells was expressed as "increment". the findings of fnc were correlated with clinical signs and serum creatinine values. results: fnc allows to judge the in situ inflammation in the rejecting kidneys within 2 h. non-rejection grafts show cell counts comparable to peripheral blood. with onset of rejection (grade 1) t-and bblasts and monocytes appear in the graft. with sever-ity of the rejection (grade 2) monocytes and lymphocytes as well as blood cells increase in numbers. acute rejection (grade 3) is heralded by high numbers of monocytes and macrophages. acute tubular necrosis (atn) and virus infections can be distinguished from rejection episodes to be treated. in all cortisone and azathioprine resistant cases where alg was used in order to suppress inflammation during rejection episodes it reduced not only the peripheral white blood cells, but also within 12 h after start of alg therapy the number of in situ cells. beside reduction of inflammatory cells typical changes in shape of the nucleus and density of chromatine in up to 10 % of the lymphocytes and granulocytes could be detected. these changes look closest to what has been described as apoptoses. conclusion: cortisone and azathioprine resistant rejection episodes could be monitored within 12-24 h using fine needle aspiration cytology. this method is safe, cheep, and it provides good information about the in situ situation of the graft, it allows to distinguish acute rejection from atn and other situations where higher immuno-suppressive therapy, especially with cortisone, could only be harmful for the patient. criteria of the border of normothermic ischemic tolerance in dog kidneys are first normal pah-and exogenic creatinine-clearances compared with unilateral nephrectomized dogs in neuroleptic analgesia under excessive water and sodium load and second perfectly normal kidneys after two weeks in pathological examination. the protective solution htk ® by bretschneider has a superior buffering capacity compared to euro-collins-solution ® , which, by itself, enhances anaerobic glycolysis by substrate stimulation (glucose) especially between 15 ° and 35°c. without preservation in normothermia oligo-anuric arf is observed at the border of ischemic tolerance (15-20 min; 36°c). the obligo-anuric arf is dependend on ischemia which will result in necrosis of the kidney after 45 rain and 36°c ischemic temperature. the border ofischemic tolerance corresponds to an intrarenal ph of less than 6.2 (outer stripe of medulla), and medullary lactate levels of 60 to 80 pmol/gdw. substrate stimulation of anaerobic glycolysis limits the effectiveness of euro-collins-solution ® above 15°c earlier than anaerobic glycolysis in pure ischemia. after warm ischemia (>--__..27°c; 60 min) all kidneys protected with euro-collins-solution ® show anuric arf, whereas all htk®-protected kidneys (60 to 240 min of warm ischemia; 32°c) are having polyuric arf. under these conditions, renal ph will not increase above 250 nmol/1 h+-concentra -tion. the border of ischemic tolerance for htk ®protection ofkidneys is 120rain-31°c and seems to be limited by a transitory secondary postischemic oliguria (3-48 h). in summary: intrarenal anaerobic glycolysis limits tolerance of pure warm ischemia by inducing anuric arf. in contrast polyuric renal failure is observed at the border ofischemic tolerance by using the protective procedure with the htk®-solution mentioned above. at least 60 % of unrelated pigs respond to liver allografting with a rejection episode which they overcome without immunosuppression; kidney transplants are rejected uniformly within 9 to 14 days. in this study, mlc responses were measured at weekly intervals in such animals. non-litter mate pigs were subjected to autograft, exchange liver allograft, or exchange renal allograft. blood from mlc responses were taken from unaesthetised pigs before and at weekly intervals after operemained the same for each pair of transplants. in 5 of 8 pairs of liver allografts, mlc responses were depressed up to 10 fold for 3 weeks post-operatively. in all these pigs, the mlc responses were initially high. in 2 pairs where the mlc response was initially low, there was no change after the transplant, and in one pair with a low pre-operative response, there was a marked increase post-operatively. in 3 pairs of kidney allografted pigs, there was a similar depression in the single post-operative sample available. in 3 liver autografted pigs, there was a slight post-operative rise in mlc response. it appears that liver and kidney transplantation but not hepatic autografting, markedly depress sequential post-operative mlc responses. the process of ischaemic kidney degeneration was estimated by measuring the adenine nucleotide (atp)levels and the effect of inosine and naftidrofuryl (praxilene) was assessed. 50 min ischaemia was induced on both dissected kidneys of wistar male rats. then, either both clamps were removed and the right kidney was excised after i0 rain reperfusion the role of mononuclear phagocytes in various disease states has been extensively studied by phagocytosis, fc and complement receptor sites, and enzyme content. chemotaxis of peripheral blood mononuclear phagocytes, however, has not been studied to any great extent. we have therefore investigated a method for quantifying chemotaxis by mononuclear phagocytes. mononuclear phagocytes were purified from peripheral blood by centrifugation over ficoll-hypaque and a discontinuous percol gradient. chemotaxis was quantified by the 'under-agarose method'. mononuclear phagocytes were allowed to migrate towards the chemotactic agent zymosan activated serum (zas), and the control non-chemotactic agent eagles medium. the distance of cell migration was measured after 20 hours incubation at 37°c in 5 % co2 with the aid of a microscope eyepiece graftcule. using non-sepcific esterase staining the purity of mononuclear phagocytes obtained was 85%___ 10% and the viability by trypan blue dye exclusion was at least 99 % preliminary results have shown selective migration by purified human peripheral mononuclear phagocytes towards zas. this method may therefore represent a means of investigating an important role of these cells in disease states. the intrathoracic pressure rises when a person exhales into a manometer to such an extent that finally the venous blood-flow to the heart stops. it is supposed that this venous stop flow pressure (vsfp), measured by an ultrasound device, is equal to the central venous pressure. in a prospective clinical trial by two independant examiners in 97 % of the cases (n= 100) the correlation was within 5 cm h20. therefore, the central venous pressure (cvp) can be measured non-invasively with an ultrasound device. we have previously reported that opiate receptor blockade with naloxone (nal) significantly improves cardiovascular function and survival in canine hemorrhagic shock [1, 2] . since species differences do exist, we investigated the effects of nal in cynomulgus monkeys anesthetized with n20/o2. blood was withdrawn to achieve a mean arterial pressure (map) of 45 mmhg (t---0) which was maintained until t= 1 h when the reservoir was clamped and the animals treated with either nal at 2 mg/kg bolus plus 2 mg/kg.h infusion i.v. or 0.9% nac1 in equivalent volumes. there were no significant differences between nal (n=5) and con (n=5) in map, left ventricular contractility (lv dp/dt max, mmhg.10vs), and survival; the nal animals, however, were acidotic (pha 7.24-----0.09) and colder (37.95-0.3°c) than con (7.42+__0.02, 38.5-+-0.1). map responses to nal were proportional to ph a complications of vascular bypass grafts, especiallyin the femoro-popliteal region are common. the most frequent of these by far is occlusion. other late complications iclude leaking and false aneurysm formation at the anastomotic site, dilatation of the graft material and stenosis ofanastomotic sites. the most commonly employed non-invasive studies render little information of value in the diagnosis of these complications. we have recently undertaken a study to evaluate arterial grafts at various intervals after implantation with the use of a duplex ultrasonic scanner. imaging of graft material with this system ist excellent and patency of the lumen can easily be established. graft diameter can be measured and accumulation of material within the lumen can be measured and quantitated. although anastomoses are not always clearly visualised they are often seen satisfactorily for diagnostic purposes. graft dilatation, false aneurysm formation and occlusions can be accurately diagnosed with this method. we have also studied the behaviour of femoro-popliteal grafts across the knee joint using the duplex scanner. as a non-invasive technique scanning with a duplex system has many advantages enabling frequent investigations and therefore early recognition of complications where synthetic material vascular grafts have been used for arterial bypass. an experimental model for dissecting aortic aneurysm has been designed to obtain much better understanding of the disease, leading to more effective methods of surgical treatment. dissection of the descending thoracic aorta was successfully performed by modified blanton's prodedure in 85 % of more than 100 mongrel dogs. fals lumens ruptured distally into true lumens to form a doublebarreled aorta in 70% of the dogs. the method of surgical treatment performed were (1) closure of entry by direct suture, (2) closure of entry by inserting dacron vascular prosthesis with a stainless steel ring, and (3) bypass grafting with vascular prosthesis and ligation of the thoracic aorta. in 10 dogs treated by methods (1) or (2) at the time of performing the dissection, cine angiography revealed that false lumens hat thrombosed within 1 month and dissection was found to have completely healed on autopsy. in 10 chronic cases treated by methods (1) or (3), false lumens were all patent with various degrees of formation of mural thrombi at observation of 3 to 6 months after surgical treatment. these results indicate that closure of entry for acute dissecting aneurysm should be a curative procedure. studies are now continuing on chronic dissecting aneurysm. the lack of controlled trials not rarely resulted in the incorrect acceptance o fan ineffective operation as an effective one 0igation of internal mammary arteries for relief of angina pectoris, for example). this failure also explains the non-stop controversies over the appropriate operation (and whether the question operate at all) for various myocardial revascularization procedures, over the thymectomy for myasthenia gravis, the limited vs radical mastectomy for breast cancer etc. several clinicians and surgeons assert that controlled clinical trials for new operations are unrealistic and naive because of some important differences between operations and drugs. compared with medical trials surgical ones are often really more difficult to be carded out. they are subject to more restrictions, ethical, statistical and practical, and may require longer or even remarkably long times. in any case, patients must nevertheless be sheltered from harmful and ineffective surgical operations since luckily not all the undoubted difficulties are unsurmountable, and prospective controlled trials of surgery, including random allocation of patients, are quite feasible. comparison between surgical and non-surgical treatment is a really particularly suitable field for these studies. comparison of effectiveness of two operations of the same type or of different types can also be carried out, but only on certain conditions and using suitable and appropriate expedients. also the doubleblind approach may be feasible, although remarkable restrictions and adequate cooperation are necessary. acceptance in trials of surgery as placebo is objectively very difficult, and obviously it has to be considered only when an effective operation for the considered disease does not exist. the ethical sound of this delicate aspect of clinical research can be minimized enough only if (1) there are substantial doubts about the effectiveness of the intended operation and (2) requested placebo-surgery is of very slight importance. posters for scientific meetings mary evans and a.v. pollock scarborough hospital, north yorkshire y012 6ql1, u.k. the display of information in posters antecedes even the,art of writing. they have been used to advertise, to educate and to inform and their function is to disseminate information to a wide audience quickly, simply and effectively. in medicine they have been used since the thirteenth century for the promotion of health education. in recent years poster sessions have been introduced into scientific meetings as an alternative to the spoken paper for the presentation of original work. following this experimental procedure hepatic coma supervenes in 6-8 h and animals die after ca. 24 h. five male pigs, aged 3-4 months, weighing 30-35 kg were studied ca. 20 h after operation. the detoxifying system consisted of activated charcoal (norit ¢ (lmm x 2ram) and ionexchange resin (dowex lx2), subsequently inserted in an extracorporeal circulation. plasma amino acids were determined serially (3, 20, 40, 60 rain) in and out the detoxifying system. plasma extraction of individual amino acids (means_ se in/zmol/min) is reported in the table a remarkable extraction was present in the first 40 rain and chiefly involved aromatic amino acids and tryptophan which did not further increase. during hemoperfusion, the molar ratios between neutral amino acids improved. the ratio of branched-chain to aromatic amino acids increased from 1 late hepatic effects of small intestinal bypass (sibp) for obesity plasmafl-endorphin (/3-end, pg/ml, by ria) was related to t:fl-end = 334 (t-37) + 597, r=0.67, p<0.05. when acid-base balance and temperature were maintained, nal (n=6) significantly (*p<0.05) improved map and lv dp pg/ml in nal and 969 + 94 pg/ml in con). r-end is released in and contributes to the cardiovascular depression of primate hemorrhagic shock. nal reverses this depression and improves survival but only when arterial ph and core temperature samples of exudates at site of infection were taken wherever possible for aerobic and anaerobic cultures. in following infections were recorded: wound, respiratory tract, thrombophlebitis, indwelling intravenous catheter, unidentified origin fever (>38 °c lasting more than 3 days) (fuo) and miscellaneous. incidences: 310 patients (41%) had postoperative septic complications. wound infection was recorded in 145 patients (19.1%), respiratory tract infection in 105 (13 %), urinary tract infection in 100 (13 %), fuo in 55 (7.2%) thrombophlebitis in 18 (2.3 %) and miscellaneous infections in 49 (6.4o/0). predisposing factors: wound infections were 53/413 (12.8 %) in clean operations, 41/215 (22.7 %) in potentially contaminated, 19/65 (29%) in contaminated and 24/65 (36.9o/0) in dirty.wound infections were diagnosed later (mean 9th day) in clean than in dirty operations (mean 5th day) (p<0.05). a statistically significant correlation was found between wound infection and leght of preoperative hospital stay: from 5.4% in patients operated on within 0-6 days to 42.8% for patients operated after 28 days or more (p<0.001). no correlation was found between wound infection and age. urinary infections were more frequent when the patients were catheterized at least once in the postoperative period (34 % vs 11%). a statistically significant correlation was found between the incidence of respiratory infections and duration of anaesthesia (3.2 %----<60 min, 120/0>60<120 min, 270/0>120 min;/~0.05). bacterology: 181 out of 295 cultures gave positive results (61%): aerobes were isolated in 129 samples (490/0); mixed aerobes and anaerobe in 29 (9.8o/0); anaerobes in 23 (7.7 %). bacteroides fr. was the commonest isolated anaerobe in all types of sample except hemocultures were propionibacterium aches was the most frequent. a statistically significant correlation was found between the incidence of recvery of anaerobes and intraoperative contamination (7.4 % in clean operations, 13.5 % in potentially contaminated, 12.5% in contaminated and 20.8% in dirty; p<0.05).in wound infections the most frequent aerobes were staphylococcus in clean and e. coli in contaiminated operations. costs: mean postoperative hospital stay of patients with septic complications was 15 days, whereas patients with no postoperative sepsis were discharged after an average of 10 days (p<0.05). mean daily cost in our hospital was extimated $100; accordingly, the mean postoperative hospital stay of patients with sepsis costs $1500 vs $1000 of that o~ the patients without postoperative infections. overwhelming postsplenectomy sepsis/infection has been accepted the highest risk with more than 50 percent mortality due to pneumococcus species. however, e. coli, pseudomonas and staph. aureus have been reported a deadful threat to the splenectomized individual also. pneumococcal challenge after splenectomy in animal experiment and after partial salvage has been well examined. staph. aureus-challenge has not been tried after splenic repair, so far. material and method: a total of 150 nmri-mice (18-20 g) have been subjected to: 1 sham-operation, 2 splenectomy, 3 peritoneal splenosis, 4 controls as non-operated group.the technique was similar to the one performed in 90 rabbits, prior reported. 6 weeks postoperatively, the mice were exposed to intraperitoneal injection of staph.aureus concentrations of 101°c/ml down to 104c/ml. the peritoneal cavity of mice has a high resistance to staph.aureus, as only no. 36835 proved lethal. results: whereas the macroscopic view post mortem seemed promissing, showing almost total salvage of the splenic particles 6 weeks postoperatively bacterial challenge with staph.aureus was contradictory: 3 x 10 s c/ml was survived by all splenectomized mice, by only 25percent of the splenosis micecand by none of the sham and nonoperated mice. due to the small number of only 48 mice within this mice-pathologenic staph. aureus species, we may not draw definite conclusions. as for one, the intraperitoneal application may not be the natural way of septicemia. on the other hand, splenic remnants may not be as effective in the protection of staph.aureus-sepsis as in a pneumococcal challenge. therefore, further studies with staph.aureus species in other animals and by other application route will be needed! studies of the coagulant effects of boomslang (dispholidus typus) venom have indicated that the coagulant effect was mainly due to its ability to activate prothrombin. it also activates prethrombin i, factor x and possible factor ix as well [1] . although boomslang envenomation is said to represent a classic model of disseminated intravascular coagulation, certain features are worthy of critical thought. the coagulation profile of a nine year old lad, who was admitted to the h.f. verwoerd-hospital, 48 h after a reputed boomslang bite, provided the impetus to explore the in vivo effect ofcurde. d. typusvenom on the thrombelastographic and other haemostatic changes in the chacma beboon. methods: 0.0005 mg, 0.0015rag and 0.05mg respectively of crude. d. typus venom were injected subcutaneously in 3 adult baboons. serial determinations of the flow parameters were done over 3-6 days. thrombelastography on whole blood an platelet this report presents the ten year survey of liver mitochondria analized in 385 patients.1. two mechanisms were of major importance in the augmentation of mitochondrial ability to synthesize atp: a) the enhancement of atp-generating capacity per unit of respiratory assemblies and b) the increase in respiratory enzyme contents. those compensatory mechanisms were functional within a certain range of contents in cytochrome a (0.5-1.5 nmol/mg protein, normal; 0.8). hepatic insufficiency was observed in patients with cytochrome a contents less than 0.5 or more than 1.5.2. in all patients with cytochrome a of 0.7-1.0 nmol/mg, the blood glucose level after an oral glucose load (50g) returned toward normal within 2 h (parabolic pattern). in 25 patients with cytochrome a of more than 1.5, the blood glucose level did not return toward normal within 2 h (linear pattern). parabolic and linear patterns were intermingled in the patients with cytochrome a form 1.0 to 1.5.in this report, we will emphasize the importance of preoperative ogtt and measurement of cytochrome a contents during operation for the prediction of operative prognosis, better than any other currently used index of liver function, in the patients receiving major surgery such as hepatectomy or operation for esophageal varices in severe cirrhosis. the reticulo-endothelial system (res) has largely been ignored in studies of liver regeneration. in this study, the res was "blocked" with colloidal carbon, or was stimulated with olive oil or with glucan. indices of liver regeneration were the thymidine kinase acitivtiy (tk) and the number of mitotic figures (mi) in liver biopsies. fibronectin was measured as a putative index of kupffer cell function.four animals in each group were sacrified at 6, 12, 24, 36, 48, 72 and 96 h after sham-operation or 68% partial hepatectomy (ph). group 1: carbon suspension (pelikan ink, wagner) 16 mg/100 g rat single injection pre-operative; group 2: olive oil (10 % in 5 % dextrose water + 0.1% tween 20) 0.25 ml/20 g rat single injection pre-operative; group 3: as group 2 given at intervals of 24 h; group 4: glucan 1.0 rag/ 100 g rat pre-op, and at intervals of 24 h.results: glucan or a single dose of oil increased tk in ph and sham-operated rats but did not influence m.i. carbon inhibited mi but did not influence tk. fibronectin levels were increased in sham-operated and ph rats after daily oil or glucan.in conclusion, no administration enhanced both tk and mi but stimulation of the res with glucan or oil did increase fibronectin levels in both sham and ph rats. it appears that the res does not have a role in liver regeneration as assessed above, but fibronectin appears to have been an indicator of res activity. anatomical abnormalities of the gallbladder include multiseptate and bilobed organs, and the more common transverse septate variant. patients with the latter type often have typical biliary symptoms which are thought to be caused initially by raised intracystic pressure and subsequently by inflammation and calculi formation in the distal lobe [1] .this study evaluate~ the long term effects of sibp on hepatic lipid and fibrous tissue accumulation. liver was obtained at the time of abdominal operation from 6 normal weight patients, 18 morbidly obese patients (mean body weight 163+21 kg) and 27 patients 4 or more years (mean 69-+ 19 months) postoperative from sibp. from wedge liver biopsies hepatic total lipid, triglyceride, cholesterol, phospholipid and protein contents were determined as well as the activities of acetyl coa carboxylase and fatty acid synthetase. histologic evaluation of needle biopsies of the liver was used to quantitatively estimate the amount of hepatic steatosis and fibrosis present.there was also a significant histopathologic increase in hepatic fibrosis present in the liver tissue from sibp patients when compared to hepatic fibrosis in liver from obese patients as well as when compared to liver specimens obtained from the sibp patients at the time of their sibp. the effect of reversal of the sibp on hepatic fat content and fibrosis was determined by transcutaneous liver biopsy 6-22 mos. following take down of sibp. post-reversal histologic quantitation of hepatic fibrosis and inflammation was significantly decreased from pre-reversal values and the improvement was greater in patients who lost weight. patients year after sibp have persistent hepatic steatosis with hepatic fibrosis and inflammation. improvement in these parameters may be anticipated following reconstruction of the intestinal tract particularly if weight loss is maintained. the aim of this paper is to study the metabolic effects of gastric bypass in dogs. we used dogs weighing between 21 and 25 kg. the animals were divided into three groups: a) five dogs without surgical operation (control); b) six dogs with a fundoj ejunal bypass, and c) six dogs with a gastroplasty. in group b and c the exclusion was 9/10 of the stomach. in all of the animals and groups were determined: cholesterol , live r function tests, calcium, magnesium-and electrolyte levels, the body weight evolution and the apparent-digestibility coefficient (adc) of fat, protein, minerals and glucosides for a type of diet. the composition of this diet in dry substance was: fat 35.4%, protein 35.2%, glucosides 26.5% and minerals 2.9%. these analyses were determined five times during the twelve months of control. the animals were kept in individual metabolism cages which allow feces and urine to be gathered separately as well as the food consumed to be controlled. the cages were housed in a room thermoregulated at 18 °c±2. the cholesterol, calcium, magnesium and electrolyte levels were normal during the twelve months of control in the three groups. the liver function tests were normal in all groups with the exception of serum aspartate transaminase initially deteriored in group b. the body weight evolution was significantly diminished in groups b and c compared with the control group (p<0.002 and p<0.002). the results of adc expressed in % ± sd were.by 15roviding a functional gastric capacity of less than 100 ml and consequently a forced reduction in food intake the gastric bypass produces an effective loss of weigth. our results suggest that the gastric bypass can be considered as an effective and safe alternative to intestinal bypass for treatment of morbidly obese subjects who have failed nonsurgical treatment; however, a great and deeper research is necessary to discover the possible side effects of 227 gastric bypass surgery; then its ultimate benefits will be fully understood. there was no operative mortality. the percent of the excess weight lost following the two operations is shown and compared with weight lost following gastric bypass. it is concluded that: 1. complications occured more often following gastroplasty, 25.5 % compared with 10.4 % of the patients treated by gastrogastrostomy. 2. the amount of excess weith lost was greater after gastroplasty, a mean of 61% for 30 patients followed for two years, than after gastrogastrostomy, a mean of 41% for 13 patients followed for two years. 3. because of the incidence of stomal obstruction requiring reoperation following gastroplasty, 14.8 % compared with 1.4% after gastrogastrostomy, is our preferred operation. 4. to improve weight loss after gastrogastrostomy the ta-55 is now used instead of the ta-90 stapling instrument. the proximal gastric pouch is reduced in size to 20-25 ml and the stoma made smaller to 9 ram. an artifical esophagus was made of silicone rubber tube covered with a dacron mesh. a segment of thoracic esophagus of 16 dogs was replaced with this graft using three different types ofamastomosis, i.e., overlayer end-to-end anastomosis, two layer end-toend anastomosis both using a flanged tube, and monolayer end-to-end anastomosis with noflange tube. seven of 16 dogs (44%) survived more than 12 months without complications, and 4 of them more than 6 years. in 6 of 7 of the prolonged survivors, extrusion of the graft was recognized in the 3rd to 6th month after operation. esophageal stenosis increased slightly up to the 6th month after extrusion of the graft, but it did not further advance until sacrifice. in these dogs, mucosal regeneration of the neoesophagus was complete with muscle layers and mucous glands in the submucosa recognized microscopically. proximal esophagus from the replaced portion was apparently dilatated more than that of the distal portion. there was no definite difference between the anastomotic techniques with regard to complication or prognosis. these results suggest a possibility for clinical trials. paper withdrawn 171 attempts to reduce nephrovascular hypertension by surgical techniques deviating renal venous blood and renin directly to the hepatic filter are at present discouraging. experimental data with portocaval transposition are contradictory, due mostly to the use of heterogeneous biological models (mongrel dogs) and collateral circulation developement. renal to portal vein end-to-side (ets) and end-to-end (ete) anastomoses in the rat were therefore tested as possible experimental models. we observed that even after right kidney decapsulation, collateral vein circulation develops through the periureteral plexus, particularly after ets shunts, one month after surgery. nevertheless, collateral cirulation in the male rat can be prevented by ligature and cutting offthis plexus near the kidney. in the female rat instead, collateral circulation is still possible. in fact, newly formed pericapsular veins join the right kidney to the right ovaric vein and therefore preventive ligature and eradication of these vessels is also necessary. in conclusion, both models (ete-ets shunt) appear feasible in the rat, and reliable for studies dealing with nephrovascular hypertension and hepatic metabolism of renin. the handling of the exocrine system is one of the main problems inpancreas transplantation. one trial to overcome this problem consists of the occlusion of the pancreatic duct system. a the theoretical disadvantage is the induction of a secretion oedema which may lead to blood flow disturbance followed by venous thrombosis. the aim of the present experiments was to study the effect of an anti-oedematous drug (cumarin and rutin sulphate (venalot ®) on the blood flow of autologous segmental pancreatic transplantation was tested by chosing the cervical region of the dog as the site of grafting. material and methods: 10 dogs weighting 20-30 kg were used. these dogs were divided into two groups (control group, n=5; treated group, n=5, venalot ® dose: 1 rag, cumarin/kg/b.w.). the body and the left limb of the pancreas was removed, perfusion with eurocollins was started wia the splenic artery and the duct system was injected with prolamine. a distal a. v. fistula of the splenic vessels was performed. this conditioned graft was transplanted at the neck of the same dog, performing the anastomoses between the carotid and the splenic vessels. the blood flow of the pancreatic graft was measured with radioactively labelled microsperes of 15/~m immediately after, as well as 4 days after grafting. the developing oedema of the graft was estimated by weight gain, histological and electron microscopic investigation. residual exocrine function of the graft was measured by determining the serum lipase and amylase levels.results: the autotransplantation of an segmental pancreatic graft to the neck of a dog is a feasable and technically easy surgical procedure, without major venalot -treated dogs local complications. in the ® there was a higher weight increase of the graft in comparison to the controls. as tested so far there was no difference in the behaviour of the i~lood flow of the grafts in both experimental groups. there was a significant increase of serum amylase and lipase values in the venalot®-trea'ted group. conclusion: the technique of cervical segmental pancreatic transplantation in dogs is recommended in cases where immunological monitoring (aspiration cytology, biopsies) is the aim of the study (good access to needles). the technique of microspheres injection is a useful method for the examination of the blood flow in segmental pancreatic grafts in dogs. the prolamine-induced secretion-oedema of ductoccluded pancreatic grafts is resistant to venalot ®treatment. graft pretreatment has been used in various organs to prolong auograft survival. we have recently demonstrated that graft pretreatment of canine renal allografts with cyclosporin a (ca a) led to improved animal and graft survival. our present study assesses the effect ofcy a as a graft pretreatment on pancreatic islet cell allografts. pancreases were removed from unrelated donor mongrel dogs and placed in iced saline (4°c) after the collagenase digestion. the tissue fragments were washed once more and then another 5 mg cy a was added to the preparation prior to intrasplenic injection of the islet cell allografts. three groups were studied: group i (n= 15) served as pancreatectomized non-transplanted controls, group ii (n = 15) received a non-pretreated islet cell allograft after total pancreatectomy and group iii (n= 10) received a cy a graft pretreated islet cell transplant after total pancreatectomy. all animals were given minimal immunosuppression with azathioprine (5 rng/kg/day x 3, followed by 2,5 mg/kg/ day). blood glucose values were monitored to determine engraftment and subsequent rejection. hyperglycemia was considered when plasma glucose values rose above 150 mg/dl. all pancreatectomized controls (group i) became hyperglycemic by the first post-operative day. non-pretreated islet cell allografts in group ii had variable function and became hyperglycemic between one and nine days after transplantation. only five of ten animals in group iii, receiving cy a pretreated islet cells, became hyperglycemic levels greater than 90 days and one died of unknown causes immediately after transplantation. the following table presents the animal survival data for the 90 day follow-up period.although the exact mechanism of action is not known, this study indicates that cy a graft pretreatmerit can be beneficial in prolonging pancreatic islet cell allograft survival, further studies will optimize the use ofcy a in this application and hopefully contribute to the improvement of pancreatic islet cell transplantation. grafting of vascularised organs has become a standard procedure in surgical research. however, only few data of the fate oforthotopicliver transplantation in the rat are available, probably because of the difficult ~iargery involved. this communication gives an account of the outcome of 40 liver allografts and 16 liver isografts in four inbred strains of rat. the following groups of allo and isografts are formed; the survival time is given in days postoperatively.the technical details of the operative procedure of orthotopic liver transplantation in the rat are described elsewhere (eur surg res 13:236 (1981). the distribution of death among allallograft groups show 3 blocks of survivors. the first block includes animals surviving up to 30 days. acute rejection and infection are the diagnosed causes of death in this compartment. the second block includes animals surviving up to 110 days. in the grafts, which belong to this compartment a intrahepatic bile duct proliferation is a frequent and dominant histological feature. because of the development of identical lesions in isograft surviving the same periode and the induction of this lesions by common bile duct ligation experiments, chronic rejection is excludet as the cause of death. the third block includes all so called long-term survivors, in which the structure of the grafts are remarkably well preserved and very few abnormalities are present. the occurence of fatal acute rejection and long-term survival in each allograft group demonstrates, that the fate of the orthotopically transplanted livers in not dependent upon key: cord-032181-gmcugd8h authors: song, jian-xin; zhu, lin; zhu, chuan-long; hu, jin-hua; sun, zi-jian; xu, xiang; xin, min-you; zhang, qiong-fang; zhang, da-zhi; shang, jia; huang, jia-quan; xu, dong title: main complications of aechb and severe hepatitis b (liver failure) date: 2019-05-21 journal: acute exacerbation of chronic hepatitis b doi: 10.1007/978-94-024-1603-9_2 sha: doc_id: 32181 cord_uid: gmcugd8h this chapter describes the clinical features, and diagnosis of complications in aechb including secondary bacterial infections, coagulation disorder, water electrolyte disorder, hepatorenal syndrome, hepatic encephalopathy, hepatopulmonary syndrome and endotoxemia: 1. patients with severe hepatitis have impaired immunity and are therefore vulnerable to all kinds of infections. after infection, these patients may experience shock, dic and multiple organ failure, all of which seriously affect their prognosis and are major causes of death. concurrent infections consist primarily of infections of the lungs, intestines, biliary tract, and urinary tract, as well as spontaneous bacterial peritonitis and sepsis. 2. severe hepatitis may reduce the synthesis of coagulation factors and enhance their dysfunction and increase anticoagulants and platelet abnormalities, leading to coagulopathy. infection, hepatorenal syndrome and complications can further aggravate coagulopathy, resulting in dic and seriously affecting patient prognosis. 3. hepatorenal syndrome, which is characterized by renal failure, hemodynamic changes in arterial circulation and abnormalities in the endogenous vascular system, is a common clinical complication of end-stage liver disease, and one of the important indicators for the prognosis of patients with severe hepatitis. 4. water electrolyte disorder (water retention, hyponatremia, hypokalemia, hyperkalaemia) and acid-base imbalance are common in patients with severe hepatitis. these internal environment disorders can lead to exacerbation and complication of the illness. 5. hepatic encephalopathy is a neurological and psychiatric anomaly syndrome based on metabolic disorder, and an important prognostic indicator for patients with severe hepatitis. 6. the hepatopulmonary syndrome is an important vascular complication in lungs due to systemic hypoxemia in patients with cirrhosis and portal hypertension. the majority of patients with hps are asymptomatic. long-term oxygen therapy remains the most frequently recommended therapy for symptoms in patients with severe hypoxemia. 7. endotoxemia, an important complication of severe hepatitis, is not only a second hit to the liver, but also leads to other complications including sirs and mods. (nacseld) for survival analysis of prospectively enrolled cirrhosis patients hospitalized with infections, bajaj found that there is 15.8% of nosocomial infection in all 507 patients [1] . domestic data showed significant difference of infection incidence in different times. it is related to raising diagnosis and treatment level. according to the data from shanghai public health clinical center, in 372 patients who were treated as severe hepatitis from january 2007 to december 2009, there were 68 patients obtained nosocomial infection with a rate of 18.3% [2] . the incidences of infection complicated with acute liver failure, acute on chronic liver failure or chronic liver failure are similar. patients with old age or long hospitalization are more easily to get infection. there was rare report about infection in children patients with severe hepatitis. in 2011, godbole et al. from uk reported that 25% in 145 children patients with severe hepatitis were complicated with infection, and mostly are sepsis, respiratory tract infection and urinary infection. most of the infections occurred within 2 weeks of admission, while patients with infection had longer hospitalization [3] . reduced innate immunity the innate immune system is the first line of defense against premier environmental challenges and injury. in liver, it is a complex system that includes nk cells, nkt cells, kcs, neutrophils, eosinophils and complement components. the innate immune response acts much more rapidly compared with adaptive immunity. monocytes and macrophages: the liver plays an important role in defense and immune function. the main cellular components of the innate immune system within the liver are the kupffer cells. kupffer cells represent 80-90% of the tissue macrophages in the human body [4] . in normal condition, kupffer cells in liver help to clear the macromolecular substance such as pathogen, endotoxin, heteroantigen and immune complex to defense infection. in severe hepatitis, due to massive hepatocytes necrosis, the number and function of monocytes/macrophages are impaired, thus the activity of fibronectin, which is opsonin of macrophages, is decreased. therefore, bacterium, endotoxin and other poison from gut directly access into circulation. subrat kumar acharya from india reported that the plasma fibronectin (fn) level in severe hepatitis patients was significantly lower than that in healthy controls (85.6 μg/ml ± 75.8 μg/ml, vs. 295.5 μg/ ml ± 88.5 μg/ml). the fn level was remarkably correlated with incidence of infection and mortality [5] . complement: liver is the organ where complements are mainly produced, such as c2, c3, c4, c5. the complements help to expand phagocytosis of phagocytes by chemotaxis, opsonization or adhesion, as well as help antibody to kill or solute some gram negative bacilli [6] . report from wyke showed that the defect of complement closely correlated with impaired opsonization [7] . defect of c3 or c5 can result to weakened movement of polymorphonuclear leucocyte. in severe hepatitis, the ability of liver to produce complement has been weakened due to massive injury of parenchymal hepatic cells, which leads to decreased activity of complement to 40% of normal condition. meanwhile, serum opsonization to e. coli or saccharomycetes are also diminished. besides, high ammonia level in severe hepatitis also restrains complement activity to impact germicidal effect. the most direct and also the most important result for decreased complement production and reduced opsonization is the susceptibility to infection. it was also reported that complement and the alternative pathway play an crucial role in lps/d-galn-induced fulminant hepatic failure [8] . neutrophils: a majority of patients with severe liver disease have altered function of neutrophil granulocytes [9] . the most common manifestation include abnormal ultrastructure or function of neutrophil granulocytes, as cytoplasm degeneration, organelle reduction, mitochondrial swelling, pyknotic nuclei, etc. decrease filtration and phagocytosis of reticuloendothelial system, as well as impaired chemotaxis of blood cells, making immunity weakened, lead to invasion of bacterium. therefore, patients with severe hepatitis are vulnerable to be infected with bacterium or fungi due to decreased phagocytosis and germicidal effect of neutrophil granulocytes, and impaired adhesion of macrophages and white cells [10] . data from liu h demonstrated pretreatment neutrophil-lymphocyte ratio was associated with the prognosis of patients with hbv-aclf, and elevated nlr predicted poor outcome within 8 weeks [11] . natural killer and natural killer t cells: natural killer (nk) and natural killer t cells (nkt) are important components of the innate immune response. natural killer cells have potent cytolytic activities that are exerted through the death receptor and perforin/granzyme pathways. activated nkt cells have both perforindependent and fas-ligand dependent cytotoxic function that are triggered upon tcr recognition of an antigen [12] . nk cells and nkt cells play an important role in many experimental models of liver injury, such as viral hepatitis, alcoholic liver disease, and autoimmune liver disease [13] . however, their role in aclf has not yet been clearly elucidated, it was reported the median percentage of nk cells in the lymphocytes of patients with acute and fulminant liver failure were significantly lower compared to healthy controls. meanwhile, patients with acute and fulminant liver failure had significantly high and comparable nkt cells compared to control group [14] . the important pathophysiological role of innate immune dysfunction in patients with acute-on-chronic liver failure (aclf) has been investigated in recent years. however, dysregulation of adaptive immunity remains poorly elucidated [15] . patients with severe hepatitis has varying degrees of impaired cellular immunity, manifested as decreased cd4+ cell number and declined cd4+/cd8+ ratio, which is pathogenesis of opportunistic infection. it was reported that there exists a reduction in cd4(+) t lymphocytes in hbv-aclf patients. these cd4(+) t cells predominantly are cd4(+) tconv, and the development of suppressive cd4(+) tregs greatly surpass tconv, which constitutes important characteristics of adaptive immune dysfunction of hbv-aclf [15] . a report from china showed total amount of lymphocytes, cd4(+) t cells, cd8(+) t cells and nk cells in circulation were lower in the hbv-aclf patients compared to the chb patients [16] . hbv-specific cd8(+)t-cell responses are considered to be of great importance in viral control and immune-mediated liver damage [17] . however, cd8(+) t cell has seldom been studied in aclf. ye [18] reported decreased activated cd8(+) t cells may be related to poor outcomes in patients with sh. the frequency of circulating th17 cells increased with disease progression from chb to aclf patients compared to healthy control. th17 cells were also found largely accumulated in the livers of chb patients. the increases in circulating and intrahepatic th17 cells were positively correlated with plasma viral load, serum alanine aminotransferase levels and histological activity index. in addition, the serum concentration of th17-associated cytokines was also augmented in both chb and aclf patients [19] . in process of diagnosis and treatment for severe hepatitis, repeatedly abdominal paracentesis, retention catheterization, venous cannula, hemofiltration, or trachea cannula are usually necessary. unthoroughly sterilization or nonstandard sterile operation will lead to pathogen invading to develop an infection. in addition, artificial liver treatment is also an important cause of infection. it was reported that the incidence of fungal infection in severe hepatitis correlated with the number of artificial liver treatment. application of broad spectrum antibiotics is also a major cause of infection in severe hepatitis. antibiotics inhibit or kill sensitive normal bacterium as well as pathogens, especially normal bacterium colonized in natural orifice, leading to flora disproportionality. this time, nonpathogen could cause infection, or mass produced pathogen become dominant colony to develop infection. it was proved that dosage, exposure time, or varieties of antibiotics used in patients was closely correlated with severity of dysbacteriosis and incidence of sbp [20] . nosocomial origin of infection, longterm of norfloxacin prophylaxis, history of recent infection by multiresistant bacteria and recent use of β-lactams were independent inducements associated with the development of multiresistant infections. bacterial translocation is defined as the migration of viable microorganisms or bacterial products (i.e., bacterial lps, peptidoglycan, and lipopeptides) from theintestinal lumen to the mesenteric lymph nodes and other extraintestinal sites [21] . there are multiple mechanisms which are involved in defective gut functions and altered microbiota in patients with cirrhosis or liver failure. these include small intestine bacterial overgrowth (sibo), increased intestinal permeability, and impaired antimicrobial defense. additionally, decreased bile acids, due to decreased synthesis and defective enterohepatic circulation, contribute to altered gut microbiota [22] . small intestinal bacterial overgrowth (sibo) was defined as ≥10 5 total colonyforming units per milliliter of proximal jejunal aspirations, which presents in 59% of cirrhotic patients and is associated with systemic endotoxemia [23] . in the condition of hepatic failure, due to impaired immunity, bacterium overgrowth and translocation happened, which increased incidence of infection [24] . in vivo study from wang showed phagocytosis of monocytes were prominently and immediately diminished after resection of liver tissue up to 90%, accompanied by decreased oxygen ingestion [24] . mass propagation of e. coli in the lower part of the small intestine and bacterium translocation were all happened 2 h after resection. in rats with ascites in severe liver disease induced by carbon tetrachloride, the bacteria easily transferred from the intestine to extra-intestine, including local lymph nodes and blood circulation. these evidences proved that liver failure prompted the proliferation of intestinal bacterial overgrowth and translocation, which may be the main reason for the endogenous infections in patients with liver failure. another reason for bacterial translocation is that patients with cirrhosis or liver failure display increased intestinal permeability. in patients with severe hepatitis, gastrointestinal mucosa membrane has impaired ability for regeneration and repair, leading to dysfunction of intestinal barrier and declined anti-infection ability. patients with severe hepatitis have weakened regeneration and repair capacity of gastrointestinal mucosa, as well as decreased gastrointestinal barrier function and resistance to infection. the intestinal mucosa often manifested as congestion, edema or erosion. especially when intestine get infection, bacteria can invade to abdominal cavity directly or through slight mucosal defect [25] . besides, ascites often present in severe hepatitis patients, which provide a good medium for bacteria to multiply. patients with severe hepatitis often have ascites combined with portal hypertension. due to lobular structural damage, abnormal construct of liver sinusoids or blood vessels, bacteria directly access into the systemic circulation without filtration and phagocytosis by liver, leading to formation of bacteremia. meanwhile, bacteria in blood circulation may access into the abdominal cavity and cause abdominal infections. hence, patients with severe hepatitis are not only prone to get endogenous infection but also prone to have intestinal endotoxemia. patients with severe hepatitis have decreased bile secretion, changes of bile composition, so that infection are prone to occur in epithelium of bile duct and gallbladder. in patients with severe hepatitis, intestinal edema and cellulitis are obvious, some may develop acute serositis. for patients with portal hypertensive gastrointestinal disease, regeneration and repair capacity of gastrointestinal mucosa are decreased, accordingly, the natural immune barrier function is reduced. once esophageal and gastric venous bleeding occur, gastrointestinal ischemia aggravate, which result in decreased resistance to infection. invasive procedures, complications, prophylactic use of broad-spectrum antibiotics, underlying diseases, long hospital stay, and old age are risk factors for infection in patients with severe hepatitis. elderly patients with other underlying diseases, decreased immune function, more severe primary disease, have a high risk of infection and may predispose to severe infections. in addition, albumin level is closely related to ascites production and sbp occurrence. irrational uses of immunosuppressive agents such as corticosteroids suppress immune function, lead to flora, and promote the formation of drug-resistant strains and pathogenic bacteria. mortality of severe hepatitis is closely related to infection, which directly affects the prognosis of severe hepatitis. to reduce the incidence of infection in patients with severe hepatitis, we should aim at a variety of risk factors, improve patients' immunity, rectify hypoproteinemia, treat the primary disease, prevent complications, strengthen disinfection and isolation, operate with strict aseptic technique and strict indications for invasive procedures, and use antibiotics rationally. once patients with severe hepatitis are infected, the consequences are often serious, as infection easily diffuse, which is difficult to control. after infection, the bacteria produce more toxins, which aggravate liver disease and cause a series of adverse effects, finally lead to severe complications (hepatic encephalopathy, hepatorenal syndrome, and etc.), which is the major cause for death in patients with severe hepatitis. bacterial endotoxin plays a major role in development and prognosis of severe hepatitis when infection happened [26] . endotoxemia may directly or indirectly worsen liver injury. intrahepatic cholestasis positively correlated with endotoxin levels. endotoxin can trigger hepatorenal syndrome. for kidney, endotoxin is a potent renal vasoconstrictor substance, which can make a strong contraction of the renal artery and renal blood flow reduction. in addition, endotoxin can cause kidney capillary thrombosis and acute renal tubular necrosis, or even renal cortex necrosis. endotoxin can activate coagulation factor vii and factor vi, making the intrinsic coagulation system startup, as well as directly damage hepatocytes which release tissue thromboplastin to start the extrinsic coagulation system. meanwhile, endotoxin can also damage the vascular endothelial cells to cause bleeding, especially upper gastrointestinal bleeding. in the event of gastrointestinal bleeding, infections are more prone to happen or primary infection aggravates. in recent years, due to extensive use of broad-spectrum antibiotics, the mortality of severe hepatitis has been significantly reduced. however, infection is still an important cause of death. nosocomial infection control directly affects the prognosis of patients with severe hepatitis, which is an important part for the treatment of severe hepatitis. once the etiology and other evidence of infection appear, patients should be treated with antibiotics. effective prevention and treatment for nosocomial infections are positive to promote recovery and reduce mortality. bacterial translocation (bt) is the key mechanism in the pathogenesis of sbp, which is because of the concurrent failure of defensive mechanisms in liver failure [30] . investigators have demonstrated pronounced impairment of gastrointestinal tract motility in cirrhosis [31] . the disturbance of gut flora microecology was associated with changes in the (ultra)structure of the gastrointestinal tract [32] . meanwhile, reduced cellular and humoral immunity make it easier for the free flow of microorganisms and endotoxins to the mesenteric lymph nodes [33] . besides, it was reported that hypoalbuminemia was related to sbp. a prospective study from runyon reported that the incidence of sbp in patients with ascites protein less than 1.0 g/l was 15%, while the number in patients with ascites protein more than 1.0 g/l was only 2%. after 3 years of follow-up, sbp incidence in patients with ascites protein higher than 1.0 g/l were negligible [34] . the level of ascites protein correlates with opsonin activity, which means ascites protein <1.0 g/l was an independent risk factor for sbp. the clinical manifestations of sbp are diverse. most patients have subtle and insidious onset, which usually manifested as abdominal pain and fever. acute onset may burst chills, fever and abdominal pain. generally the body temperature of patient is around 38 °c, sometimes as high as 40 °c. fever type usually presents as remittent fever. abdominal pain is always around the navel or lower abdomen, paroxysmal or persistent. nausea and vomiting are obvious, sometimes with diarrhea. ascites can occur in patients without ascites, or increased significantly. patients may also have significant bloating, abdominal muscle tension, tenderness, rebound tenderness, diminished or disappeared bowel sounds, and so on, or even shock in severe cases. according to clinical manifestations, sbp can be divided into five types: 1. common type: acute onset, protruding abdominal pain, followed by fever. or irregular fever take place followed by abdominal pain, abdominal tenderness and rebound tenderness, mild to moderate abdominal tension and growing ascites. increased wbc count and nuclear left shift. routine examination of ascites showed acute inflammatory changes. 2. shock: septic shock break out in a few hours to one day after abdominal pain or fever. the clinical manifestations include low temperature, lip cyanosis, abdominal tenderness, and hardly relieved shock. wbc count increases, blood culture is positive. 3. encephalopathy: fever and abdominal pain are often obscure, the early emergence of trance and other neuropsychiatric symptoms rapidly develop into a coma. careful examination of the abdomen in patients with light coma may still find in patients with pain expression. jaundice is deep, liver damage is serious. 4. refractory ascites: ascites progressively increase, which is difficult to subside with invalid diuretic therapy. abdominal distension is obvious, often without pain. carefully check the abdomen may still find slight peritoneal irritation. 5. asymptomatic: symptoms are mild, exhibited as slight bloating, occasional fever. mild tenderness can be found by deep palpation. in addition, a considerable part of the patients showed non-specific symptoms and signs, such as deepened coma, deepening jaundice, oliguria, azotemia or dramatically increased ascites. diagnosis of sbp should be considered if following conditions exist: (1) fever, which can't be explained by other reasons or other parts of infection; (2) abdominal pain, abdominal tenderness or rebound tenderness, but not serious; (3) sudden increased ascites or poor diuretic effect manifested as refractory ascites; (4) sudden onset of septic shock; (5) rapid deterioration of general condition, or deteriorated liver and kidney function in a short term, deepening jaundice, or hepatic encephalopathy. the diagnoses of sbp mainly rely on ascites puncture. easl clinical practice guidelines recommend that a diagnostic paracentesis should be performed in all patients with new onset grade 2 or 3 ascites, and in all patients hospitalized for worsening of ascites or any complication of cirrhosis (level a1) [34] . for patients with severe hepatitis, diagnostic paracentesis indications are: 1. in liver cirrhosis patients with ascites that paracentesis should be performed after admission to determine whether sbp exist. 2. if the following conditions happened during hospitalization, diagnostic paracentesis should be performed: (1) abdominal sighs suggest abdominal infections, such as abdominal pain, rebound tenderness and gastrointestinal symptoms (such as vomiting, diarrhea, intestinal paralysis); (2) systemic infection signs such as fever, leukocytosis, or septic shock; (3) no clear incentive for hepatic encephalopathy, or rapidly emerged renal dysfunction. 3. in patients with ascites and gastrointestinal bleeding, paracentesis should be performed before prophylactic antibiotics. once ascites was acquired, polymorphonuclear cells (polymorphonuclears, pmn) count and ascites culture should be performed. diagnosis of sbp is made according to international guidelines [34, 35] in patients with liver cirrhosis if the ascites polymorphonuclear (pmn) cell count exceeds 250 cells/μl and other forms of peritonitis have been excluded. an ascites fluid polymorphonuclear (pmn) leukocyte count ≥250/mm 3 should be considered as sbp, if pmn >500/mm 3 can be confirmed as sbp. if there is bloody ascites (erythrocytes more than 10,000/mm 3 ), pmn count are as 1/250 of red blood cells. the leukocyte esterase reagent strips (lers) test is based on the esterase activity of the leucocytes. since 2000, 26 studies have examined the validity of using leukocyte esterase reagent strips (lers) in sbp diagnosis. lers appeared to have low sensitivity for sbp. on the other hand, a high negative predictive value (>95% in the majority of the studies)supported the use of lers as a preliminary screening tool for sbp diagnosis [31] . there is bacteria colonization in ascites but no inflammation. the diagnosis is based on positive ascites culture, ascites pmn countless than 250/mm 3 , without evidence of systemic or local infection. bacterascites have two outcomes: a shortterm or transient bacterial ascites (mostly asymptomatic), or the development of sbp (mostly with symptoms). once the diagnosis was established, paracentesis examination should be conducted again after 2-3 days. take appropriate action under the circumstances. if the second sample has a pmnl count >250/mm 3 , treat as for sbp. if the pmnl count is<250/mm 3 and a second set of cultures is positive, treat as for sbp. if the pmnl count is <250/mm 3 and the second set of cultures is negative, no further action is recommended [36] . if ascites culture is positive but ascites pmn <250 /mm 3 and there were signs of abdominal infection, it usually progress to sbp within a few days. these patients should be given appropriate antibiotic therapy. brolin first proposed the concept in 1982. the mechanism: in early stage of severe hepatitis, ascites has not been exist yet, however because of necrosis of hepatocytes, dysfunction of kupffer cell, impairment of liver immune barrier, intestinal bacteria easily invasive into the systemic circulation through liver, causing spontaneous bacteremia, and then sbp. diagnostic criteria: (1) primary disease was severe hepatitis, no ascites was detected by strict examination and ultrasound. (2) clinical manifestation include fever, varying degrees of abdominal pain, diarrhea, diffuse abdominal tenderness and rebound tenderness, increased peripheral blood leukocyte, positive rivalta's test, ascites fluid leukocyte count>500/mm 3 , or pmn > 250/mm 3 . (3) exclude abdominal organ perforation or primary foci. it was used to describe the clinical situation when the ascitic pmnl count is >250/mm 3 but cultures fail to grow any bacteria. however, the term is now considered obsolete. in severe hepatitis with secondary infection, pneumonia is most common. patients with hepatic encephalopathy are susceptible to pulmonary infections as pneumonia since bed-ridden, impaired cough reflex and inadequate ventilation, especially comatose patients with intubation and tracheotomy. inpatients underwent thoracentesis, paracentesis and other invasive procedures, non-specific damage to immune barrier provide conditions for the bacterial invasion. reported in patients with invasive procedures, lung infection risk increased significantly, which demonstrated blood infection is an important way for pulmonary infection. furthermore, there was a significant increase of pulmonary infection in patients with intestinal infections or abdominal infection. pathogen: in recent years, pneumonia was classified as "community acquired pneumonia" (community acquired pneumonia, cap) "and" nosocomial pneumonia (hospital acquired pneumonia, hap). cap is the pneumonia acquired outside hospital [37] . although cap can be caused by a wide variety of micro-organisms, the pneumococcus, mycoplasma pneumoniae and chlamydophila pneumoniae, staphylococcus aureus and certain gram-negative rods are more usual pathogens encountered [38] . hap is the pneumonia that develops 48 h or more after hospital admission and that was not incubating at hospital admission. hap infected with gram-negative bacilli accounts for more than 60%, of which pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, other aeruginosa are common bacteria. the primacy is pseudomonas aeruginosa, while gram-positive cocci accounted for about 20%, mainly are staphylococcus aureus, coagulase-negative staphylococci, viridans and streptococcus pneumoniae. anaerobic is rare. clinical manifestations and diagnosis: clinical manifestations are characterized as fever, cough, expectoration, dyspnea, and cyanosis. diagnostic criteria: 1. chest percussion of dullness, auscultation of rales, along with one of the following conditions: (1) purulent sputum or change of phlegm; (2) positive pathogens in sputum culture. 2. the chest x-ray and/or ct examination revealed new or progressive exudative lesions, and the emergence of one conditions above. urinary tract infection is divided into upper urinary tract infection and lower urinary tract infection. upper urinary tract infections are mainly pyelonephritis, which can be manifested as acute pyelonephritis and chronic pyelonephritis. lower urinary tract infections include urethritis and cystitis. the most common pathogen of urinary tract infection is escherichia coli, followed by enterococcus faecalis, klebsiella pneumoniae and streptococcus agalactiae. urinary tract infections often occur in patients with indwelling catheter, therefore, aseptic urethral catheterization and management and timely replacement of catheterization are effective to prevent such infections [39] . 1. acute pyelonephritis: (1) acute onset; (2) chills, chills; (3) fever; (4) malaise, headache, fatigue; (5) loss of appetite, nausea, vomiting; (6) urinary frequency, urgency, dysuria; (7) low back pain, kidney area discomfort; the (8) tenderness of upper ureter point; (9) tenderness of rib waist point; (10) percussion pain in kidney area or bladder. 2. chronic pyelonephritis: (1) acute onset of acute pyelonephritis with the same, but usually much lighter, even without fever, malaise, headache and other systemic manifestations, urinary frequency, urgency, dysuria and other symptoms are not obvious; (2) edema; (3) hypertension. 3. urocystitis and urethritis: frequent urination, urgency, dysuria, urinary bladder pain. urethral secretions. laboratory tests and diagnosis: diagnosis elements include: (1) tenderness in rib waist point, percussion pain in kidney area. (2) urine leukocytosis, pyuria. (3) urinary sediment smear find bacteria. (4) positive in urine culture. (5) urinary colony counts>10 5 /ml; in patients with urinary frequency and other symptoms, colony counts>10 2 /ml are meaningful; counts 10 3 -10 4 /ml also helpful in diagnosis; (6) one hour urine wbc count>200,000. (7) blood test showed leukocytosis, neutrophil nucleus left. (8) increased esr. biliary tract infection is a common complication in severe hepatitis, which is often in company with cholelithiasis to reinforce each other. hepatitis b virus can directly violate bile duct cells and cause cholecystitis. on this basis, cholelithiasis and secondary bacterial infections are easy to happen and become a major focus, which can cause other parts of infection in severe hepatitis patients. furthermore, severe hepatitis patients often have reduced gastric acid secretion, so that e. coli in duodenum easily reproduce and cause ascending infection. biliary tract infection is usually a mixed infection of aerobic and anaerobic infections. enteric gram-negative bacteria include escherichia coli, klebsiella, enterobacter, proteus and enterococcus. anaerobic bacteroides include clostridium and fusobacterium, in which bacteroides is common, about 80-90%, particularly bacteroides fragilis. common symptoms of biliary tract infection include chills, fever, nausea, vomiting, right hypochondrium pain and tenderness in gallbladder area. clinical performance of concurrent biliary tract infection in severe hepatitis is not always very clear, often unconfirmed by pathogen test. symptoms were epigastric or right upper quadrant pain, radiating to the right shoulder, hours after a heavy meal or a high fat meal. the patient may have severe colic, often accompanied by nausea and vomiting. patients with chronic biliary tract infection have indigestion symptoms as heartburn, belching, acid reflux and bloating, or sometimes fever and right upper quadrant pain. severe hepatitis patients have weakened intestinal resistance, especially for reduced immunoglobulin a secretions, which creates good invasion opportunities for bacterium. in addition, as mentioned above, intestinal flora are prone to happen in severe hepatitis patients, which makes intestinal infection very common [24] . few patients exhibit cellulitis like colitis, which can further result in peritonitis and septicemia, and finally lead to death. pathogens in intestinal infections could be shigella spp., salmonella, campylobacter jejuni, clostridium difficile, and salmonella typhi etc. clinical manifestations are nausea, vomiting, abdominal pain, diarrhea, watery stool or bloody mucopurulent stool. some patients may have fever and tenesmus. depending on the pathogenesis and clinical manifestations, intestinal infections are classified as enterotoxigenic bacterial enteritis and invasive bacterial enteritis. pathogenesis of enterotoxigenic bacterial enteritis is that bacteria adhere but not invade intestinal mucosa. intestinal toxins are secreted in the process of bacteria growth and reproduction, which stimulate small intestinal epithelial cells secreting large amount of water and electrolytes. overuptake of water and electrolyte and retention in intestine makes watery stool, which is called "secretory diarrhea." secretory diarrhea is exhibited as frequent, large amount stool with no pus, usually without pain or tenesmus. it is often accompanied by vomiting, which is prone to bring out dehydration, electrolyte imbalance and acidosis, but less severe systemic toxic symptoms. stool examinations show less erythrocytes or leukocytes. invasive bacterial enteritis refers to pathogenic bacteria adhere and invade the intestinal mucosa and submucosa, causing significant inflammation. different pathogens violate different parts of intestine, small intestine, or colon, and sometimes cause inflammation both of small intestine and colon. the basic clinical manifestations include obvious systemic sepsis, high fever, and even septic shock in severe patients. stool can be mucus bloody or purulent, less amount and more frequency. abdominal pain are often severe, paroxysmal colic. if the lesion invades the rectum and distal colon in particular, there may be tenesmus. sigmoidoscopy examination showed diffuse inflammation and ulceration. if only the small intestine or upper part of colon are invaded, the stool is more moisture, and without tenesmus. stool examination show large amount of leukocytes. although diagnosis of intestinal infection is not difficult, it should be careful to distinguish the site of infection, make sure of pathogens, and pay particular attention to water, electrolyte imbalance and acid-base imbalance. therefore, in addition to routine examinations and stool culture, keeping abreast of the general condition is also important to avoid disturbance of the internal environment which aggravate liver damages. in severe hepatitis, the more severe hepatic damage and immune dysfunction, the higher of incidence of sepsis. bacterium most commonly enters through intestine into the portal vein and then into the systemic circulation, followed by skin, respiratory tract, urinary tract or other intrusion. pathogens are often opportunistic bacteria, in which gram-negative bacteria are more than gram-positive bacteria, especially escherichia coli. clinical manifestations of nosocomial infection concurrent with severe sepsis are not specific, easily masked by the primary disease and complications. in some cases primary focus is not obvious, therefore the diagnosis mainly rely on blood culture. clinical manifestations include: (1) unexplained sudden chills, fever, shock, increased peripheral blood leukocytes or neutrophils; (2) deepening jaundice, an increase in ascites, or appearance of hepatic coma, hepatorenal syndrome in a short term. when complications appear in severe hepatitis patients, sepsis should be alert. the mortality of sepsis in severe hepatitis is high. nosocomial infection not only aggravates liver damages, but also induces hepatic coma, hepatorenal syndrome, upper gastrointestinal bleeding and other serious complications, leading to multiple organ failure. nancy rolando reported mortality of septicemia in patients with liver failure was as high as 59%, in which 98% with septic shock [40] . fungal infections can be classified into endogenous and exogenous infection. according to the source of the fungus, the former belongs to opportunistic pathogens, while the latter is in the environment, being infected through various routes of exposure. fungal infections in severe hepatitis are invasive fungal infection in most occasions, and the majority are nosocomial infection and endogenous pathogenic fungi infection [27] . candida infection is most common, followed by aspergillus, again neoformans and histoplasma monocytogenes. candida albicans is widely present in normal human digestive tract. other fungi such as cryptococcus neoformans, aspergillus are widely found in nature, which can colonize in body surface, or non-enclosed cavity. they also exist in hospital work environments, increasing the chance of nosocomial infection in hospitalized patients. clinically, severe hepatitis with fungal infections are mostly superinfection. the rate of fungal infection in severe hepatitis is increasing in recent years. nancy rolando reported fungal infection was present in 16 of 50 acute severe hepatitis patients (15 candida, 1 aspergillus) and in seven cases was considered the major cause of death [41] . domestic data reported that in a group of patients with liver failure, fungous infection was found in 143 cases in which the rate of nosocomial infections was 86.71%. in 155 separated fungous strains, 90 strains (58.06%) were candida albicans, 17 strains (10.97%) were aspergillus fumigatus and 25 (16.13%) strains were non-candida albicans. the main sites of fungus infection were lungs (94 cases) and oral cavity (53 cases) [29] . for severe hepatitis complicated by fungal infection, the mechanism is complex. there are many influencing factors such as immune dysfunction and reduced defensing ability, besides, application and abuse of broad-spectrum antimicrobial drugs are also related [42] . because of broad-spectrum antibiotics destroy the imbalance between bacteria and fungi in digestive system, which inhibit the growth some gram-negative bacteria that had an anti-fungal effect and some bacteria that are able to synthesize vitamin b family. lack of vitamin b can lead to inhibition of oxidation coenzyme, enabling weakened immunity, which is conducive to fungal growth. research and experience have demonstrated that repeatedly use of glucocorticoid is also an important factor to induce fungal infection in patients with severe hepatitis [28] . and therefore, the use of corticosteroids also need special care of. currently the use of glucocorticoids in patients with severe hepatitis is still controversial. the majority do not advocate glucocorticoids, or consider a short-term use in the early stages of the disease and be removed as soon as possible. otherwise it will cause a large increase of possibility of fungal infection. fungal infections are among the leading causes of death in patients with severe hepatitis. according to an analysis from rolando, among 11 cases of severe hepatitis, seven cases of deaths directly related to fungal infections [41] . a recent domestic research analyzed outcomes of 115 patients with severe hepatitis, it was found that the mortality of patients with fungal infection was significantly higher than those without fungal infections (59.1% compared to 34.8%) [28] . according to statistics, fungal infections often occur eight days after admission (0-24 days) or 5.5 days after broad-spectrum antibiotics usage (1-14 days). symptoms of fungal infection are often covered by severe liver injury related symptoms, while clinical systemic manifestation such as fever is also difficult to identify with the bacterial infection. the site of infection often occurs in mouth, respiratory, digestive or urinary tract. severe immunocompromised persons may appear disseminated infection. oropharyngeal is the site that fungal infections mostly occur, and candida albicans is the most common pathogen, followed by non-candida albicans and aspergillus infection. bedridden patients with severe hepatitis can not properly maintain oral hygiene, which results in change of oral local environmental ph and blood flow. candida albicans easily retains in the mouth, and multiplies, causing oral flora and opportunistic infections. usually general symptoms are mild. patients often have abnormal taste or loss of taste, followed by xerostomia, mucosal burning and other symptoms. candida stomatitis may have pseudomembrane formation which is not easy to peel, accompanied by angular cheilitis, or sometimes manifests as mucosal congestion, erosion or clumps shrink of tongue papillae, thickening of coating on the tongue. there are clear lines between oral pseudomembranous damages. if remove the pseudomembrane it will leave a bright red base, sometimes thick film is like a layer of cheese. take the film directly under microscopic examination shows hyphae and spores. oral fungal infection is often a prelude of deep fungal infection, which should be on the alert. simple oral candida albicans infection is not always accompanied with fever. oral fungal infection is easy to be found, therefore, if oral fungal infection exists with fever and increased leukocytes, it should be paid attention to merger of deep fungal infections such as the lungs or other organs [43] . aspergillus is ubiquitous in nature which can be spread through air flow. the most common pathogen that causes invasive aspergillosis is aspergillus fumigatus, while aspergillus flavus, aspergillus niger and aspergillus soil are less common. inhalated aspergillus spores can reproduce in healthy or immunity weakened people. respiratory tract is an infective route of invasive aspergillosis, accounted for 95%. once tissue infection exists, blood vessels violation and bloodstream invasion are common. invasive aspergillosis infection has three characteristics: tissue necrosis, hemorrhage, dissemination [44] . the mortality of invasive pulmonary aspergillosis accompanied by severe hepatitis is up to 90% [44] . it is lack of specific clinical manifestations. in most patients fever is the first arisen symptom, mostly with middle or low heat, sometimes with sudden high fever. hemoptysis occurs with fever simultaneously or 1-8 days later, often accompanied with purulent tan sputum or bloody sputum, sometimes with a pinhead size of gray-green particles in it. shortness of breath and chest tightness often exist in late stage of infection, as well as dyspnea, cyanosis, hypoxemia, and expectoration of a lot of bright red bloody sputum or blood clots. pulmonary signs appear later, manifests as pulmonary wet or dry rales, occasional pleural friction sound. in some cases there are no pulmonary signs. x-ray examination of invasive pulmonary aspergillosis showed patchy infiltrates and (or) nodular lesions. typical nodules were like cotton, which can occur in unilateral or bilateral lobes. the lesion progresses rapidly to cause expanded infiltrates, and segmental or lobar consolidation. ct examination showed mass shadow, nodules, or exudative lesions. there are two typical imaging performance: (1) a characteristic finding on chest ct is the halo sign: a solid nodule surrounded by a halo of ground-glass attenuation [45] . (2) the formation of voids, which appear in late infection. signs are hollow cavity lesions, the balloon "crescent" sign was seen around necrosis tissue [46, 47] . in order to prevent hepatic encephalopathy, patients can be given high doses of laxatives such as lactulose, which can reduce the intestinal ph value, creating an environment for growth of fungi, thereby increasing the chances of fungal infection of the intestine. in particular, some antibiotics combination increases the incidence of intestinal candidiasis. patients usually have watery or jerry-like diarrhea, with foam or blood. diarrhea is accompanied by bloating, sometimes with vomiting and fever, however, abdominal pain is not obvious. in patients with severe hepatitis, due to decreased intestinal mucosal defense capability, candida may invade the muscle and cause intestinal bleeding, or even perforation. in part of patients, it may even progress to fungal peritonitis, which is similar to clinical manifestations of bacterial peritonitis. in patients with oral candidiasis, if there is dysphagia or pain, especially retrosternal burning, it should be considered that esophagus is invaded. incoordination motor of the upper and lower esophageal can be found by esophageal barium enema. gastroscopy helps to confirm the diagnosis. urinary fungal infection usually involves bladder and kidney, among which candida infection is the most common. however, all pathogenic fungi (such as cryptococcus neoformans, aspergillus species, mucor species, histoplasma, blastomycete, coccidioides) can spread to the urinary system as a systemic or part of disseminated fungal infection, which is more related to usage of broad-spectrum antibiotics and indwelling catheters. clinical manifestations include fever and urinary tract irritation, while some patients were asymptomatic with candidiasis urine. candida and bacterial infections often occur simultaneously. candida infections of the kidney, mostly secondary, are caused by the spread of blood candida. renal cortex and medulla abscesses can occur, which affect renal function in severe cases. patients may have low back pain, abdominal pain, fever and chills, accompanied by urgency, urinary frequency, proteinuria and hematuria. urine tests may find hyphae and fungal spores, culture for candida is positive. candida albicans is common, but now there is a growing trend of candida glabrata [27] . the main pathogen of fungal sepsis is saccharomyces. most patients have high fever, often above 39 °c. the thermal type varies, of which intermittent fever or remittent fever is more common. wbc count and neutrophil are usually increased. disease in patients with fungal sepsis followed by severe hepatitis is deteriorating rapidly, even progressing to shock. fungal septicemia may invade all the tissues and organs. involved organs have corresponding performance, such as fungal pneumonia, oral fungal infections, intestinal and urinary tract infections. disseminated fungal infection often occurs in severe immunocompromised patients who have long-term use of antimicrobial agents. candida, cryptococcus, aspergillus can disseminate along with blood to all of the organs, such as kidneys, lungs, heart, and liver. the condition is dangerous, which often lead to death in a short term. in addition to common bacterial and fungal infections, severe hepatitis can also be complicated by other pathogens, such as viruses, tuberculosis, protozoa and others. cmv (cytomegalovirus, cmv), herpes simplex virus (herpes simplex virus, hsv), or varicella -zoster virus (varicella-herpes and zoster virus, vzv) infections are three of common herpes viruses infections in severe hepatitis. their common characteristics rely on that once the host is infected, the virus can persist for long periods in the host. when the host immunity is weakened, the virus can re-proliferative, which leads to disease resurgence [48] . hsv infections manifest as perioral or external genital herpes, oral and esophageal mucosa inflammation and ulcers, also viremia which leads to pneumonia and encephalitis. common clinical symptoms of herpes simplex are mild, only a few people show fatigue, fever and other symptoms. local manifestations are single or multiple blisters on skin or mucous membrane, with tingling. due to reduced immunity, skin rashes in patients with severe hepatitis perform as varicella-like rash, vaccination herpes, herpes keratoconjunctivitis and disseminated herpes simplex. severe herpes usually manifests as herpes simplex virus encephalitis with high mortality. there are fever, headache, mental disorders, coma and other clinical symptoms, often without skin herpes lesions. the sites of infection are commonly in the frontal and temporal lobes. elevated serum antibodies help confirm the diagnosis. cytomegalovirus infections are common in cirrhosis of the liver [49] . interstitial pneumonia is the most common clinical manifestation in severe hepatitis concurrent with cmv infection. chest ct examinations mainly perform as diffuse interstitial or alveolar infiltrations, very few case show as nodular shadows, occasionally as pleural effusion [50] . pulmonary consolidation reminds complicated bacterial or fungal infections. pathology manifestations show alveolar interstitial edema, with varying degrees of fibrosis, lymphocyte infiltration and epithelial cell proliferation. blood examination shows leukopenia. because viral pneumonia shares a certain similarity in clinical manifestations, diagnosis mainly depends on pathologic examination. pathogenic examinations for cmv often use methods below: (1) detect of cmv inclusion body cells and viral particles: eosinophilic nuclear inclusions giant cells are found in respiratory secretions and bronchoscopy lung biopsy specimens. respiratory secretions, saliva, urine, cervical secretions, liver or lung biopsy specimens were inoculated to human embryonic fibroblast cell culture medium, where cytomegalovirus was separated. (2) immunological methods: cmv antigen from secretions was detected by fluorescent antibody assay, or elisa, which is conducive to the early diagnosis. serum antibodies can also be detected by complement combined experiment, in which acute and convalescent serum antibody titer more than 4 times are positive. (3) molecular biology methods: pcr technology and nucleic acid hybridization, which helps to make distinction between a variety of different subtypes of the virus. most of vzv infections in patients with severe hepatitis are due to latent virus reactivated. in patients who have had chickenpox, there is a small amount of virus lurking in the spinal cord dorsal root ganglia or cranial nerve sensory ganglia. when severe hepatitis happened, latent virus is reactivated in ganglia due to decreased immunity. the activated viruses spread along with sensory nerve axons to downstream disposal areas, proliferate and cause shingles. in early time, there is paresthesia, itching, and pain in local skin. and then rashes and herpes break out, chaining into a strip, which distribute in denomination or trunk, unilateral, with duration of about three weeks or several months. part of patients with severe hepatitis show severe disseminated herpes zoster. varicella-like rashes appear in a few days, often accompanied by fever, which may be complicated by lung, brain damage with a high mortality rate. latent tuberculosis can burst to tuberculosis and extrapulmonary tuberculosis when cellular immune function gets weakened. under normal host immune function, lymphocytes, macrophages and common langerhans cell may promote granuloma formation and make infection localized. when host immunity is dysfunctional, tissue reaction is very small or even disappeared, leading to mycobacterium growing rather than formation of granulomas, nor any effective defense against infection. severe hepatitis patients with m. tuberculosis infection may develop acute miliary tuberculosis that manifest as fever, cough, expectoration, bloody sputum, chest pain, and shortness of breath in addition to deteriorated liver function. moreover, tuberculosis easily spread in patients with severe hepatitis, with poor anti-tuberculosis efficacy. common extrapulmonary tuberculosis can be lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, renal tuberculosis, epididymal tuberculosis, genitourinary tuberculosis, nervous system tuberculosis, tuberculous meningitis [51] . in patients with severe hepatitis concurrent with tuberculous mycobacterial infections, tuberculin reaction is negative in about half of patients, especially in those applied with glucocorticoid. diagnosis relies on sputum acid-fast staining or pcr, but the diagnostic yield is not high. interferon gamma release assays (igras), including quantiferon ® -tb gold in-tube, and the t-spot tb, have been extensively used for the auxiliary diagnosis of tuberculosis infection in adults. igras detect circulating t-cells responsive to specific mycobacterium tuberculosis antigens, which are absent in bcg and other non-tuberculosis mycobacteria, and exhibited similar sensitivity and higher specificity than tst in adults [52, 53] . however, these igra tests are also affected by host immune status [54] . in addition, the decision regarding whether to treat ltbi should be dependent not only on igras results but also on clinical histories. ntm generally causes local wound infections. however, in severe hepatitis patients with impaired immune function, non-tuberculous mycobacteria can invade the lungs, causing tuberculosis-like diseases, but rarely causes hematogenous dissemination. histological examination of the lesion is mainly characterized by epithelioid cell granulomas and foam cell-like balls of tissue proliferation, detection of non-tuberculous mycobacteria [55] . protozoa and worms, such as toxoplasma gondii, giardia lamblia, cryptosporidium and stercoralis, can also infect patients with severe hepatitis, especially those with immunosuppression drugs or combined with cancer. main lesions of toxoplasmosis manifest as lymphadenopathy, hepatosplenomegaly, encephalitis and pneumonia [56] . clinical manifestations of giardia lamblia infection are chronic diarrhea and malabsorption, also fever and cholecystitis [57] . pathological changes are deformation of small intestine villi and lymphoid hyperplasia. parasites present in small intestinal surface and gallbladder, and the detection of parasites from the stool and duodenal drainage fluid that is eligible confirmed. strongyloides stercoralis is a weak virulent worm, there is few clinical stercoralis infection [58] . but this worm infection in patients with severe hepatitis is a serious threat, even causing death. clinical manifestations include long-term nausea, vomiting, diarrhea, bloating, intestinal paralysis, dehydration, electrolyte imbalance, edema, weight loss and difficulty breathing in cases with extensive lung lesions. all patients have hypoproteinemia and anemia. patients with increased eosinophils have good prognosis, whereas eosinophils reduction often is a dangerous signal. varieties of complications, such as system or local infections, coagulopathy, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, acid-base imbalance and water-electrolyte imbalance, are main causes of hbv-associated mortality during deterioration of liver function of aechb. reducing and effective treatments of these complications are still nodules in therapy of severe phase of aechb. infection is one of usual complications of aechb, which shows 18-52% morbidity in civil china. the most common localities are respiratory system, especially lungs, and abdomen, which shows the incidence of spontaneous bacterial peritonitis (sbp) as much as 49%. others include urinary tract and bloodstream. gram-negative bacteria are the predominant causes including aerobic gram-negative bacilli, escherichia coli, klebsiella, enterococcus and anaerobic bacteroides fragilis, etc. however in recent years, gram-positive bacterial infections are found increasing in patients with aechb, including pneumococcus and other streptococcus, but staphylococcus aureus infection is relatively infrequent. fungal infections are usually secondary to bacterial infection. candida, aspergillus, sporotrichosis, histoplasmosis and coccidioidomycosis infection, especially candida albicans, are most frequent in occurrence. but, infections of cryptococcus and mucormycosis are relatively rare. beyond of bacterial and fungal infection, other infectious pathogens in aechb are virus, mycobacterium tuberculosis, mycoplasma, chlamydia, parasites and protozoa. bacterial infection is the most frequent complication in aechb. infections are more likely occurred in abdomen, including abdominal cavity, biliary tract, gastrointestinal tract, or other parts like respiratory tract, and urinary tract. keeping oral and perineal asepsis, unobstructed respiratory tract and urine tract, anti-bedsore care for patients with limited mobility, rational use of antibiotics, avoiding long-term use of broad-spectrum antibiotics, strict controlling usage of glucocorticoid, aseptic practices in invasive operation, parenteral nutrition and protecting the intestinal mucosa are all necessities to prevention of bacterial infection in aechb. empirical use of antimicrobial agents could be determined by the localities of infection without antibiotic susceptibility testing results. gram-negative infections are more frequent in peritoneal or biliary tract, in which cephalosporin or quinolone could be the first choice. penicillin and vancomycin could be considered in pulmonary infection. azithromycin and quinolone could be adopted in urinary tract infection. metronidazole or tinidazole could be used in anaerobic infection. broad-spectrum antibiotics can be used in serious infection, such as ceftriaxone, cefoperazone, cefepime, and carbapenems, but the secondary infections need to be highly concerned. initial antibiotics should be adjusted according to antibiotic susceptibility testing results as soon as possible. non-specific immune enhancer agents, such as thymosin, are well used in treatment of infection during aechb, but it currently still lack of evidence-based support. the incidence of fungal infection ranks secondly in aechb associated infection. respiratory tract, gastrointestinal tracts and genitourinary system are the major sites. keeping wards dry and ventilated can help to reduce environmental fungal growth. oral and perineal cleaning and regularly dental check are necessary. if oral fungal spots are found, alkaline mouthwash and nystatin with glycerol can be used in treatment. for patients with limited mobility, anti-bedsore care is terribly necessary. rational use of antibiotics, especially avoiding long-term usage of broadspectrum antibiotics, can prevent secondary fungal infection. glucocorticoid should be used strictly according to indication. during invasive procedures, aseptic operations are obligated. regular check of sputum, lungs, urine and feces will facilitate early diagnosis. empiric antifungal treatment is generally not recommended, but to patients with aechb with aids, especially with count of peripheral blood cd4 + t cell-less than 200/μl or oropharyngeal candidiasis found, the sulfamethoxazole (smz-tmp) should be chosen to prevent pneumocystis pneumonia. fluconazole should be considered when moderate amount of candida albicans has been indicated by sputum culture. empirical treatment. if candida albicans infection is highly susceptible, the initial treatment choice is fluconazole (200-400 mg/days). but if aspergillus infection is preferred, amphotericin b liposome, itraconazole, caspofungin or voriconazole could be considered as the initial treatment, in which process liver and kidney function should be intensively monitored. to patients with cryptococcal encephalopathy combining severe liver damage, fluconazole or flucytosine combined with intrathecal injection of amphotericin b treatment could be implemented. evidence based treatment. initial antifungal strategy should be adjusted according to antibiotic susceptibility testing results. for certain invasive pulmonary aspergillosis, combination treatment of several different types of antifungal agents should be considered under monitoring of liver and kidney function. however, for pulmonary mucormycosis infection, the combination of amphotericin b and flucytosine is the only effective strategy. amphotericin b is a type of polyene antifungals, mainly for aspergillus, candida, cryptococcus, histoplasma infection, but is invalid in aspergillus terreus or ringworm fungus infection. intravenous or intrathecal injection of amphotericin b should be administrated because of non-digestive absorbance. the recommended initial dose of intravenous administration is 1-5 mg/days, and gradually increases to 0.5-1 mg/kg.days. the infusion track needs to be dark and process needs not less than 6 h. the initial dose of intrathecal injection is 0.1 mg/days and gradually increases to 0.5-1 mg/days. amphotericin b has an extra toxicity to liver and kidney, and also causing hypokalemia. intensive monitoring of liver and kidney function and serum potassium levels is necessary during treatment, and be sure largely avoiding combination with other agents with liver and kidney toxicity. itraconazole is one of triazole antifungal agents, mainly for aspergillus, candida, cryptococcus and histoplasma infection, but it is invalid in fungi fusarium or zygomycetes infection. the intravenous dose for the initial 2 days is 400 mg/days, administrated by twice, and then followed by 200 mg/days for 2 weeks. the oral doses of 200 mg bid could be subsequent. the total curative course could be determined by the improvement of symptoms and largely absorption of radiographic lesions of infection. the monitoring of liver function is necessary and recommended, especially in long duration treatment. furthermore, combination treatment with other hepatotoxic drugs should be avoided. fluorocytosine is one of bacteriostatics, mainly for cryptococcosis and candida infection, frequently based on the combination with amphotericin b. the daily dose for adults is 2.5 g with intravenous dripping speed of 4-10 ml/min. a half dose should be conducted in renal insufficiency. the inhibited administration includes severe liver or kidney dysfunction and allergy to fluorocytosine, and also cautious treatment for pregnant or breastfeeding women. no combination treatment of fluorocytosine with cytarabine or bone marrow suppression agents is recommended. fluconazole is a triazole antifungal, mainly for candida albicans or cryptococcus infection, but not as good in candida glabrata infection, totally invalid in aspergillus or candida krusei infection. the recommended daily dose for adult is 200-400 mg, and the initial dose should be doubled. voriconazole belongs to triazole antifungals, mainly for candida, cryptococcus, aspergillus, fusarium and histoplasma capsulatum infection, especially used for invasive aspergillosis and invasive fluconazole-resistant candida infection, but is invalid for mucor or rhizopus infection. the recommended intravenous initial dose in adult is 6 mg/kg, q12h, with infusion rate of 3 mg/kg within 1-2 h. the maintenance dose from the second day is 4 mg/kg, q12h. to patients without tolerance to normal dose, it could be reduced to 3 mg/kg, q12h. the reduction of daily dose could not be necessary in mild to moderate liver dysfunction. but intravenous administration should be avoided in patients with severe renal dysfunction. the side effects of voriconazole include transient visual disturbances, mental disorders, thrombocytopenia and so on. caspofungin belongs to echinocandin antifungals with antibiotic spectrum of pathogenic aspergillus, candida and pneumocystis, without in cryptococcus neoformans, fusarium and mucor. it is mainly used for invasive aspergillosis. the initial dose for adults is 70 mgqd, and with subsequent 50 mg qd. the infusion time is no less than 1 h. no fixation curative course is suggested. caspofungin should be avoided for patients with severe liver function, because of highly hepatic distribution and metabolic pathways during treatment. liver is the largest solid organ in the body, and it plays a central role in the clotting process, except for general function such as metabolism, detoxification and choleresis [59] . a majority of the coagulation factors are synthesized almost exclusively in the liver. in the pathogenesis of severe hepatitis, massive hepatic necrosis leading to reduced production and dysfunction of coagulation factor. in addition, the increased levels of anticoagulation and platelet abnormalities also contribute to occurrence of coagulopathy, which were further exacerbated by severe complications such as spontaneous bacterial peritonitis (sbp) and hepatorenal syndrome (hrs). coagulation abnormalities, even disseminated intravascular coagulation (dic) often occur in those patients with severe hepatitis. therefore, the changes in coagulation factors were usually used to evaluate prognosis of severe hepatitis [60] . the normal procedure of coagulation includes two independent coagulation process, namely intrinsic and extrinsic pathway, which initiated by coagulation factor xii, and factor viia/tissue factor, respectively. the two pathways reach a confluence at the points of factor xa and va. wherein factor xa activates prothrombin to thrombin in the presence of ca 2+ and factor va bound to membrane surfaces, and then thrombin converts fibrinogen to fibrin. thus, blood becomes clotted and the "y" shaped pathway was established [61] . simultaneously, there are some anticoagulation systems existing in our body, which prevents the formation of thrombus, then keeps normal circulating of blood flow. it also participates in maintaining of normal permeability of the blood vessel [62] . among anticoagulation systems, the most important one is the fibrinolytic system, whose basic process can divided into two stages, i.e. plasminogen activation and fibrin degradation [63] . plasminogen activators include tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator (upa), and the principal function of these two plasminogen activators is to convert plasminogen to plasmin, then plasmin cleaves fibrin into soluble small peptides, namely fibrin degradation products [64] . moreover, the process of fibrinolysis was affected by fibrinolysis inhibitors, such as plasminogen activator inhibitor (pai) and alpha2antiplasmin (α2-ap). those fibrinolysis inhibitors can either inhibit plasminogen activation, or reduce the function of plasmin [65] . thus, there is a dynamic balance between coagulation and anti-coagulation systems under physiological conditions ( fig. 2 .1) [66] . many factors involved in coagulation process, i.e. clotting factors, thrombin inhibitor, fibrinolytic system and so on, are synthesized almost exclusively in the liver. thus, liver plays a pivotal role in remaining the balance between hemorrhage and coagulation under physiological conditions (table. 2.1) [67] . it is the important basis for pathogenesis of coagulopathy that massive hepatocyte necrosis occurs in severe hepatitis patients, resulting in reduced production and dysfunction of those blood clotting factors [68] . coagulation abnormalities, such as decreased production and dysfunction of coagulation factors, are commonly found in severe hepatitis, there are at least 14 types of factors participated in the coagulation process, including 12 classical coagulation factors, namely factor i/fibrinogen, factor ii/prothrombin, factor iii/tissue factor, factor iv/calcium ions, as well as factor v, vii, viii, ix, x, xi, xii, and factor xiii. some bradykinin factors, such as rk (prekallikrein) and hmwk (high molecular weight kininogen) are also associated with coagulation. among above factors, the others belong to proteins excepting factor iv. plasma coagulation factors are synthesized exclusively in the liver, excepting factor iii/tissue factor, factor iv, factor vi/activated factor v, factor viii and factor viiia [69] . massive hepatic necrosis leads to the decreased production of clotting factor in patients with severe chronic hepatitis. moreover, the coagulation factors are sensitive indicators for clinical evaluation of severe hepatitis. it is common that the serum levels of factor v, vii, ix, x, xi and prothrombin decreased in the patients with severe hepatitis. on the contrary, clotting factor viii is synthesized, and then excreted increasingly by the mononuclear phagocytic cells with stimulation of various inflammatory cytokines in the patients with severe hepatitis [70] . anorexia and antibiotics overuse lead to obstacles in assimilation, absorption, and application of vitamin k in these patients. vitamin k dependent coagulation factors includes factor ii, vii, ix, and x. the function of γ-hydroxylation was weaken by the deficiency of vitamin k and damage of hydroxylase, then the abnormal clotting factors without γ-hydroxy glutamate were synthesized. finally, these abnormal clotting factors lead to dysfunction of the vitamin k dependent coagulation factors [71] . coagulation factor vii eliminated significantly in the initial stage of liver injury was usually used as an early diagnosis index [72] . in addition, coagulation factors ii and x, then factor ix decreased markedly, along with exacerbation of liver injury. the deficiency of vitamin k induced by obstructive jaundice, malabsorption syndrome, can be rescued after intravenous injection of vitamin k, which is different from coagulopathy caused by liver injury. the plasma level of fibrinogen is within normal range in patients with compensatory cirrhosis. however, patients with severe hepatitis or liver failure have a significant reduction in the level of fibrinogen, and then develop dysfibrinogenemia [73] . fibrinogen, as the main hydrolysis substrate of thrombin, is the crucial factor in the coagulation process. thus, decreased levels or abnormal structure of fibrinogen leads to coagulation abnormalities in patients with severe hepatitis [74] . there are two clotting factors associated with thrombin except for fibrinogen, i.e. factor v and factor xiii. factor xiii induce a crosslink of sfm (soluble fibrin monomer), resulting in the formation of insoluble fibrin [75] . it reports that the levels of circulating coagulation factor xiii were decreased in 30% of patients with liver disease, resulting in fibrin decline or abnormality. these patients with liver disease will receive a bad prognosis if the plasma level of fibrin was at level below 35% of normal concentration [76] . it is uncommon that the plasma level of plasma factor v decreased markedly in patients with severe hepatitis, except for those complicated with dic or hyperfibrinolysis. the low level of factor v cannot induce the formation of the enzyme-substrate complex, which delay the activation of prothrombin [77] . contact factors include classical coagulation factor xii and xi, as well as some bradykinin factors, i.e. pk and hmwk. these factors contact with vascular intima damage or abnormal surface in blood vessel walls, then active the intrinsic coagulation pathway [78] . moreover, the contact factors can connect with bradykinin, fibrinolysis and complement system. in addition, coagulation factor xiii also activates fibrinolysis system as the initiating factor. factor xii deficiency does not cause excess bleeding, but induce thrombus due to its decreasing activation of fibrinolytic system [79] . there is a dynamic balance between pro-and anti-coagulation systems in physiological conditions, which keep normal blood circulation in the body. the anticoagulation system includes physiological anti-coagulation factors (e.g. antiprothrombin-iii, protein c, and so on) and fibrinolysis factors (e.g. plasminogen, α-2 plasmin inhibitor, and so on) [80] . at-iii, with a half-life period of 2.8 days, synthesized mainly in hepatocytes and partly in endotheliocyte, is responsible for about 70% of anticoagulation in plasma. it is the main reason for low plasma level of at-iii that decreased production and increased consumption of at-iii caused by hepatocytes necrosis in patients with severe hepatitis. the activity of at-iii obviously decreased in severe hepatitis [81] . therefore, heparin, thrombin, activated coagulation factors (e.g. factor x, ix, xi, xii) cannot be inhibited, due to rare at-iii combining with them [82] . there is a negative correlation between at-iii: a (the activity of at-iii) and pt (prothrombin time), which indicate that the level of at-iii: a decreased obviously along with exacerbation of liver injury [83] . the plasma levels of pro-and anti-coagulant factors are low in patients with liver injury. when the necrotic tissue and cytolysis are released in the blood, the balance of pro-and anti-coagulant is destroyed. finally the depletion of at-iii by massive activated coagulation factors will lead to dic [84] . massive hepatocytes necrosis, vitamin k deficiency, and protein c without γ-hydroxyglutamic acid result in blocking activation of protein c. thus, va, viiia and pai cannot be degraded, and the plasma level of them will rise up, due to reduction of activated protein c (apc) in the patients with severe hepatitis [85] . plasminogen synthesis will decreased by about 70% in the liver when sever hepatitis occurs. as the activators of plasminogen, tpa and upa are synthesized by vascular endothelial cells, and their production will increase after vascular endothelial cells initiated with virus, immunocomplex or endotoxin in the patients with severe hepatitis b [86] . with exacerbation of liver injury, pai synthesis decreased significantly in the liver. the main physiological activity of pai is to inhibit tpa induced plasminogen activation. therefore, the activity of tpa will increase with reduction of pai synthesis, resulting in promoting the conversion of plasminogen into plasmin [87] . plasmin, as a kind of powerful proteolytic enzyme, can hydrolyse fibrinogen into fdps, degrade coagulation factors, and inhibit platelet aggregation, resulting in the aggravation of bleeding [88] . hyperfibrinolysis can be caused by congenital or acquired reason, and it commonly leads to a rapid depletion of coagulation and anticoagulation factors, especially in those patients with dic [89] . synthesis and secretion of tpa and upa markedly increase, while pai, namely tpa or upa inhibitor, has been a decrease in the plasma of severe hepatitis patients, resulting in hyperfibrinolysis [90] . at the same time, mononuclear phagocyte system cannot degrade plasminogen activator, also leading to hyperfibrinolysis. it is not necessarily accompanied by hemorrhagic tendency, although there are many risk factors that can cause hyperfibrinolysis, even hyperfibrinolysis occurred in severe hepatitis [91] . in addition, fdps inhibit fibrin monomer polymerize function, and also block platelet aggregation, then further worse the deficiency of hemostasis and coagulation, finally leading to the aggravation of bleeding tendency [92] . studies have shown that low-level endogenous small molecule heparin in patients with chronic hepatitis may be associated with many factors, such as increased mastocyte, decreased production of heparinase in the liver and reduced activity of ph4 (platelet factor4) [93] . when the disease progress to cirrhosis or chronic severe hepatitis, pt was significantly prolonged, while endogenous heparin wasn't increased markedly, indicating that low-level endogenous heparin has little effect on pt elongation and hemorrhagic tendency [94] . however, if the patient is undergoing the following condition together, the endogenous small molecule heparin will increase significantly. (1) esophageal variceal bleeding with serious infections (such as abdominal infection, biliary tract infection and pulmonary infection) or portal hypertension. (2) combining with the significant increased white blood cell count. (3) percentage of neutrophils >75%. the level of endogenous small molecule heparin in plasmin will decrease after those infections were cured. taken together, these data indicate that increased level of endogenous small molecule heparin in blood circulation was closely related to severe hemorrhagic tendency when combining with infection [95, 96] . platelet significantly promotes blood coagulation through connecting with many coagulation factors (such as fibrinogen, factor v, factor xi, factor xiii and so on). α-granule includes fibrinogen, factor xiii, and some platelet factor such as platelet factor2 (pf2) and platelet factor3 (pf3), which promote coagulation activation process of factor xii and factor xi can be accelerated by activated platelets [97] . it is estimated that pf3 provided by platelets could accelerate activating of thrombin by twenty thousand times. xa and factor v could not be inhibited by at-iii and heparin if they were linked with pf3. when platelets aggregated and formed platelet plugs, the process of coagulation was initiated at this site, and then platelets reveal a large number of phospholipid surfaces, which were helpful for activating of factor x and fibrinogen [98] . various platelet factors were released from α-granule after platelet aggregating, and then hemostyptic fibre was produced increasingly. those hemostyptic fibre finally produced blood clot formation after capturing the other hemocytes. thus, platelet plugs would progress independently of platelet disintegrating gradually [99] . two aspects of platelet abnormalities consist of depletion and dysfunction. in patients with chronic hepatitis and cirrhosis, the occurrence of decreased platelet level is usual through hypersplenism accompanied with other hemocyte decreasing [100] . the ratio of which reaches 37-77%. its incidence rate in severe hepatitis and explosive hepatic failure also reaches approximately by 50%. when severe hepatitis occur, myelosuppression, decreased megacaryocyte replication leading short-life platelet, lacking of hemopoietic material such as vitamin b, folate and so on, all of these can initiate thrombocytopenia [101] . other reasons leading to thrombocytopenia contain low expression and metabolic disturbance of thrombopoietin (tpo), as well as platelet antibody production. researches show that the serum level of tpo related positively to platelet expression [102] . we next studied platelet associated immunoglobulin (paig) and its sorts such as paigg, paiga, paigm etc., with chronic hepatitis. in line with previous reports, we found that serum levels of paig and paigg negatively correlated with blood platelet count, corroborating the crucial role of the paig-mediated autoimmunization in controlling thrombocytopenia in viral hepatitis [103] . various factors impact platelet function forwardly or passively. increased expression of oxide and prostacyclin, two kinds of platelet inhibitor derived by endothelium, may inhibit platelet activation in vivo [104] . on the other hand, increased serum level of von willebrand factor (vwf) can promote platelet adhering and aggregation in patients with cirrhosis. when severe hepatitis occur, 66.7% and 77.8% of patients have decreased levels of platelet adhesion rate and platelet aggregation rate (par), respectively. in addition, reduced effectiveness of pf3 and abnormal clot contraction occur in patients with severe hepatitis by an incidence of 65.6% and 77.8%, severally [105] . patients with terminal liver disease significantly exert hemorrhagic tendency, especially in the digestive tract [106] . the latest report indicated that basic laboratory examinations for coagulation function testing in common use at present, such as pt, aptt, international normalized ratio (inr) etc., have little correlation with occurrence of gastrointestinal bleeding in these patients, thereby revealing the importance to search and pay close attention to those complicating disease upregulating bleeding risk, such as bacterial infection, renal failure, hemodynamic change after portal hypertension, dysfunction of endotheliocyte as well as macrophagocyte and so on [107] . it is common to see renal failure occurrence in advanced hepatopathy. when it happened, acquired platelet dysfunction, abnormal activation of platelet and vascular wall, anemia and so on, all of them significantly promote hemorrhage [108] . as another severe complication, bacterial infection is also very important and common [109] . when tumor necrosis factor (tnf) were injected into healthy individuals, we found that endotoxin have an important role to exert its function directly in clotting cascade reaction. early researches indicated that the body can express endogenous heparin-like substance through stimulation by endotoxin in patients with cirrhosis [110] . furthermore, some studies revealed the relevance of endotoxin with prothrombin fragment, indicating that infection promoted occurrence of dic-like status in laboratory examination in cirrhosis [111] . coagulation system became weaker in patients with chronic hepatitis. it can hardly mediate factors due to the relative deficiency of pro-and anticoagulation factors. when the balance was destroyed, it may tend to hemorrhage or thrombosis depending on related risk factors which were in the ascendant. to assess abnormality of blood coagulation in patients with liver disease, we should be in consideration of hypercoagulability, one state may easily be overlooked. otherwise, it will be unfair. current literatures indicated that, unlike previous concept that the body of patients initiated anticoagulation state automatically when infected by the hepatitis virus. various clinical evidences revealed that hepatitis virus infecting cannot inhibit thrombus forming. furthermore, it may increase dangerous to thrombus forming especially in the portal system, for existence of individuals having hereditary mutation to promote thrombus forming [112] . slower bloodstream, abnormal fibrinolysis initiated by blood stasis, and decreased activity of anticoagulant accelerate formation of venous thrombosis. moreover, the changes of platelet phospholipids membrane activity were also helpful to the formation of thrombosis in patients with chronic liver disease [113] . it is reported that the incidence of periphery deep venous thrombosis and pulmonary embolism was 0.5 and 1.0% in patients with cirrhosis, respectively [114] . the rate of portal vein thrombosis is about 1% in patients with compensated cirrhosis, but it reaches 8-25% in the candidates waiting for liver transplantation [115] . for mutations (such as leiden mutation of factor v, g20210a mutation of prothrombin, c677 mutation of methylenetetrahydrofolate reductase) or the existence of antiphospholipid antibody syndrome, the hypercoagulable state will be represented in patients with liver disease [116] . the hypercoagulable state represents diverse modes in the body of patients with chronic hepatitis. among them, thrombosis is more traditional and common. the mechanism of dic complicated with severe hepatitis may contain several aspects as follows. 1. with attacks from endotoxin, virus and immune complex etc., endotheliocyte was injured, and then it activated plasmakinin system and complement system, leading to the aggravation and participation of dic progress. damaged endotheliocyte can simultaneously activate factor vii, intrinsic coagulation pathway and platelets, and participate in micro-thrombosis with platelets adhesion and aggregation beneath endothelium [117, 118] . 2. in patients with severe hepatitis, massive necrotic hepatocytes activated extrinsic coagulation system with the releasing of various tissue thromboplastin-like substances [119] . 3. hepatocellular necrosis or dysfunction decreased expression of anti-coagulation factors such as at-iii, pc, protein s and so on, and then it enhanced activation of thrombin and plasmin [120, 121] . 4. impaired function of the mononuclear phagocyte system. mononuclear phagocytes can express activated tissue factor with the releasing of tnf, il-1 and platelet activating factor (paf) on its surface after stimulation by endotoxin, inflammatory cytokines and complement activation. tnf and il-1 can decrease the activation of protein c through its function to increase the expression of plasminogen activator and plasminogen activator inhibitor-1 and to inhibit the production of thrombomodulin (tm) [122] . activated clotting factor and other factors with promoting coagulation can lead to the occurrence and aggravation of dic, since it cannot be promptly removed [123] . 5. the onset and enhancement of fibrinolysis. massive clotting factors and platelets were depleted in the extensive formation in vivo microthrombus. thrombin promoted the conversion of fibrinogen into fibrous protein [124] . simultaneously, it activated fragments which dropped in the formation of activated factor xiii, factor xa and factor xiia. all of them can activate plasmin, and it enhanced fibrinolysis with tpa, one factor released by damaged blood vessel endothelium [125] . fibrinogen and fibrous protein were degraded after plasminogen activation to generate the corresponding fdps, one factor inhibit blood coagulation and also block platelet aggregation [126] . taken together, the above process exacerbates bleeding initiated by coagulation factors depletion and platelet deficiency. coagulopathy, characterized by prolongation of blood clotting time, tendency of hemorrhage, or even dic, were commonly found in severe hepatitis patients. it may cause uncontrolled external or internal hemorrhage. as common clinical symptoms, the external hemorrhages include gingivale or nasal mucosal bleeding, skin petechiae, the punctures or injection-site ecchymosis, and so on. the internal hemorrhages include esophagogastric varices, intracranial, subcutaneous, and muscle interval bleeding. except for esophagogastric varices hemorrhage, the other internal hemorrhages rarely occur in those patients. in addition, massive hemorrhage from esophagogastric varices is a serious medical emergency, can potentially cause death and cardiac arrest if proper medical treatment is not received quickly [127] . when dic occur, a wide range of thrombogenesis in microvasculature can cause circulatory collapse, characterized by low blood pressure and shock. microcirculatory dysfunction occur after microthrombosis producing, resulting in hypofunction of multiple organs (e.g. kidney, liver, lung and pancreas), which perform from dysfunction to failure, with illness progressing [88] . crushed by the fibrous protein in the vessels, red blood cell destruction can lead to intravascular hemolysis. at the early stage of disease, it shows hypercoagulability. the blood in the needle is easy to coagulate, when getting blood sampling from the vein. after that, it comes with the stage of consumed hypocoagulation. the consuming of a great number of blood coagulation factors leads to significant tendency of hemorrhage [107] . it is difficult to stop bleeding in many parts of the body, including visceral organs, operative sites, injection sites, puncture sites, and mini-invasiva sites [128] . when the third stage, namely secondary fibrinolytic stage comes, a great number of the blood coagulation factors have already been used up, resulting in severe bleeding under the condition of low coagulation. shock, acidosis and mods make the patient's condition continue deteriorating, and are also the main reasons for death [129] . the latest study indicates that thrombosis is also a noticeable state in cirrhosis and end-stage liver disease. portal thrombosis and peripheral vein thrombosis (e.g. deep vein thrombosis and lung embolism) are commonly seen. the deep vein thrombosis is more danger than lung embolism. anyway, the incidence rate of thrombosis in cirrhosis and end-stage liver disease is still very low. the clinical symptoms depend on the embolizing position after the thrombosis occurs [130] . presently blood clotting factors test is the most maturely and frequently used test in the term of blood coagulation function in the world. the indicators including prothrombin time (pt), international normalized ratio (inr) and prothrombin time activity (pta) have always been chosen in the patients with severe hepatitis. prothrombin time (pt) reflects whether there are anticoagulant substances in the extrinsic blood coagulation system and blood circulation or not. the elongation of prothrombin time (pt) presents the declined activity of several blood clotting factors including factorii (fii), factorv (fv), factor vii (fvii), factorx (fx) or the existence of anticoagulant substances. in severe hepatitis patients, the incidence rate of prothrombin time (pt) elongated can reaches to 90%, thus it is regarded as a sensitive and frequently used indicator in the term of liver function [131] . another index international normalized ratio (inr), a reckoned ratio calculated from prothrombin time (pt) and international sensitivity index (isi), making prothrombin time (pt) between different laboratories and different reagents comparable, is an international general indicator. the guides about acute-on-chronic liver failure and acute liver failure take the index, inr ≥ 1.5, as one of the most significant diagnostic criteria in the american association for the study of liver diseases (aasld) and the asian pacific association for the study of liver (apasl). international normalized ratio (inr) can also be used as a monitor on blood coagulation function [132] . hepaplastin test (hpt), reflecting not only blood coagulation mechanism in hepatitis patients but also the function of hepatocytes to synthetize vitamin k dependent clotting factors including factorii (fii), factor vii (fvii), factorix (fix), factorx (fx) synthetically, is a test about liver reserve function; but this indicator can't reflect the change of factorv (fv). when severe hepatitis occur, the function of liver to synthetize above-mentioned clotting factors declines and the time of hepaplastin test (hpt) elongates. with illness progressing, hpt continues elongating. survivors undergoing effective therapies can have gradual recovery in the time of hepaplastin test (hpt). so this test is regarded as the specific test of liver diseases or the optimal indicator reflecting liver reserve function [133] . the severity of liver damage is positively correlated with the decline degree of prothrombin time activity (pta): the more severe damage occurs in hepatocytes, the more significantly prothrombin time activity (pta) will decline. therefore prothrombin time activity (pta) <40% and total bilirubin (tbil)>171 μmol/l have always been used as the main laboratory indicators to diagnose severe hepatitis domestically. pta <40% is also regarded as diagnostic criterion of blood coagulation dysfunction in the guideline of acute-on-chronic liver failure in the asian pacific association for the study of liver (apasl) [134] . in severe hepatitis, total bilirubin (tbil), total cholesterol, prothrombin time activity (pta) and complications (e.g. rectory hyponatremia, hepatic encephalopathy, hepatorenal syndromes and so on) are all independent risk factors to evaluate prognosis. the lack of any factors including factori (fi), factorii (fii), factorv (fv), factorvii (fvii), factor x (fx), can lead to the decline of prothrombin time activity (pta). moreover the half-life time of those factors are extremely short, factorii (fii) 50 -80 h, factorv (fv) 12 -24 h, factorv (fv) 2 -6 h, factorv (fv) 48-60 h, respectively. it means that when hepatocytes suffer from severe serious damage and necrosis in severe hepatitis, prothrombin time activity (pta) will have dramatic decline just in a few days. as a result, prothrombin time activity (pta), characterized by significant advantages in evaluating patients condition and judging prognosis over other laboratory indicators, has been widely used. elongation of aptt prompts the lack of any clotting factor belonging to intrinsic coagulation system or the existence of anticoagulant substances. the incidence of the elongation of aptt reaches to 80-100% in severe hepatitis patients [135] . fxii:c reflects liver synthetic function. fvii, characterized by the shortest halftime 6-8 h, is the first one to be affected when facing liver synthetic dysfunction. on the contrary, fv:c, characterized by a relatively long half-time, is one of the latest factors to be affected and is correlated with the degree of liver damage. it prompts severe liver failure, bad prognosis and even easy death when plasma levels of fv:c under 20%. some literatures report that fv:c and pta can be used as significant prognostic factors in liver failure and significant screening indicators in liver transplantation. however, the indicators-fxii:c and fv:c-are not listed as routine examination items. they are just selected on the condition of illness demand [136, 137] . the main test about anti-clotting factors is the determination of antithrombin iii activity (at-iii:a). in the state of pathosis, the decline of antithrombin iii activity (at-iii:a) is not parallel with the decline of antithrombin iii (at-iii) content, namely the depletion of antithrombin iii activity (at-iii:a) more apparent. owe to this, it has more clinical value to determine antithrombin iii activity (at-iii:a) rather than antithrombin iii (at-iii) content. moreover, anti-clotting factors tests include the determination of protein c activity (pc:a) as well [138] . serum levels of fdps are very low in normal people. significantly elevating of fdps indicates the existence of hyperfibrinolysis and reflects the occurring of dic indirectly. there are many assay methods including immunization fi test (namely latex particle agglutination test, normal titer<1:8), fdps flocculation test, radial immunodiffusion staphylococcal clumping test, indirect hemagglutination inhibition test, enzyme-linked immunosorbent assay (elisa) and so on. if the serum levels of fdps elevate, it indicates acute dic may occur [92] . plasma protamine paracoagulation test (3p test) and ethanol gel test (egt) reflect the soluble fibrin complexes in plasma. soluble fibrin complexes, combination of fdps and fibrin monomer, can't be solidified by thrombase. but protamine is able to make the complexes isolate and then fibrin monomers separate out again. the paracoagulation test means self-polymerization between fibrin monomers and fdps, then forming macroscopic flocks. ethanol gel test (egt) has the same principle with plasma protamine paracoagulation test (3p test), whereas the former has a lower positive rate. the two methods may have false negative results and false positive results. in contrast, egt has a relatively lower sensitivity, while 3p test has a relatively lower specificity. for example, relative small molecular mass of the shreds of fdps may lead to negative result using 3p test. so it is more valuable to compare the two indicators simultaneously [139, 140] . euglobulin, a protein (including fibrinogen, plasminogen and other activins except for fibrinolysis inhibitor) separating out from plasma in acid circumstances, can be used to determine whether levels of plasminogen activators increase or not [141] . when hyperfibrinolysis occurs, plasma levels of plasminogen decline, plasma levels of plasmin increase, and euglobulin suffer from accelerated dissolution by a great number of plasmin. the normal value of euglobulin lysis time (elt) is above 2 h. that is to say dissolution within 2 h means the occurrence of hyperfibrinolysis. domestic population data report the positive rate of elt test reaches 25-42.9%, when dic occur [142] . furthermore there are other tests about fibrinolysis including tissue-type plasminogen activator test (t-pa), plasminogen activator inhibitor test (pai), plasminogen antigen test (plg:ag), plasmin activity test (pl:a), α2-plasmin inhibitor test (α2-pi) and so on [143, 144] . blood platelet count reflects the absolute number of platelet in peripheral blood circulation. according to the reports at home and abroad, platelet count have a significant decline in patients with chronic severe hepatitis. moreover, studies on domestic population find that platelet count ranges from 68 × 10 9 /l to 130 × 10 9 /l in peripheral blood of severe hepatitis patients. some studies have already compared the platelet count among early stage, typical stage and late stage in severe hepatitis, and they have turned out to be 130 × 10 9 /l, 109 × 10 9 /l and 87 × 10 9 /l respectively. as a result, it indicates that platelet count is positively correlated with the severity of hepatitis [145] . except for platelet count, other routine indicators including mean platelet volume (mpv), plateletcrit (pct) and platelet distribution width (pdw) also have significant reference value. when severe hepatitis occurs, the above-mentioned three indicators will be dramatically lower, and they have the tendency to continue declining with illness progressing. what's more, platelet quality tests include platelet aggregation rate, platelet factor 3 validity tests and blood clot retraction test (table 2. 2) [146] . diagnosis of blood hypercoagulability in the early stage of dic relies on several molecular marks including plasma thrombinogen segment 1 + 2 (f1 + 2), thrombinantithrombin complex (tat) and d-dimer, due to no significant changes in general laboratory tests. this stage is characterized by the elevated levels of those three molecular marks, and levels will increase more significantly with the occurrence of typical dic symptoms. dynamic monitoring of above-mentioned indicators is helpful for early diagnosis of dic [147] . the stages are mainly characterized by the decline of blood clotting factors (including factorv (fv), factorvii (fvii), factorxii (fxii), factorix (fix), fac-torx (fx)), platelet count and plasminogen, and increasing of fibrinolytic the elevating levels of fibrin(−ogen) degradation products (fdps) and d-dimer; the shortening of euglobulin lysis time (elt) and the positive reaction of 3p test [148, 149] . with the occurrence of dic, there will be a wide range of blood coagulation and highly-activated fibrinolysis in the patient's body [150] . what's more, abnormal increased soluble fibrin monomer and fdp fragments will exist in plasma [151] . the level of soluble fibrin monomer complex (sfmc), a complex combined fibrin monomer with fdp, is determined by 3p test. the 3p test shows positive with the occurrence of secondary fibrinolysis, whereas it shows negative with the occurrence of primary fibrinolysis. this means 3p test is negative when there is no blood coagulation. domestic population data report that the positive rate of 3p tests reach 50-60%. however, the test can't be used as the ideal diagnostic indicator for dic, owing to many affected factors. false positive reactions are mainly found in the following conditions: gastrointestinal bleeding, massive hemoptysis, malignant carcinoma or blood sampling reserved improperly, whereas false negative reactions are usually found at the late period of the stage of secondary fibrinolysis [152, 153] . as mentioned above, plasma thrombinogen segment 1 + 2 test (f1 + 2), thrombinantithrombin complex test (tat) directly reflects production of intracorporeal thrombase, which increased in the early stage of dic. plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the d-dimer antigen, which reflects production of intracorporeal plasmin. d-dimer will have an apparent increase with the occurrence of secondary fibrinolysis, but d-dimer test shows negative when primary hyperfibrinolysis occurs. monitoring the above molecular markers dynamically is helpful to estimate therapeutic efficacy and guide treatment [154] . as mentioned above, clinical manifestations of blood coagulopathy in patients with severe hepatitis are lack of specificity. the most common manifestation is bleeding, not only little hemorrhage from superficial sites, but also massive hemorrhage from internal sites, such as esophagogastric varices [155] . the diagnosis of severe hepatitis complicated with blood coagulopathy is mainly based on the results of laboratory tests and clinical manifestations [156] . according to current guidelines, basic diagnose conditions of dic contain the following points [157] . 1. severe or multiple bleeding tendency. 2. microcirculation collapse or shock which is difficult to explain using protopathy. 3. a wide range of embolism in skin and mucosa, focal ischemic necrosis and ulcer, or unexplained dysfunction of kidney, lung, brain. 4. anticoagulant therapy is effective. if severe hepatitis b patients have one of the above-mentioned points except for (1) and exhibit blood coagulating easily or prothrombin time (pt) shortening over 3 s simultaneously, it can be considered as the early stage of dic in which the tendency of bleeding is not obvious. in addition, if severe hepatitis b patients have two of the above-mentioned four points, dic can be considered as the preliminary clinical diagnosis. furthermore, it can be definitely diagnosed when combined with the aforementioned items of laboratory tests ( table 2. 3). firstly, we should use anti-viral therapy as a mainly method to treat primary disease of severe hepatitis b [158] . then, we must eliminate the incentive, maintain the balance of water and electrolyte, and correct hypoxia and acidosis. it is very important to focus on massive upper gastrointestinal hemorrhage and disseminated intravascular coagulation when treating coagulation disorders [159] . gastrointestinal bleeding, including esophageal varices bleeding and non-bleeding esophageal varices, were correlated with coagulation dysfunction and portal hypertension. prevention is still focused on improving the coagulation function, including adequate vitamin k1 supplements, coagulation factors, fibrinogen, fresh plasma or platelets supplements. it is particularly critical to control diet in patients with severe hepatitis. in which, light and easily digestible diet was recommended, but rough, hard and too greasy food was prohibited. for these patients, appropriate antacids can be used to protect the gastric mucosa [160] . 1. general treatment: bed rest is necessary, meanwhile vital signs were closely monitored. the patients can eat liquid diet when bleeding mildly or having no active bleeding. however, abrosia is required when the patients have a heavy bleeding. 2. fluid resuscitation: firstly, intravenous access should be established rapidly in patients with gastrointestinal bleeding, then, the patients were given intravenous infusion of normal saline, lactated ringer's solution, plasma, whole blood or plasma substitute [161] . 3. vaso-active agents: vaso-active agents such as dopamine and alamin may be given to maintain normal blood pressure, if blood circulation is still not stable after fluid resuscitation [162] . 4. hemostatic: if mucosal bleeding was caused by portal hypertension, oral administration of norepinephrine and ice normal saline can promote mucosal vascular contraction and hemostasis. in addition, oral administration of yunnan baiyao may be effective [163] . 5. acid suppression therapy: the proton pump inhibitors, such as omeprazole, pantoprazole and esomeprazole, are commonly used to inhibit gastric acid secretion [164] . 6. reduction of portal pressure: patients with severe hepatitis complicated by gastrointestinal bleeding often accompanied with portal hypertension, so it may be considered to give them drugs to decrease portal pressure. especially in patients with portal hypertension gastropathy, reduction of portal pressure is more important than acid suppression therapy [165] . 7. compression hemostasis via using three-chamber double-balloon catheter: after the above treatment, if there is still active bleeding in patients with bleeding esophageal varices, it can be considered to use three-chamber double-balloon catheter [166] . 8. endoscopic hemostasis: if the above treatments have no effect in upper gastrointestinal bleeding, endoscopic hemostasis, including endoscopic hemostatic agents spray, endoscopic ligation or endoscopic injection sclerotherapy may be used [167] . 9. others: surgery or interventional therapy may be considered, if internal medicine therapy is ineffective [168] . firstly, we should treat original disease, then improve coagulative function in those patients. in addition, prevention and control of infection, correction of electrolyte disturbance, avoiding the hemorrhage and reduction of allergies and transfusion reactions should be considered [169] . the key is early diagnosis and early treatment. 1. anticoagulant drugs: low molecular weight heparin is the most commonly used drug in earlier stage of dic. it is recommended to periodic test blood routine and coagulation, and dynamically observe coagulation status during medicine therapy. others such as dextran, anti-platelet aggregation ticlopidine, salvia injection, urokinase may be effective, but it should be support by more evidence-based medicine [156] . 2. plasma and blood products: during the process of dic formation, the patients should be transfused with fresh plasma, each 10-20 ml/kg. when they developed the stage of secondary fibrinolysis, prothrombin complex containing coagulation factors, cryoprecipitate and platelets can be supplemented due to a large consumption of coagulation factors [170] . 3. others: such as hemodialysis, anti-fibrinolytic 6-aminocaproic acid and tranexamic acid should be supported by more evidence-based medicine. hepatorenal syndrome (hrs) is a common and serious complication occurring in patients with decompensated cirrhosis and liver failure, who have overt circulatory dysfunction. the 1-year incidence of hrs in patients with ascites is about 20% [171] . hrs may predict a poor prognosis in spite of it being functional reversible [172, 173] . the clinical characteristics of hrs were first described in 1963 [174] . in 1979, hrs was defined by a group of international investigators as a progressive renal dysfunction that occurred in severe liver diseases, with features of prerenal failure (low urine sodium concentration and hyperosmolar urine) but without any improvement following volume expansion [175] . the international ascites club (iac) developed hrs definition in 1996, as a syndrome that occurs in patients with cirrhosis, portal hypertension and advanced liver failure, characterized by impaired renal function with marked abnormalities in the arterial circulation and activity of endogenous vasoactive systems [176] . hrs is a potentially reversible syndrome that occurs mainly in patients with liver cirrhosis, ascites and all kinds of liver failure [172] . it's clinical features include impaired renal function, marked changes in cardiovascular function and over activity of the renin-angiotensin systems and the sympathetic nervous. progressive hrs with severe renal vasoconstriction is able to cause a decrease of gfr [177] . clinically, hrs can be divided into two types (1 and 2). type-1, so-called acute hrs is a rapid progressive form of renal failure defined by doubling of the initial serum creatinine concentrations to a level higher than 220 mmol/l (2.5 mg/dl) or a 50% reduction of the first 24 h creatinine clearance to <20 ml/min within 2 weeks [178] . it appears mainly in patients with acute liver failure, but often develops after a precipitating event, such as bacterial infections (especially spontaneous bacterial peritonitis, sbp). the prognosis of type 1 is poor with the median survival about 1 month [177] [178] [179] . type-2, so-called chronic hrs is a steady or slowly progressive form of renal failure defined by serum creatinine from 133 to 226 mmol/l or from 1.5 to 2.5 mg/ dl [175] . type-2 hrs is mostly related to refractory ascites. survival of patients with type-2 hrs is generally around 6-7 months, which is better than that of patients with type-1 hrs but shorter than that of non-azotaemic cirrhotic patients with ascites. patients with type-2 hrs tend to develop type-1 hrs while infections or other trigger events occurred [171, [177] [178] [179] ]. portal hypertension is the essential factor of haemodynamic changes, which resulted from the development of cirrhosis associated with distortion, compression and even obliteration of the hepatic sinus and vessels [175] . in patients with portal hypertension, bacterial translocation is increased and intrahepatic hypercontractile stellate cells activated [180, 181] . this overall increased resistance to portal hypertensional causes increased local production of various vasodilators such as nitric oxide, leading to splanchnic vasodilation [182, 183] . there are several other factors contributing to the splanchnic vasodilation, including hyporesponsiveness of the splanchnic vessels and mesenteric vascular hyperplasia [181] . in addition, portal hypertension per se can cause renal vasoconstriction by activating sympathetic nervous. for example, when tips is used to reduce portal hypertension and improve renal blood flow, a corresponding reduction in sympathetic nervous activity has been observed [184, 185] . as a result of splanchnic vasodilation, blood is accumulated in the splanchnic vascular bed just like a splanchnic steal syndrome [186] . the combined effect leads to reduction in the effective arterial blood volume (relative hypovolemia) causing a relative inadequacy in the systemic circulation, which triggers a hyperdynamic circulation in these patients [187, 188] . vasodilatation induces activation of neurohumoral systems including the reninangiotensin-aldosterone system (rass); sympathetic nervous system (sns); and non-osmotic release of antidiuretic hormone (adh) [189] . relative hypovolemia initially causes sodium and water retention, increases intravascular volume, and simultaneously increases cardiac output. as cirrhosis progresses, vasodilatation aggravates, which activated vasoconstrictive systems, causing renal vasoconstriction and decreased renal blood flow [181, 190] . local release of potent vasodilators such as nitric oxide (no) leads to splanchnic visceral vasodilation, as well as enables the splanchnic circulation against a variety of vasopressor agents, including norepinephrine, vasopressin, angiotensin ii and endothelin [191] . the resistance of the splanchnic circulation to these vasopressor agents makes the control of arterial pressure during cirrhosis dependent on the extra-splanchnic effects produced by the endogenous vasoconstrictor systems. the role of vasoconstrictors in maintaining haemodynamic stability becomes pivotal as arterial vasodilatation increases during cirrhosis, which makes clear why cirrhotic patients with hrs are prone to develop renal, hepatic and cerebral vasoconstriction [189] . the reduction of effective arterial blood volume leads to the compensatory activation of various vasoconstrictor systems. normally, the kidneys increase the production of renal vasodilators including prostaglandins and kallikrein to maintain blood flow. however, renal vasodilator production is generally reduced in patients with cirrhosis, thus contributing to renal vasoconstriction. this type of renal hypoperfusion further increases the production of various intrarenal vasoconstrictors such as angiotensin ii and endothelin, causing further decline of renal haemodynamics and renal function, occasionally accompanied by glomerular ischaemia and mesangial constriction [178] . when blood pressure fluctuates, renal auto-regulation regulatory mechanisms initiate to make sure that the kidneys receive a relatively constant blood supply. when the critical threshold is below 65 mmhg, renal blood flow is proportional to renal perfusion pressure which, in turn, is dependent on mean arterial pressure. in cirrhosis, with the development of liver disease in patients of cirrhosis, the renal auto-regulation curve gradually shifts to the right -which means as liver disease advances, the renal blood flow gradually decreases for each given renal perfusion pressure [178] . furthermore, lumbar sympathetic blockade increases renal blood flow in patients with hrs, suggesting that the renal sympathetic activity is involved in this outgoing hepatorenal arm. insufficient cardiac output is considered one of the leading causes for renal hypoperfusion in patients with hrs in recent years. despite control of infection, the cardiac output of cirrhotic patients with sbp who developed progressive renal failure was lower than that in similar sbp patients without renal failure. similarly, when patients with non-azotaemic cirrhotic patients who developed hrs are compared with similar patients who did not, it is observed that low cardiac output and high plasma renin activity (pra) were independent predictors of hrs. in addition, in patients developing hrs, the evolvement of circulatory dysfunction leading to arterial hypotension and renal failure occurs in the setting of a continued decline in cardiac output and increase in pra. therefore, effective hypovolaemia occurs when cardiac output decreases, resulting in renal hypoperfusion and hrs [192] . to summarize, the principal mechanisms leading to renal vasoconstriction include systemic circulation changes, accompanying portal hypertension which are characterized by decreased peripheral vascular resistance with subsequent vasodilatation, hyperkinetic circulation and the activation of compensatory mechanisms, i.e., the sns, raas, and adh. with the progression of cirrhosis, the combined effective of all the above factors will result in the gradual deterioration in renal function. any event that leads to a sudden deterioration in hemodynamics can cause a rapid renal dysfunction, precipitating type 1 hrs [193] . the diagnostic criteria for hrs have been first defined by iac in 1996 [176] . the main findings include reduced glomerular filtration (creatinine clearance) less than 40 ml/min or serum creatinine increased more than 135 μmol/l after excluding the other causes of renal dysfunction. however, estimation of renal function by using creatinine clearance is not reliable, because these patients have lower levels of serum creatinine and higher renal tubular creatinine secretion compared with filtered creatinine. furthermore, it is often incomplete for the collection of a 24 h urine. iac developed the new definition and diagnostic criteria for hrs in 2005, which (1) excludes creatinine clearance because of its inaccuracy of renal function estimation and the complicity to perform; (2) includes renal failure at the time of combined bacterial infection (but absence of septic shock), indicating that hrs can be diagnosed before antibiotic treatment; (3) determines by using albumin for plasma volume expansion better than saline. (4) excludes minor diagnostic criteria (urinary indices) because of its poor sensitivity and specificity for the diagnosis [178, 189, 194] . the diagnostic criteria of hrs for patients with liver cirrhosis are as follows: 1. cirrhosis with ascites; 2. serum creatinine >133 mmol/l (1.5 mg/dl); 3. no improvement in serum creatinine (decrease to a level of ≤133 mmol/l or 1.5 mg/dl) after at least 2 days of diuretic withdrawal and volume expansion with albumin. the recommended dose of albumin is 1 g/kg body weight/day up to a maximum of 100 g/day; 4. absence of shock; 5. no current or recent treatment with nephrotoxic drugs; 6. absence of parenchymal kidney disease as indicated by proteinuria >500 mg/ day, microhematuria (>50 red blood cells/high power field) and/or abnormal renal ultrasonography [177, 194] . there are some other causes of renal failure in patients with cirrhosis that were not included, such as membranoproliferative glomerulonephritis and/or iga nephropathy in patients with chronic liver diseases. these chronic forms of kidney disease can cause acute rises in serum creatinine. determining whether it is a potential kidney disease or hrs that causes a sudden increase in serum creatinine in patients with cirrhosis and chronic kidney disease could be difficult [195] . naturally, liver transplantation is the only rational solution in cases of advanced liver disease while it is also the treatment of choice for both type-1 and type-2 hrs. as calcineurin inhibitors (ciclosporin and tacrolimus) may induced gfr impairment, it is recommended to delay their administration until a partial recovery of renal function is recorded, usually 48-72 h after transplantation [177] . clinically, the haemodynamic associated with hrs as well as neurohormonal abnormalities fade away within one month of transplantation, and the patients recover their ability to excrete sodium and free water. compared with patients without hrs, patients with hrs tend to have more complications, take more days in intensive care units and have higher in-hospital mortality rates after liver transplantation. nevertheless, their 3-year survival rate is acceptable (60% vs. 70-80% in liver transplant patients without hrs [196] . the primary limitation of liver transplantation is that most patients with type-1 hrs die before transplantation due to the shortage of donor liver and their extremely short survival time. reference to the model of end-stage liver disease (meld, including scr, tbil, inr) for organ prioritisation has partially addressed this problem, since patients with hrs have a high priority on the waiting list. in addition, the use of vasoconstrictors and albumin in the treatment of type-1 hrs can improve survival rate of these patients, and increase the likelihood of their transplantation [177] . in patients with advanced liver disease and the renal insufficiency, simultaneous liver and kidney transplant (slkt) is taking into consideration. however the reversibility of renal function in some patients when they receive slkt should be taken into account. therefore, to ensure allocation of transplants only to those truly in need, the transplant community proposed an evaluation algorithm in 2006, whose purpose is to determine the presence of kidney disease with structural damage (preferably on biopsy) before giving slkt. in the case of chronic kidney disease, a decreased creatinine clearance at 30 ml/minute or less is considered an indication of slkt. slkt should not be performed for the patients with simple hrs, but for the patients with hrs who become dialysis dependent and without any recovery after 6 to 8 weeks of dialysis [179, 189] . vasoconstrictors combined with albumin are the first line of therapy for type-1 hrs patients. it was recognized long time ago that the effective plasma volume was reduced when patients of advanced liver diseases complicated with hrs, and this led to many attempts to improve the patients' renal function by expanding their plasma volume, including a large dose of albumin or saline perfusion. with the advent of safer compounds including terlipressin, a vasopressin analogue with longer activity, and the α2-agonist midodrine combined with octreotide the analogue, vasoconstrictors is widely used in the patients with hrs. these vasoconstrictors are able to ameliorate vasodilatation while increasing effective arterial blood volume, improving renal vasoconstriction and improving renal flow. in order to further increase effective blood volume, vasoconstrictors have been used in conjunction with intravenous albumin. the clinical results from 12 uncontrolled studies including 258 patients with hrs (type-1, 240) showed that a total of 60% were observed complete response (mostly defined as a decrease in scr to 1.5 mg/dl). interestingly, once the treatment is stopped, hrs relapses only in a few "responders" [177, 189] . there were several randomized controlled trials (rcts) published, suggesting that terlipressin was associated with an increase in gfr compared with albumin alone or with a placebo. the rate of hrs reversal in the terlipressin group was higher than that in the control group (46% vs. 11%). as survival rate was not improved in the two largest rcts, liver transplantation is still the preferred treatment for hrs, but terlipressin seems to serve as a "bridging" treatment. two recent small, open-label rcts suggested that the incidence of hrs reversal and the rate of side effects showed no significant difference between the two groups of norepinephrine and terlipressin [189, 197, 198] . the initial dose of terlipressin recommended in many studies ranged from 0.5 to 1 mg per 4-6 h [199, 200] . if the creatinine level did not decrease by 25% on the third day, the dose could be increased to 2 mg every 4 h or 12 mg/days by continuous intravenous infusion, respectively. in some studies, the daily dose of albumin was generally 20-40 g by a load of 1 g/kg body weight. some mentioned central venous pressure to establish albumin doses and to prevent body fluid from overloading. this treatment was maintained until hrs is reversed, but did not exceed 2 weeks [177] . about 20% of patients relapsed after the treatment withdrawal. however, these patients should be given repeated treatment with terlipressin, which is often effective [177] . several studies have evaluated the role of transjugular intrahepatic portosystemic stent-shunt (tips) in hrs. these studies show that tips help decreasing in scr in most patients, even in a minority of organic renal failure, but it is slower compared to those obtained using terlipressin combined with albumin [201] . recrudescence of hrs is rare provided the shunt remains patent, while hepatic encephalopathy often comes. it is worth noting that the vast majority of patients included in these studies suffered from alcoholic cirrhosis, many of whom have active alcoholism, and therefore the improvement observed may be caused by the improvement in an acute-on-chronic process. in addition, since all these studies excluded patients with a child-pugh score ≥ 12, resulting in a lack of data, the efficacy of tips should be further explored in rcts [189] . extracorporeal albumin dialysis molecular adsorbent recirculation system (mars) is designed for making clearance of water-soluble cytokines (i.e., il-6) and albumin-bound toxins (i.e., bile acids) which is implicated in the pathogenesis of hrs. two studies showed that mars was ineffective in improving survival rate and emic haemodynamics in type-1 hrs [193, 202] . another clinical observation including 32 patients with type-1 hrs reported a rate of complete renal response of 28% [201] . extracorporeal albumin dialysis (ecad) reduces serum creatinine levels, but it is not clear whether this effect is due to a real improvement of renal function or merely a filtration process. several studies demonstrated that patients' systemic haemodynamics improved after ecad, manifested as an increase in systemic vascular resistances and arterial pressure, as well as a decrease in cardiac output, pra and levels of norepinephrine. however, there were too few studies on the effect of ecad on survival in type-1 hrs patients to draw any definitive conclusions [203, 204] . in addition, ecad is very expensive and therefore not suitable for wide and rapid clinical application [177] . the treatment of type-2 hrs should take into account the survival rate as well as controlling the ascites. both hrs-1 and hrs-2 are indications for the tips treatment. the therapeutic effect of tips is excellent for its better controlled of complications of portal hypertension compared with other treatments. tips have been reported not only to improve renal function in patients with type-1 hrs but also to treat refractory ascites in patients with type-2 hrs [205] [206] [207] . the contraindications to the creation of tips are shown in the followings [195] . • contraindications to placement of a tips: there were only a few studies evaluated the role of tips in type-2 hrs and the number of cases was quite low. in most patients, tips could decrease scr, even in some with organic renal failure [208] . hrs recurrence is rare as long as the shunt remains patent, but hepatic encephalopathy often occurs [189] . nine patients were followed-up for 1 month after the treatment of tips in a study, eight cases were found with decreased scr decreased and notably controlled ascites. four patients died, two of them died within 1 month, the other two died at 12 months and 14 months respectively. the remaining five patients survived for a long time. although tips can be used in improving refractory ascites which often contributes to type-2 hrs, data on the effect of tips on survival are still insufficient. therefore, the efficacy of tips should be further explored in randomized controlled trials (rcts) [189] . the information about combining albumin and vasoconstrictive agents treated in type-2 hrs is limited. only a few patients with type-2 hrs have been specifically treated with terlipressin and albumin. in one clinical study, 39 patients with hrs-2 were assigned to receive this treatment and 21 of them achieved improvement of renal function. however after the treatment withdrawal, 11 hrs patients showed relapsed during the follow-up. the most common side effects during terlipressin therapy are cardiovascular and ischemic and reported as an incidence of nearly 12%. the high recurrence rate of hrs after terlipressin and albumin treatment discontinuation suggests that they are less effective in treating type-2 hrs compared to type-1hrs [209] . prevention of hrs is important because it develops at a constant frequency in cases of spontaneous peritonitis (sbp) and advanced liver disease [210, 211] . it becomes possible to prevent hrs if sbp is diagnosed and treated promptly [212] . according to current data, using albumin in combination with antibiotics for the treatment of patients with sbp seems to be warranted but only for those with jaundice or renal dysfunction. the prophylactic use of antibiotics in cirrhosis with gastrointestinal bleeding also seems to be necessary, because the use of antibiotics contributes to reducing incidence of infection and rebleeding whereas improving survival rate. furthermore, the incidence of hrs in sbp patients decreases by albumin administration, and prevention of hrs can also be related to increased survival. the recommended dose of albumin is 1.5 g/kg body weight on the first day then 1 g/kg body weight on the third day, a maximum of 150 g and 100 g, respectively. albumin administration is strongly recommended in sbp patients with serum bilirubin levels higher than 68.4 mmol/l (4 mg/dl) or serum creatinine more than 88.4 mmol/l (1 mg/dl). a placebo-controlled rct that enrolled the patients with low (<1.5 g/l) ascites protein who also had advanced liver diseases or "renal dysfunction"(defined as scr ≥ 1.2 mg/dl or blood urea nitrogen≥25 mg/dl, or serum sodium level ≤ 130 meq/l) suggested that oral norfloxacin contributed to a reduced hrs incidence within 1 year (28% vs. 41%) and an improvement in survival at the end of 3 months [213] . norfloxacin may ameliorate or prevent vasodilatation by reducing bacterial translocation and overt infections, as well as suppressing plasma renin activity, thereby prevent these patients from developing hrs. the concept that the severity of the clinical course of patients with cirrhosis complicated with serious bacterial infection is related to the degree of an impairment of circulatory function, which has led to new and effective approaches in the prevention and treatment of these complications. in patients with severe hepatitis, multiple causes may lead to disorders of internal environment, mostly manifesting fluid and electrolyte imbalance as well as acidbase imbalance, usually resulting in deterioration, greater complexity and even death. accurately recognizing the occurrence of severe hepatitis with complications such as fluid and electrolyte imbalance and/or acid-base imbalance, and therefore giving appropriate treatment to maintain balance of internal environment, is of great importance for improving prognosis of the patients [214] . water is the major component of human body. electrolytes are substances that dissociate in solution to form charged particles, orions. body fluid comprise mainly of water and electrolytes and electrolytes comprise mainly of na + , k + , ca 2+ , mg 2+ , cl − , hco 3 − , hpo 4 2− and so 4 2− . the primary function of electrolyte include: (1) to maintain osmotic pressure and acid-base balance of body fluids; (2) maintain nerve, muscle, cardiac cells resting potential, involved in the formation of action potentials; (3) involved in metabolism and physiological activities [215] . body fluid include intracellular fluid and extracellular fluid, the latter can be divided into plasma and interstitial fluid. intracellular and extracellular fluid differ in ion components. na + is major cation in extracellular fluid and its main anions are cl − and hco 3 − . k + is major cation in intracellular fluid and its major anion is hpo 4 2− . the total number of ions in body fluids is called osmolality, its unit is mosm/l. if the osmolality on both sides of a semipermeable membrane is not equal, water moves toward the side with the higher osmolality. this phenomenon is called osmosis [216] . osmosis of water can be opposed by applying a pressure across the semipermeable membrane in the direction opposite to that of the osmosis. the amount of pressure required to oppose the osmosis is defined to be osmotic pressure. in spite of various solute concentrations are different in extracellular and intracellular fluid, the osmotic pressure remain equal. normal plasma osmotic pressure is 280-310 mosm/l. steady osmotic pressure is the basic guarantee to maintain the fluid balance across the cell membrane. multiple mechanisms in nervous and hormonal system are involved in the regulation of body fluid and electrolyte balance [217] . (1) there exists sensation of thirst in central nervous system, which plays an important role in regulating body water. (2) there is a powerful feedback system for regulating plasma osmolarity and sodium concentration that operates by altering renal excretion of water independently of the rate of solute excretion. a primary effector of this feedback is called antidiuretic hormone (adh) which plays an important role in regulation of renal concentration and dilution to maintain the body fluid homeostasis. (3) reninangiotensin-aldosterone system (raas) is an important regulator of sodium reabsorption and potassium secretion by the renal tubules. human body fluid environment must be suitable ph value for maintaining normal metabolism and physiological function, under normal conditions, human body take in acidic and basic food and drinking water, produce acids and bases during metabolism and eliminate acidic or basic substances by the kidneys and lungs. in the plasma, the normal ph value ranges from 7.35 to 7.45 with an average value at 7.40. the regulation of the acid-base balance is accomplished by the buffer system of the body fluid, the respiration of the lungs and the excretion of the kidney [218] . (1) blood buffer system is composed by a weak acid and its corresponding buffer base, includes bicarbonate buffer system, phosphate buffer system, plasma protein buffer system, hemoglobin and oxygen synthetic hemoglobin buffer system. (2) the role of the lung in acid-base balance is to adjust the concentration of plasma carbonic acid by changing the amount of co 2 , so that the ratio of hco 3 − and h 2 co 3 in the plasma is close to normal, so that the ph is relatively constant. (3) the major role of the kidneys in maintaining acid-base balance is to conserve circulating stores of bicarbonate and to excrete h + . the kidneys maintain ph by increasing urinary excretion of h′ and conserving plasma hco 3 − when the blood is too acidic, or increasing urinary excretion of hco 3 − , and decreasing urinary excretion of h + when the blood is too alkaline. patients with severe hepatitis are prone to develop water retention, with the main manifestation of seroperitoneum (ascites) as well as body weight gain. with the acatharsia of water becoming more serious, oliguria and edema of lower extremities occur. sbp (spontaneous bacterial peritonitis) can also occur, which is manifested with symptoms such as fever and abdominalgia. several factors contribute to ascites include an increase in capillary pressure due to portal hypertension, obstruction of venous and lymph flow through the liver, decrease in colloidal osmotic pressure due to impaired synthesis of albumin by the liver, salt and water retention by the kidney [219] . some theories have been used to explain the increased salt and water retention by the kidney. because of vasodilation or an actual loss of fluid into the peritoneal cavity, the effective blood volume maybe reduced, which may in turn decrease the renal blood flow leading to a lower glomerular filtration rate (gfr) and an activated rennin-angiotensin-aldosterone system (raas). the diagnosis of water retention depends on typical clinical symptoms such as ascites, pleural effusion, and edema of lower extremities, when the body begins to excess water, blood pressure is increased, which leads to many complications such as congestive heart failure and pulmonary edema. hyponatremia is a common complication in severe hepatitis patients, and always incorporate with the retention of water and sodium, but the total sodium can be decreased, normal or even increased, that is dilutional hyponatremia. in laboratory test, serum sodium is below 135.0 mmol/l. the mechanism of hyponatremia probably depends on the following factors: (1) the decreased function of adh inactivation in liver brings adh increase, enhancing the reabsorption of water in renal tubule, which causes the formation of water retention. this is the main cause of dilutional hyponatremia. (2) water retention brings about volume extension, causing the aldosterone secretion decrease, which leads to the sodium egestion increase in urine. (3) some severe hepatitis patients frequently vomit, and can not eat, bringing about a major loss of body fluid and electrolyte. (4) severe hepatitis patients, serum albumin reduces, combining with the factors such as poor appetite, anorexia, fasting or limit sodium, bring about a state of low permeability in the cell, which causes the extracellular na + moving into the cell. (5) iatrogenic factors, exhaust potassium diuretic such as hydrochlorothiazide and furosemide and spironolactone have a strong role in the excretion of sodium, so a large number diuretic is liable to hyponatremia in ascites patients, and in the treatment of cerebral edema, a large infusion of mannitol may cause hyponatremia either [220] . hyponatremia due to the osmotic pressure of extracellular fluid decreased, water moves to the cells, causing cell edema, especially brain edema. so the symptoms of nerve system are the main manifestation in hyponatremia patients [221] . generally dilutional hyponatremia in severe hepatitis patients develops slowly and progressively, and the symptoms are often covered by primary disease symptoms, like weak, feeble, nausea, vomiting, lethargy, significant body weight increase, pale and moist skin and sometimes saliva, tears increase. improper treatment will bring about a sharp serum sodium decrease in the short term, such as serum sodium rapidly decreasing to below 125 mmol/l, acute hyponatremia syndrome comes, the manifestation include convulsions, coma, hypotension, pulse narrowing, tachycardia, oliguria even respiratory arrest and death. if cerebral hernia happens, corresponding nerve location signs will follow. hypokalemia refers to the condition in which the concentration of potassium (k + ) in serum is less than 3.5 mmol/l. it could occur during the whole period in severe hepatitis patients and is more common in early metaphase of disease. hypokalemia can be the result from one or more following medical conditions: (1) insufficient intake of potassium due to the poor appetite or anorexia in the patients with severe hepatitis. (2) frequent vomiting leads to excessive loss of stomach acid, which causes alkalosis and extracellular potassium is transferred into cells. (3) the reduce of effective circulating blood volume can cause to high aldosterone levels and excessive urinary losses of potassium. (4) the decreased function of aldosterone inactivation in liver brings aldosterone increase, enhancing urinary losses of potassium. (5) some medications such as diuretics can also cause urinary losses of potassium. the clinical syndromes of hypokalemia are related to the degree of the shortage of intra/extracellular potassium and disorders of other electrolytes and acid-base, but more depends on how soon it occurs. in the early time it shows muscle weakness, first in limbs, then develops to the torso and respiratory muscle. deficiency of potassium also can lead to weak peristalsis, poor appetite, sick and constipation in mild hypokalemia but abdominal distention and paralytic ileus in severe situation. in cardiac syndromes, it mainly presents atrioventricular block and arrhythmia which including premature ventricular contraction or atrial premature beats, sinus bradycardia, paroxysmal auricular tachycardia or junctional tachycardia, even ventricular fibrillation. in hypokalemia state an increasing shift of potassium from extracellular fluids into cells and an obligate loss of potassium from kidney can cause metabolic alkalosis and abnormal acidic urine. long-term hypokalemia also can lead to hypokalemic nephropathy with proteinuria and cylindruria syndrome. the changes of ecg [222] : in the early stage, flattened t wave and an obvious u wave, st-segment depression can be found, qu interval is widen. in severe situation, a wide pr interval, low voltage, wide qrs interval and ventricular arrhythmia can occur. hyperkalemia refers to the condition in which the concentration of potassium (k + ) in serum is higher than 5.5 mmol/l. it is more common in the middle and late period of severe hepatitis. the mechanism of hyperkalemia: (1) the most usual way lead to hyperkalemia is oliguria or uroschesis which are caused by renal dysfunction among the patients have severe hepatitis with hepatorenal syndrome. (2) metabolic acidosis and na + -k + -atp enzyme inactive lead to a shift of potassium out of cells also contribute to develop hyperkalemia. (3) long-term and high dose potassium-sparing diuretics applied during the treatment lead to hyperkalemia is not rare, and easy to get sudden death in patients. hyperkalemia mainly influences myocardium and skeleton muscle. the most dangerous situation is fatal arrhythmia. when the concentration of k + is higher than 5.5-6.5 mmol/l, there are peaked t waves. when it is over 7-8 mmol/l, pr interval is widen and p wave is flattened even vanish. when it is up to 9-10 mmol/l, t waves and qrs complex can evolve to sinusoidal shape and cardiac arrest. hyperkalemia is a common critical and severe symptom in clinic. when it happens, all of the potassium-sparing diuretics and potassium uptake should be stopped. at meantime, the treatment against the toxicities to myocardium and skeleton muscle should be taken to accelerate a shift into cells and potassium excreting. there also can happen hypocalcemia, which shows neuromuscular excitability, cardiac electrical instability and instable emotion. the concentration of calcium in serum under 2 mmol/l is significant in diagnosis. hypomagnesemia can be found too. it mainly presents similar symptoms as hypocalcemia such as weakness, muscle cramps, increased irritability, tetany and chvostek positive. hypomagnesemia can cause cardiac arrhythmia, when it occurs, the concentration of magnesium is less than 0.75 mmol/l, and it should be urgently treated. severe hepatitis patients prone to acid-base imbalance [223] , mainly to alkalemia. the main types of acid-base imbalance include respiratory alkalosis, metabolic alkalosis, respiratory alkalosis plus metabolic alkalosis, secondly include respiratory alkalosis plus metabolic acidosis, triple acid-base disorders (tabd), metabolic alkalosis plus metabolic acidosis [224] . history and clinical manifestations provides important clues for the judgment of acid-base imbalance. the result of blood gas monitoring is the decisive basis for judging the type of acid-base imbalance. serum electrolyte examination is an important reference. anion gap (ag) has important diagnostic value in determining the type of acidbase imbalance [225] . ag = na + -(cl − + hco 3 − ) is a simple formula for the value between the number of cations and anions in serum. its normal value was 12 ± 4 mmol/l. ag can not only help diagnose "potential" metabolic acidosis and to distinguish different types of metabolic acidosis, can also help determine special types of mixed acidosis, and has its unique role in the judgment of tabd. sometimes the indicators of blood gas analysis are normal, the calculation of ag value become the only evidence of diagnosis of metabolic acidosis. in addition, ag value can be used as reference for correction of acid-base imbalance. in severe hepatitis, the change of ag values can be used as an indicator to estimate the complications and prognosis. clinical observations indicate: ag value significantly increased often suggestive of severe infection, kidney dysfunction or severe bleeding, and the prognosis is poor. respiratory alkalosis refers to arterial paco 2 decrease and ph > 7.45 as well as compensatory decrease of blood hco 3 − . respiratory alkalosis occurred in early stage of severe hepatitis. in severe hepatitis, respiratory alkalosis related to hyperventilation: accumulated ammonia and other vasoactive peptides excited respiratory center, ascites and pleural fluid increase respiratory rate, hypoxemia excited respiratory center. compensatory mechanisms: co 2 reduction, breathing shallow and slow, so that co 2 retention, h 2 co 3 compensatory rise; when last longer, reduce renal row h + , hco 3 − excretion increased, hco 3 − /h 2 co 3 equilibrium at a low level. most patients have the performance of shortness of breath and heart rate increase. can have vertigo, hand, foot and mouth numbness, muscle tremor, hand and foot convulsions. convulsions associated with low calcium. dysfunction of nervous system is related to the damage of brain function and cerebral blood flow decrease. respiratory alkalosis diagnosis relies on the following: (1) ph is normal when fully compensated, increased underdecompensation. (2) paco 2 lower (typically <35 mmhg or 4.67kpa). (3) hco 3 − compensatory decline. (4) ag value may have a slight increase. (5) blood cl − may increase. metabolic alkalosis refers to the type of acid-base imbalance characterized by an increase hco 3 − in extracellular liquid. the inappropriate application of basic drugs, potassium-sparing diuretics, dehydrating agents, hormones can often induce or aggravate metabolic alkalosis. severe gastrointestinal symptoms, anorexia, vomiting or diarrhea are also the reasons for the occurrence of metabolic alkalosis. compensatory mechanisms: when alkaline substances increased in body, buffer system instantly transfer strong base into weak base, increase hco 3 − consumption, h 2 co 3 increased. inhibit respiratory center, decrease pulmonary ventilation, co 2 retention, hco 3 − compensatory increase. renal carbonic anhydrase activity decreased and h + formation and excretion decreased, nahco 3 reabsorption is also reduced, so hco 3 − /h 2 co 3 compensatory restore to 20:1, ph value is normal. patients with mild metabolic alkalosis usually have no obvious symptoms. many disorders can occur in severe metabolic alkalosis. (1) functional changes in the central nervous system, the patient may have irritability, confusion, delirium, consciousness disorders. (2) slow and shallow breathing, hypoxemia. brain tissue is particularly sensitive to hypoxia, thus neurological symptoms first appeared. (3) hypocalcemia and neuromuscular stress increased, the performance of tendon hyperreflexia, face and muscle twitching, and limbs twitching. (4) hypokalemia can cause neuromuscular symptoms and arrhythmias. according to ph value, paco 2, hco 3 − , level of k + and cl − , effective circulating blood volume and performance of primary disease, diagnosis of metabolic alkalosis is no difficult to make. metabolic alkalosis should be divided into two categories based on the urinary level of cl − . (1) chloride positive metabolic alkalosis: supplement sodium chloride can correct the alkalosis. it indicates that the body has cl − deficiency, urinary cl − < 10 mmol/l. (2) chlorine negative metabolic alkalosis: alkalosis can not be corrected by supplement sodium chloride, urinary cl − >20 mmol/l. respiratory alkalosis plus metabolic alkalosis tend to occur on the early stage of severe hepatitis. in most cases, there is no obvious complication, more often metabolic alkalosis happens on the basis of respiratory alkalosis, or the other way around. due to respiratory and metabolic factors are inclined to alkaline change, name as reduce paco 2 and elevated plasma hco 3 − , there is no mutual compensation between them, so it is easy to present as severe decompensation and poor prognosis. main point of diagnosis: (1) ph value of blood increase significantly. (2) paco 2 decrease. (3) hco 3 − increases, the value should be greater than 0.5×(40-paco 2 ) + 2.5. (4) hypokalemia and hypochloremia are common phenomenon. respiratory alkalosis plus metabolic acidosis is relatively rare. metabolic acidosis can be divided into 3 types: (1) value of ag is normal (high chlorine acidosis), commonly seen at long-term diarrhea, combined with renal tubular acidosis and a large amount of physiological saline input in patients or water intoxication. (2) high ag value type (normal blood chlorine acidosis), regularly present in combination of hepatorenal syndrome, lactic acidosis and patients with ketoacidosis. (3) a hybrid type (high ag merged high blood chlorine), mainly in patients with severe diarrhea following lactic acid or ketoacidosis. when respiratory alkalosis plus metabolic acidosis happens, paco 2 and plasma concentration of hco 3 − are higher than scope of compensation to each other. its characteristics as follows: (1) the range of blood ph change is not large, normal, slightly higher or slightly lower. (2) paco 2 reduce to less than 1.5 × hco 3 − + 6 or hco 3 − < 24-(40-paco 2 ) × 0.5-2.5. (3) ag values can be normal or elevated, the latter is more common. if the elevated blood cl − value is equal to the hco 3 − decrease, ag value normal metabolic acidosis type can be diagnosed; the cases that the rising value of ag is equal to the decline of hco 3 − values can be diagnosis as ag increased metabolic acidosis type. on the third occasion, the increase value of ag is equal to the sum of hco 3 − and cl − drop, the diagnosis is mixed metabolic acidosis. for this type of offset mixed acid-base balance disorders, treatment should be moderate, the measure of the metabolic factors correcting should be precede to respiratory factors, avoid paco 2 quickly returning to normal in the process of treatment, which would lead to blood ph drop rapidly and acidosis more worse. to patients with severe hepatitis, the harm of alkalosis is greater than acidosis, thus the blood ph should be kept slight acidic in a normal state. generally, the target of alkali supplement can be arterial blood ph value recovered to 7.20. respiratory alkalosis tabd refers to respiratory alkalosis, metabolic acidosis and metabolic alkalosis three primary imbalances coexist in the same patient, which is one sort of serious acid-base imbalance, mostly develops in the late stages of severe hepatitis, fatality rate is high. respiratory alkalosis tabd characteristics as follows: (1) blood ph value depends on the relative severity of these three primary imbalances, which can be normal, or slightly high generally. (2) reduce paco 2 , its value is less than 1.5xhco 3 − + 6. (3) hco 3 − can raise, normal or lower. (4) value of ag rise significant, and extent of ag raise is greater than the hco 3 − lower. (5) cl − often lower than normal. the occurrence of metabolic alkalosis plus metabolic acidosis in patients with severe hepatitis is not uncommon. usually it is accompanied with existing lactic acidosis or ketoacidosis, and the patient may manifest frequent vomiting. since the causes for raising and lowering hco 3 − coexist, they tend to cancel one another. the ph and hco 3 − concentration can be normal, increased or decreased, depending on the relative severity of the two kinds of imbalances. severe hepatitis can also be accompanied by metabolic acidosis, metabolic acidosis plus respiratory acidosis, tabd of respiratory acidosis, and etc. pure metabolic acidosis refers to arterial blood ph < 7.35 and compensatory decline of paco 2 due to primary decrease of hco 3 − . typical manifestation is known as kussmaul breathing, characterized by deeper and faster breathing, as well as obvious contraction of respiratory muscle, and also ketone-smelled exhaled breath. the patients often flush, companied by increased heart rate and decreased blood pressure. there may be reduced or disappeared tendon reflexes, confusion or stupor. due to respiratory and metabolic factors both towards to acidic changes, there is no respiratory compensation for decrease in hco 3 − , nor renal compensation for increase in paco 2 , hence presenting severe decompensated status. the resulting distinct decrease in ph and vicious circle are the characteristics for metabolic acidosis plus respiratory acidosis. the characteristics for tabd of respiratory acidosis include significantly increased paco 2 , elevated hco 3 − , ag > 16 mmol/l, and significantly decreased cl − . the incidence of the above types of acid-base imbalance is very low in patients with severe hepatitis. once it happens, it should be actively treated with corresponding methods, so that the blood ph quickly restores to the safety range. during therapeutic process against various pure acid-base imbalance, interactions among various treatments need to be taken into consideration, in order to avoiding the possibility that the treatment for one type of acid-base imbalance causes or aggravates another type. in summary, water-electrolyte imbalance and acid-base imbalance have a relatively high morbidity in patients at various stages of severe hepatitis. this often results in deterioration and complication of the disease, evermore, the death of the patient. therefore, the functions of heart, lung, kidney, blood circulation as well as changes of body weight in the patient need to be intently monitored. regular detection of k + , na + , cl − , carbon dioxide combining power (co 2 cp), blood urea nitrogen, creatinine, ph, data about arterial blood gas analysis, and also detailed records of patient's input and output are demanded. during the process of diagnosis and treatment, careful analysis of the history, clinical manifestations and laboratory examination are necessary to achieve correct diagnosis, early prevention and prompt treatment. in normal condition, proper amount fluids can make a lubrication action on organs in peritoneal cavity. but in those patients who have severe hepatitis, especially with cirrhotic portal hypertension, too many fluids over 200 ml can lead to ascites. the ascites can be categorized into uncomplicated ascites and refractory ascites. there is no infection in uncomplicated ascites and won't lead to hepatorenal syndrome, but refractory ascites is in the contrast. the refractory ascites includes diureticresistant and diuretic-intractable ascites. the diuretic-resistant ascites shows no response to diuretic and diuretic-intractable ascites limits the application of diuretic due to the complications induced by diuretic. 100 mg antisterone per day as an initial dose can be given to those patients with moderate ascites, if it goes to no satisfied effect, 100 mg can be added after every 7 days till the maximum dose to 400 mg/d. if the patients show a hyperkalemia or aldosterone antagonist-resistant, nicorol can be combined and with an increasing dose from 40 to 160 mg/d gradually. the patients without edema losing weight should be less than 0.5 kg and those with edema should be less than 1 kg per day to avoid electrolyte disturbance or hepatorenal syndrome during the whole treatment period. diuretics should be withdrawn on the patients with severe hepatic encephalopathy, severe hyponatremia, progressive renal failure or severe muscle spasm. the patients should only take the minimum dose of diuretics to maintain the state after the syndrome controlled, or withdraw when it is necessary. due to the poor outcome and living quality, the median survival time of refractory patient is half year. so liver transplantation can be considered in the patients with refractory ascites induced by cirrhosis, which required more cautious before make a diagnosis. generally, if the patients meet the following conditions when they are receiving 400 mg/days antisterone and 160 mg/days nicorol treatment over 1 week and sodium uptake limited in 90 mmol/days, refractory ascites can be diagnosed. (1) losing weight less than 0.8 kg/days over 4 days (2) sodium uptake is more than elimination (3) grade 2-3 ascites is arisen again after 4-week long treatment (4) hepatic encephalopathy, hepatorenal syndrome and severe electrolyte disorder induced by diuretics are shown up. refractory ascites patients without complications can be treated with abdominocentesis, but it will possibly induce circulation failure, and increases the risk of hepatic coma and hemorrhage. so low rate infusion of albumin with abdominocentesis is combined to avoid circulation failure, meantime diuretics also need to give after abdominocentesis. aldosterone antagonist combined with nicorol is a suitable strategy: the dosage of antisterone is increased from 100 to 400 mg/days and nicorol from 40 to 160 mg/days gradually. the goal of this strategy is to maintain the situation without ascites under the minimum dosage. but when severe hepatic encephalopathy and electrolyte disorder show up, which means serum sodium concentration is less than 120 mmol/l, serum potassium concentration is less than 3.0 mmol/l, nicorol should be withdrawn, if serum potassium concentration is more than 6.0 mmol/l, antisterone should be withdrawn. to those patients who need abdominocentesis repeatedly, transjugular intrahepatic portosystemic shunt (tips) can be considered, but the risk of hepatic encephalopathy will be higher and the outcome is poor. so the patients with severe liver and renal failure, cardiorespiratory function failure or active infection should be in cautious. the mean survival time of refractory ascites patients complicated with hepatorenal syndrome is 3 months, preventive antibiotics combined with albumin is an option for these patients. to those patients who have already showed hepatorenal syndrome, terlipressin combined with albumin could be useful. meantime, abdominocentesis, tips or artificial liver supporting treatment can improve patients' living quality in short term, but long-term outcome won't be good, so liver transplantation should be execute as soon as possible. in general, medicine can improve the symptoms in short term but with poor outcome, liver transplantation is more meaningful. patients with hypovolemic hyponatremia can have a supplement with sodium and decrease the dosage of diuretics, patients with hypervolemic hyponatremia can restrict fluids uptake (less than 1000 ml/d) and combined with vasopressin v2 receptor blocker or antidiuretic hormone receptor blocker. currently, vaptans, tolvaptans, conivaptan and satavaptan have already applied in clinical practice. there was research showed that vaptans could improve 45-82% patients' symptoms significantly after patients took it for 1 week to 1 month, and main side reaction was thirsty. the patients complicated with hepatic encephalopathy should be used vaptans with cautious due to its high risk in dehydration and hypernatremia. meantime, vaptans is metabolized by cyp4a, so rifampin, barbital and phenytoin can decrease its effect, and ketoconazole, clarithromycin can increase its plasma concentration. tolvaptans can give some relief but increase the risk of hemorrhage. satavaptan will decrease patients' survival rate. so the proper treat period and side reactions of these drugs in long-term using need to make clear. potassium uptake should be withdrawn immediately after hyperkalemia occurs, emergency treatment for detoxicating potassium should be taken to protect cardiac. the treatment depends on the plasma concentration of potassium. the treatment for patients with fulminant hepatitis b with cirrhosis complicated with hyperkalemia is to restrict the uptake of potassium, improve the microcirculation, correct renal filtration decrease induced by hepatorenal syndrome and increase the elimination of potassium. there are also some patients have abnormal distribution of potassium due to hypoxia, acidosis, catabolism, and deficiency of energy supplies, which leads to intracellular potassium is transferred into extracellular. the treatment for these patients is to correct hypoxia and acidosis, high glucose, insulin and atp are administered to boost glycogen synthesis to transfer potassium from extracellular to intracellular. peritoneal dialysis and plasmapheresis can be given to the patients with intractable hyperkalemia. hypokalemia can present during the whole period of fulminant hepatitis b, it will occur more often on the early and middle stage. long-term inappetency and abdominal distension lead to insufficient potassium uptake. nausea, vomiting and diarrhea lead to increase of potassium losing. renal filtration rate decreasing and aldosterone increasing lead to potassium elimination increase. complicated with alkalosis and anabolism increase also can cause hypokalemia. the treatment for hypokalemia should focus on comprehensive therapy, correcting alkalosis, increasing potassium supply, improving microcirculation. disturbance of acid-base balance occurs quit often in hepatitis b patients, especially with alkalosis. during the early stage, it can present in pure respiratory alkalosis, also can complicated with metabolic alkalosis. during the middle and late stage, both of above symptoms and metabolic acidosis occurs concurrently. treating idiopathy and correcting hyperventilation is a treatment for respiratory alkalosis. arginine hydrochloride injection is used to treat metabolic alkalosis to avoid secondary metabolic alkalosis. the general regulation is prefer acid to base, till ph value of arterial blood back to 7.2. in prevention of disturbance of acid-base balance, the effective strategy is to correct hypokalemia and hypochloremia, control vomiting. meantime, controlling infection, endotoxemia and upper gastrointestinal hemorrhage are necessary. hepatic encephalopathy (he) due to metabolic disturbance is a complex neuropsychiatric syndrome caused by severe liver dysfunction or disorder and is one of the common complications and causes of death in severe liver diseases. patients with he mainly present with neuronal or mental abnormalities and disturbance of consciousness, even coma and death. the clinical manifestations and the severity of the disease vary because of its complex pathogenesis. hepatic encephalopathy is the result of acute and chronic hepatic failure caused by cirrhosis or various kinds of portosystemic shunt (pss) created. a diagnosis of he can be made after excluding encephalon diseases. the syndrome is caused by metabolic disorders and is potentially reversible. he clinical features differ due to the wide degree and range of neuropsychiatric symptoms that vary from subtle abnormalities detected only by intelligence tests or electrophysiological methods geared for detecting personality changes to abnormal behavior, intellectual impairment, and even different degrees of consciousness disorders. he was previously known as hepatic coma, but that is only one of the worst severe signs of he and does not represent all types of he. in 2003, the world congress of gastroenterology (wcog) suggested that based on the cause he can be divided into three types (a, b, and c) [226, 227] . type a: type a is acute liver failure-related he and the symptoms occur within 2 weeks. in subacute liver failure-related he, the symptoms of he occur within 2-12 weeks with or without predisposing factors. type b: patients with type b he have obvious pss and normal liver histology without associated intrinsic liver disease. these clinical manifestations are similar to those in patients with he and cirrhosis. the pss may be spontaneous or caused by surgical or interventional procedures [228] . common causes of pss include congenital vascular malformation, intrahepatic or extrahepatic portal vein obstruction (including trauma, carcinoid, and bone marrow hyperplastic disease caused by a high coagulation state due to portal vein branch embolization and thrombosis) and generation of portal hypertension by oppression of lymphoma, metastatic tumors, and bile duct carcinoma. type c: type c he is related to chronic liver diseases, with cirrhosis being the most common type, and is generally accompanied by portal hypertension and pss. type c he is mainly caused by liver function failure, rather than by pss. according to the clinical manifestations, duration and characteristics, type c can be divided into three types: episodic he, persistent he, and minimal he [226] . episodic he, related to chronic hepatic disease, is defined as a disturbance of consciousness and cognitive change in a short time and can be alleviated by spontaneous remission or drug treatment in the short term, which cannot be explained by a relevant preexisting mental disorder. episodic he can be divided into three types according to the presence of known risk factors: (1) incentive type: there is a clear history of predisposing factors; (2) spontaneous type: there is no history of predisposing factors. (3) recurrent type: he attacks more than two times within a year. persistent he related to chronic hepatic disease is defined as an occurrence of continuous neural mental abnormality, including cognitive decline, disturbance of consciousness, coma and even death. persistent he can be further divided into three types according to the severity of the disturbance in the patient's self-control and self-discipline: 1, mildest type, namely west haven level 1; 2, severe type: namely west haven level 2-4; and 3, therapeutic resistance type: medication can alleviate he quickly, but withdrawal can aggravate he rapidly. patients with minimal he, with normal clinical manifestations and routine biochemical tests, have mild cognitive and psychomotor deficits detected by neuropsychology and neural physiology tests, and these patients usually have a history of chronic hepatic disease [229] . the prevalence of minimal he in patients with cirrhosis is 30-80%. patients with minimal he with reduced physical and mental ability have gained more and more attention recently because they have a high risk of accidents when engaged in occupations involving mechanical, or driving work. the pathogenesis of he has not been fully elucidated so far, and many theories have been put forward. it is generally believed that he is caused by acute and chronic liver failure and/or pss. when toxic substances absorbed by the intestines cannot be detoxified and cleared by (or through) the liver, they directly enter into the systemic circulation and pass through the blood-brain barrier to reach the brain tissue and cause central nervous system dysfunction. a variety of the risk factors mentioned above can result in he. hyperammonemia is still recognized as one of the most important factors, especially in he related to chronic liver disease, liver cirrhosis and/or pss. according to the ammonia intoxication theory several factors including false neurotransmitters, such as γ-aminobutyric acid/benzodiazepine (gaba/bz) receptor complex, an imbalance in the ratio of branched chain amino acids to aromatic amino acids, brain cell edema, astrocyte dysfunction, mercaptan, short chain fatty acid toxicity and manganese deposition are all involved in the occurrence of he [230] . ammonia intoxication caused by an ammonia metabolism disorder is the most important factor in the pathogenesis of he [231] . ammonia comes mainly from the gut and the generation and absorption of ammonia increase in a serious liver disease when excess ammonia cannot be cleared sufficiently by ornithine cycle due to serious damage to liver parenchyma. when pss occurs, intestinal ammonia directly enters the systemic circulation without liver detoxification, resulting in increased blood ammonia. high levels of blood ammonia can enter the brain through the blood-brain barrier and generate central nervous system toxicity by interfering with cerebral energy metabolism, neurotransmitter and nerve cell membrane ion transport; increasing cerebral edema; and changing gene expression (such as stellate cell glutamate carrier, stellate cell structural protein, glial fibrillary acidic protein, peripheral benzodiazepine receptor and aquaporin-4) and inducing the mitochondrial permeability transition (mpt). the main way of removing ammonia from the brain is through urea cycle. during glutamine synthesis, glutamic acid is formed from ammonia and α-ketoglutaric acid and the glutamic acid combines with ammonia to generate glutamine. this process requires atp and consumes a large amount of α-ketoglutaric acid, which interferes with the brain energy metabolism and causes an energy supply shortage in brain cells. glutamate is an important excitatory neurotransmitter in the brain, and lack of glutamate increases inhibition in the brain. glutamine synthetase is present in astrocytes, where glutamic acid is detoxified to glutamine. glutamine is a strong intracellular osmotic agent, and increases in glutamine can lead to brain cell swelling. reports have identified a strong correlation between the content of glutamine in cerebrospinal fluid (csf) and the degree of he [232] . during he, excess ammonia under the effect of glutamine synthetase, not only reduces the formation of active glutamate but also consumes a lot of energy, leading to the accumulation of glutamine, which increases intracellular osmotic pressure and causes brain cell swelling. swollen astrocytes with impaired function further affect ammonia metabolism, reduce the ability of neurons to efficiently uptake or release extracellular ions and neurotransmitters, and stimulate glial cell synthesis of neurosteroids by upregulating their expression of the peripheral-type bz receptor (translocator protein, 18 kda). neurosteroid is an endogenous bz that can enhance gaba nerve tension and cause symptoms in patients with he [233] (fig. 2.2 shown that the metabolic rate of cerebral ammonia in he patients is increased. increased levels of blood ammonia enter the brain through the blood-brain barrier. brain dysfunction also occurs even if blood ammonia levels appear normal; this partially explains the occurrence of he in the case of normal blood ammonia and invalidates he treatment by simply reducing blood ammonia. in addition, increasing evidence suggests a synergistic effect between blood ammonia and its metabolic disorders with systemic inflammation, nerve steroids, oxidative stress, nitrification stress, manganese poisoning, and gaba/bz [233] . the main inhibitory neurotransmitter in the mammalian brain is gaba. plasma gaba is derived from the conversion of glutamic acid by glutamate decarboxylase in intestinal bacterial. notably, gaba has dual role. on one hand, during liver function failure and pss, the removal of gaba in liver is significantly decreased; on the other hand, gaba can directly enter the systemic circulation bypassing the liver, resulting in increased concentration of gaba in blood. the concentration of gaba in csf and brain tissue increases as more gaba crosses the abnormal blood-brain barrier. in addition, endogenous bz was found in the blood and csf, and the gaba receptor on the membrane surface of the brain's postsynaptic neurons increased significantly in some patients with he and in animal models. this receptor not only combines with gaba but also binds to barbiturates (barb) and bz on different parts of the receptor surface; thus, it has been named the gaba/bz complex receptor or the super receptor complex. when liver function is severely impaired, the binding affinity of this complex receptor to its three ligands is also increased. binding of gaba, barb, or bz with the complex receptor can promote entry of chloride ions from neuronal membrane ion channels into the cytoplasm of postsynaptic neurons, causing membrane hyperpolarization and nerve conduction inhibition. he symptoms were relieved in about 30% of patients treated with a gaba receptor antagonist or bz receptor antagonist, and gaba/bz and ammonia were reported to act synergistically in he. recently, some studies focused on peripheral type bz receptors, which are different from central gaba [234, 235] . some questions, including the source of endogenous bz, and the correlation between the increased degree of gaba or bz and the disease, remain to be answered. therefore, therapy targeted at reducing the blood ammonia concentration in patients with he and significantly reducing the increased gaba nerve tension seems reasonable [236] , but may not be completely effective. treatment effects of reducing ammonia vary, because of the different levels of ammonia in he patients that can be produced by the interaction between various known or unknown factors and the different effects of bz receptor antagonists. this theory is related to the metabolism of aromatic amino acids (aaa), the precursors of true neurotransmitters, including norepinephrine and dopamine. due to the reduction in the liver's detoxification function or formation of pss, the amines (phenylethylamine and tyramine) produced in the intestine cannot be cleared completely, resulting in elevated concentrations of these amines in the systemic circulation and increased levels in the brain through the blood-brain barrier. under the effect of β-hydroxylase, phenethanolamine and β-hydroxytyramine (β-dopamine) are generated from phenylethylamine and tyramine, respectively and are similar to norepinephrine and dopamine in chemical structure. these amines can be taken up, stored and released by adrenergic neurons in the brainstem reticular structure. phenethanolamine and β-hydroxytyramine are called false neurotransmitters because of their low physiological effects on the postsynaptic membrane, which is about 1/10 of norepinephrine. when these false neurotransmitters accumulate in the nerve synapse, they can outcompete or replace normal neurotransmitters, resulting in a disorder of nerve conduction. it was reported that plasma aaa (such as phenylalanine, tyrosine, and tryptophan) increased and branched-chain amino acids (bcaa, such as valine, leucine, isoleucine) decreased in patients with decompensated liver cirrhosis, leading to an imbalance of amino acid metabolism. aaa are decomposed and metabolized in the liver, and liver failure decreases aaa decomposition resulting in an elevated concentration of aaa in the plasma. insulin can promote bcaas entering muscle, which is then broken down and metabolized in the skeletal muscle instead of the liver. insulin inactivation is decreased in patients with liver failure, promoting a large number of bcaas entering the muscle tissue and decreasing the concentration of bcaas in plasma. finally, the bcaa/aaa ratio is reduced from a normal 3-3.5:1 to 1:1 or lower. the above process reduces the bcaa concentration, but increases the aaa concentration, leading to an increase in synthesis of false neurotransmitters and reduction of the normal neurotransmitter [237] [238] [239] . the epidemiological data suggests that manganese poisoning and he extrapyramidal have common clinical symptoms. the liver is an important organ for manganese excretion. the concentration of blood manganese can be increased when liver function is affected, during pss, or when excretion of bile is reduced. manganese content in plasma was sharply increased in more than 80% of patients with acute hepatitis and liver cirrhosis and the density of globus pallidus increased in the brain basal ganglia of he patients (partially 2-7 times higher by mri). based on histological results, the above changes were caused by manganese deposition, which disappears after liver transplantation. it has been suggested that manganese deposition may cause dopamine dysfunction. deposition of manganese not only cause direct brain injury, it can influence the function of 5-hydroxytryptamine (5-ht), norepinephrine and gaba neurotransmitters; impair astrocyte function; and have a synergistic effect with ammonia. however, there is no reliable correlation between the concentration of serum manganese and he severity, which may be due to the chronic deposition of manganese [240] . the characteristic change in mri imaging as the deposition of manganese remains to be verified. the effectiveness of manganese removal to improve the symptoms and neurological signs of patients with he needs further validation. the synergistic toxic effects between toxins (ammonia and mercaptan) and short chain fatty acids [241] , the 5-ht hypothesis, the effect of helicobacter pylori urease, opioids, endotoxin, tumor necrosis factor, melatonin, and hepatitis b virus termed additional theories of he syndrome. this theory also suggests the same hypothesis mentioned in the above theories. due to the extensive amount of liver cell damage caused by acute liver failure in type a he, the residual liver cells cannot effectively remove toxins leading to central nervous system dysfunction. type a he, known as non-ammonia encephalopathy, is endogenous he without clear causative agents. simple type b he is rare in mainland china; the liver can clear limited metabolic toxins in patients with chronic liver failure or pss, but once these toxins exceed the compensatory capacity of the liver, type c he occurs. the occurrence of type c he is largely related to the following risk factors, which are the most important factors in the prevention and treatment of he. patients with chronic liver failure or pss are less tolerant to the protein found in food, especially animal protein. a large amount of ammonia and aaa are produced by the decomposition of intestinal bacteria, which can induce he. oral ammonium salts, urea, and methionine can induce he by increasing the absorption of nitrogenous substances and elevating blood ammonia. intestinal production of ammonia can be increased by hemorrhage in the intestine (100 ml of blood contains 15-20 g protein). at the same time, because of the lack of isoleucine in the blood, after digestion and absorption of a hemorrhage, extra blood leucine and valine increase bcaa decomposition by enhancing the activity of bcaa dehydrogenase, thereby exacerbating the imbalance in the bcaa/aaa ratio. loss of blood volume, cerebral ischemia and hypoxia also increase the sensitivity of the central nervous system to ammonia and other toxic substances [230] . infections such as spontaneous peritonitis, pneumonia, and urinary tract infection can increase tissue decomposition and production of ammonia. secondary sepsis or sirs induce he through tnf-α, il-1, il-6 and other inflammatory factors, exacerbates oxidative stress, and increases the blood-brain barrier permeability of ammonia and other toxic molecules to liver and brain [242] . studies have shown that sirs is directly related to the deterioration of he in patients with liver cirrhosis, and its extent and mortality increase with the deterioration of sirs [243] . similarly, sirs is a common factor in triggering chronic liver failure characterized by he and renal failure. in a study of patients with liver cirrhosis, artificially-induced hyperammonemia by oral administration of glutamine may have worsened the results of psycho-mental testing in 10 cases of sepsis patients; while brain toxicity was not obvious after the inflammation was relieved, the observation of decreased cytokine levels indicated that infection and induced inflammatory mediators enhanced brain toxicity of hyperammonemia. accordingly, some researchers suggested that sirs could be an independent pathogenesis of he rather than a risk factor [243] . hyponatremia can affect the intracellular osmotic pressure and lead to brain edema, which induces he. hypokalemia is often associated with metabolic alkalosis [244] . mass use of diuretics or extraction of ascites can also cause alkalosis. ammonia is easily absorbed by the intestinal tract or through the blood-brain barrier inducing he [245] . a variety of reasons can cause pre-renal azotemia such as hypovolemia, anorexia, diarrhea, limiting the amount of liquid, mass use of diuretics, or extraction of ascites. hepatorenal syndrome or other causes can result in renal azotemia. pre-renal azotemia and renal azotemia caused by hepatorenal syndrome or other causes can increase the concentration of ammonia in the blood. several other predisposing factors can contribute to he such as constipation, hypoglycemia, the use of sedatives and proton pump inhibitors, and epilepsy. after the occurrence of constipation and intestinal obstruction, the patient's intestinal mucosa is exposed to ammonia longer thus increasing the absorption of ammonia. hypoglycemia can reduce brain deamination. the binding of sedatives, hypnotics and the brain gaba/bz receptor produce an inhibitory effect on the brain. it was reported that proton pump inhibitors increase the risk of he in patients with cirrhosis in a population study [246] . another study also suggested that epilepsy was associated with an increased risk of he in patients with cirrhosis [247] . patients with type a he often have no obvious anatomical abnormalities in their brains, but 38-50% of patients have brain edema, which may be a secondary change of the disease. hypertrophy and hyperplasia of the original plasma astrocytes in gray matter and subcortical tissue can be found in patients with type c he. patients with longer course of the disease will exhibit brain atrophy (especially in patients with alcoholic cirrhosis) of different degrees, thinning of the cerebral cortex, loss of neurons and nerve fibers, and deep cortical sheet necrosis, even the cerebellum and the base may also be involved. the majority of patients with cirrhosis may have different degrees of he at some stage in the course of the disease. the incidence of he in patients with liver cirrhosis is at least 10-50% in mainland china while the incidence of post-tips (transjugular intrahepatic portosystemic shunt) he is 25-45%. if patients with chronic liver disease have he, the outcome is poor; the one year survival rate is lower than 50% and the 3 year survival rate is less than 25% [248, 249] . the incidence of mild he is 39.9% in mainland china in patients with liver cirrhosis, 24.8% in patients with child-pugh a, 39.4% in patients with child-pugh b, and 56.1% in patients with child-pugh c. the incidence of mild he is not significantly associated with cirrhosis; however, with the increased degree of decompensated liver cirrhosis, the incidence of mild he increase. several studies have found that the incidence of depression and anxiety in patients also increased, with the increase of liver function damage, the incidence also increased, and the outcome is poor [250, 251] . the clinical manifestations of he vary, because of the difference in the nature of underlying disease, the degree of liver cell damage, the speed of injury and incentives. they are not specific to he compared with other metabolic encephalopathies. early pathological changes of he are mild he. the neuropsychological and intelligence tests detect mild form of he, which exhibit no clear clinical symptoms and often develop symptomatic he. the main clinical manifestations seen in acute liver failure induced by type a he are rapid-onset jaundice, bleeding, decrease in prothrombin, and eventually, change in mental status that can start as mild confusion but progress to coma and even death. type c he is characterized by chronic recurrent episodes of changes in personality and behavior [252] , stupor and coma, which is often accompanied by increased muscle tone, hyperreflexia, hepatic flap, ankle clonus or positive babinski sign and nervous system abnormalities. most patients in the early stages relapse, but then their symptoms become persistent. he often has a variety of risk factors such as consuming a high-protein diet or discontinuing treatment of he. patients with type c he not only have the clinical manifestations of encephalopathy, they also have chronic liver injury, cirrhosis and other clinical manifestations [226] . observation of encephalopathy dynamic changes is beneficial for early diagnosis, treatment and analysis of treatment efficacy. he can be graded and quantified according to the degree of disturbance of consciousness, nervous system performance and eeg changes. according to the 2009 edition of the "consensus on the diagnosis and treatment of hepatic encephalopathy" in china, he is divided into 0-4 periods, but each period can be overlapping or distinct but each period can be overlapping (table 2 .4). at present, scholars have stressed that the occurrence of he is a continuous progression of the disease and should be viewed as a continuum of a wide range of neuropsychiatric abnormalities, rather than isolated clinical stages. according to the traditional west haven criteria diagnosing grade 1 he is based on clinical signs and physician assessments, resulting in diagnostic criteria confusion [253] . (fig. 2.3) . covert he is diagnosed by a variety of neuropsychological and intelligence tests; the evaluation of overt he widely uses the modified west haven semi-quantitative grading table for the analysis of patients with neuropsychiatric state (table 2 .5), the glasgow coma scale for the analysis of the degree of consciousness of patients, and the simple he severity rating scale for the disease in addition to abnormal liver function (such as increased bilirubin, enzyme bile separation, and decreased prothrombin activity) commonly used auxiliary examinations for he diagnosis include: determination of ammonia, amino acid analysis of plasma and csf, psychological intelligence test, neurophysiological test, electroencephalogram and neuroimaging. the normal level of fasting venous ammonia is 6-35 μg/l (serum) or 47-65 μg/l (whole blood) and arterial ammonia concentration may be 0.5-2 times that of venous ammonia. generally, the determination of arterial ammonia is common in clinical practice than intravenous determination; however, if venous blood has been transported on ice box and detected in a timely manner after proper collection, the result is expected to be as effective as arterial detection. ammonia levels are increased in type b and c he, but are normal in type a he. thus, he cannot be ruled out based on having a normal ammonia level. the increased level of ammonia was reported to be associated with the degree of type a he, but significant overlaps in different clinical stages of patients were also found [255, 256] . therefore, ammonia detection is not routinely recommended in the diagnosis of he. notably, we need to rule out falsely elevated levels of ammonia caused by lab error, renal failure, complete parenteral nutrition, gastrointestinal bleeding, the use of steroid hormones and other extrahepatic factors. the fischer ratio (bcaa/aaa) is used as a marker of he, the plasma bcaa levels decrease while aaa levels increase; resulting bcaa/aaa: < 1 (normal >3). it was reported that the concentration of glutamate in csf in he patients is increased compared to healthy controls. the concentrations of phenylalanine and tyramine in csf were also significantly increased, and the level of phenylalanine was closely related to the degree of he [257] . recently, it was reported that h-nuclear magnetic resonance spectroscopy could select biomarkers for these diseases, such as in patients with he [258] , but this is not commonly used clinically because the the characteristic manifestations of cognitive dysfunction in patients with covert he are lack of attention, working memory problems, and deficits in executive function. therefore, various intelligence tests are used to assess the subtle changes in a patient's cognitive or precise movement, which is important for the diagnosis of covert he, but not for overt he. [259] . at present, computer-aided psychological tests such as information and communication technology (ict), cognitive drug research test (cdr), and critical flicker fusion test (cff) are not influenced by the factors mentioned above and easily operated, which can be used as an alternative choice for pen and paper tests. ict with sensitivity 87% and specificity 77% was one of the most commonly used tests to diagnose minimal he. cff was originally used to detect the critical flicker frequency of alert patients, reflecting brain conduction dysfunction. based on a spanish study of 217 cases, including patients with cirrhosis and healthy controls, cff was a sensitive method to diagnose covert he with sensitive, simple and reliable advantages [260, 261] . because the diagnosis of minimal he has just started, the related diagnostic value still needs to be further evaluated. an abnormal eeg is often observed before biochemical abnormalities or mental abnormalities [262] . the main abnormalities by eeg are slowed rhythm, sporadic or universal θ wave (4-7 times/s) and the occasional α wave (1-3 times/s). with the deepening of consciousness, symmetrical δ-wave and three-phase waves appear on both sides simultaneously. this change usually occurs on both sides of the forehead and the top, gradually moving backwards. although these eeg changes are not specific to he and can appear in uremic encephalopathy and other metabolic encephalopathy, the severity of changes have a good correlation with clinical stages of he. computer analysis of eeg frequency distribution, such as artificial neural network-expert system (aness) and short epoch dominant activity cluster analysis (sedaca), is more objective and valuable in diagnosing minimal he than conventional eeg [263] . there are many kinds of evoked potential tests, including visual evoked potential (vep), brainstem auditory evoked potential (baep), somatosensory evoked potential (ssep) and endogenous event evoked potential (event-related potentials, erps) p300, of which the p300 is the most sensitive test for the diagnosis of he. compared with intelligence tests, neurophysiologic tests, independent of age and education background, are more objective. however, they are only used in clinical studies and are limited by equipment, and professional operation. based on cerebral ct and mri, brain edema can be found in patients with type a he while brain atrophy in the frontal cortex, and the t1-weighted signal enhancement in the globus pallidus can also be found, which may be associated with manganese deposition. detected by h-magnetic resonance spectroscopy (h-mrs), the metabolic changes of he patients in the brain include increased levels of glutamate and glutamine, and decreased levels of inositol, taurine and choline [263] . using fluid attenuation inversion recovery (flair) and diffusion weighted imaging (dwi) techniques, diffuse t2-weighted signal enhancement is found in the hemisphere white matter and corticospinal tracts, which may be associated with cerebral ischemia. however, the sensitivity and specificity of the above-mentioned imaging abnormalities remain unknown, and the correlation with he staging is not clear. therefore, the main significance of cranial nerve imaging is to exclude cerebrovascular accident, intracranial tumors and other diseases, rather than diagnose he. there is no gold standard diagnostic criteria of he, and diagnosis is mainly based on the exclusion of other diseases. but one should consider the following five factors [232]: several forms of hepatic diseases may lead to different kinds of he. type a he is caused by acute hepatic failure, but without chronic hepatic disease. type b is caused by pss, but without any history of hepatic diseases. type c is caused by serious hepatic diseases and/or widespread pss, such as cirrhosis, liver cancer, post-tips and so on. psychiatric symptoms can be found such as change of mood and personality, dementia, behavior disorder and disorientation. drowsiness alternating with excitability, hypermyotonia, asterixis, ankle clonus, insanity and coma are physical signs could be present in progressed patients. some patients may lack related physical signs and psychiatric symptoms, but have deficits in ability of learning, understanding, concentration, and quick verbal response. upper gastrointestinal hemorrhage, ascites tapping, excessive diuresis, high protein diet, medicine (such as sedatives) and infection could lead to he. previous he symptoms could be helpful for the diagnosis. type a usually does not have any risk factors. metabolic encephalopathy includes ketoacidosis, hypoglycemia, uremia, pulmonary encephalopathy, serious electrolyte disturbances and toxic encephalopathy. nervous system diseases include intracranial hemorrhage, infection or tumors, mental diseases and excessive use of sedatives [264] . but one should also watch out for the coexistence of he in these situations. an overt he should be considered if (1), (3), (4), and (5) coexist; and covert he is based on (2), (3), (4), and (5) [228] . then, based on the degree of neuropsychiatric symptoms, determine the stage of he, or for he classification refer to the west have semi-quantitative classification table or ishen scores. the flow chart of diagnosis is shown in fig. 2.4. he is a complex metabolic disorder caused by many factors and comprehensive measures should be taken to cure it from different aspects. according to the clinical type, inducements and the severity of the disease, different plans of treatment should be designed for he. at present, the treatment of overt he generally includes the following aspects: (1) supportive treatment; (2) identification of possible concurrent encephalopathy and removal of other precipitants; (3) cause of treatment; and (4) empirical treatment (fig. 2.5 ). the point of nutritional therapy is to promote anabolism, inhibit catabolism, and maintain a positive nitrogen balance, rather than simply limiting protein intake. to reduce the source of ammonia, it has been suggested that patients with he should limit protein intake. in critically ill patients, it has been suggested that they should stop all protein intake and, after the disease improves, gradually increase protein intake to the maximum clinical tolerance. these recommendations are now being questioned because most cirrhotic patients are malnourished and all long-term protein-restricted diets increase the severity of malnutrition. in addition, a negative nitrogen balance increases mobilization of skeletal muscle, resulting in a reduction in ammonia metabolism that may increase blood ammonia levels. recent studies have shown that normal ingestion of protein 1.2 g/(kg • d) can also improve the health-related quality of life (hrqol), especially in mhe [265] ; and have no adverse effects on the recovery of blood ammonia and he fig. 2.4 the flow chart of diagnosis of he compared with the restricted protein intake. according to the guidelines of the european society of enteral nutrition in 2009, the intake of protein should be guided by the following principles: patients with acute phase he on the first day should be put on a prohibited protein diet and given glucose to ensure energy supply and those who cannot eat food may be fed through a nasogastric tube without short-term fasting; patients with chronic he do not need to fast and their intake of protein should be 1-1.5 g/(kg • d); oral or intravenous use of bcaa and essential amino acid preparations can be administered to adjust the balance of aaa/bcaa, promote the balance of nitrogen, and also reduce the risk of he recurrence [266] ; probiotics and prebiotics can enhance the body's tolerance to protein; plant protein is superior to animal protein because it contains methionine, has less aaa, and more bcaa, but it also contains cellulose, which is conducive to maintain the normal flora in the colon and acidize the intestinal tract, shortening the transit time of the colon and reducing absorption of ammonia. the above points need further verification. additional supportive treatments include: maintaining adequate hydration, electrolytes and acid-base balance; ensuring an energy supply of 30-35 kal/(kg • d), which should be composed of 50-60% sugar, 20-30% protein, and 10-20% fat; administering appropriate vitamins and trace elements; treating for hypokalemia, hyperkalemia, hyponatremia, hypocalcemia, hypomagnesemia and metabolic alkalosis as needed; strengthening the basis of treatment with the appropriate infusion of fresh plasma or albumin, increased plasma colloid osmotic pressure; treating for hypoxemia and cerebral edema; and preventing and treating any bleeding and bacterial infection. type c he has a variety of precipitents. actively finding and eliminating triggers can effectively prevent the development of he, such as esophageal variceal bleeding that can develop into he. active hemostasis, anemia correction, and removal of intestinal blood are also conducive to controlling he. in addition, active control of infection, correction of water and electrolyte imbalance, elimination of constipation and improving renal function are essential to control he. anesthetics, painkillers, sedatives, sleeping pills, and other drugs should be strictly controlled. patients with mania or convulsions can reduce the use of diazepam and scopolamine, and the frequency of administration can also be reduced for promethazine, chlorpheniramine, and other antihistamines. toxic substances causing he mainly come from the intestine. thus, in order to prevent and control he, it is very important to clean the intestinal tract to reduce the generation and absorption of ammonia and other toxic substances. saline or weak acidic solution enemas (such as a dilute acetic acid solution), or oral or nasal feeding of 25% magnesium sulfate (30-60 ml) can be used to clear intestinal hemorrhage, intestinal impaction, and other toxic substances. an enema composed of non-absorbable lactulose (300-500 ml) plus water (500 ml) is also useful, especially when applied for type b he. a recent clinical trial suggested that polyethylene glycol was more effective than the current standard first-line therapy in treating these patients [267] . another two studies showed that polyethylene glycol was more effective than the standard lactulose therapy in treating patients with acute he by cirrhosis [268, 269] . other available drugs include pear liquors, mannitol, rhubarb, and so on, but excessive use of these substances may lead to dehydration and aggravate he. non-absorbable disaccharides include lactulose and lactitol. lactulose, a kind of synthetic ketone disaccharide, cannot be broken down in the stomach and small intestine due to a lack of enzymes that can break down galactose in the digestive tract. after entering the colon, lactulose can be broken down into acetic acid and lactic acid with the help of gut bacteria, leading to a reduction in the colonic ph and inhibition of the absorption of ammonia in the intestine. these non-absorbent disaccharides are decomposed into organic particles in the intestinal tract, which can increase the osmotic pressure of the intestine, and their acidic products stimulate the intestinal wall and can slightly promote intestinal excretion. these non-absorbent disaccharides, acting as prebiotics in the intestine, can inhibit the growth of bacteria, which can produce ammonia and urea, finally reducing the production of ammonia and reversing low-grade cerebral edema when combined with rifaximin [270] . however, probiotics can benefit patients in the long-term [271] . oral or nasal feeding (15-30 ml, 2 or 3 times daily) was recommended to adjust the daily defecation appropriately, about 2-3 times daily. main adverse reactions include abdominal discomfort, abdominal distension, abdominal pain, loss of appetite, nausea, vomiting, and diarrhea. lactulose can even be used in patients with diabetes or lactose intolerance when the purity of non-absorbable disaccharide was high (≥ 98%), but is not used in patients with intestinal obstruction. numerous randomized controlled studies showed that lactulose or lactitol can significantly alleviate overt he and improve the patient's cognitive function and quality of life [272, 273] . lactulose is still the first-line therapy of anti-he, although its effect on improving the survival rate of patients is uncertain. antimicrobial agents can be used as a substitute for non-absorbable disaccharides in treating acute and chronic he. in the past, oral aminoglycoside antibiotics, such as neomycin, which are rarely orally ingested, were used to inhibit the overgrowth of bacteria in the intestine. however, recent randomized placebo-controlled studies have shown that neomycin may not benefit patients with he compared with placebo-treated patients and that long-term use of neomycin may lead to increased ear and renal toxicity risk and impair the function of small intestinal mucosa [274] . metronidazole can inhibit anaerobic bacteria in the intestine and alleviate he, but long-term use may lead to disruption in the intestinal flora, gastrointestinal discomfort, or neurotoxicity. rifaximin, a derivative of rifamycin with a broad-spectrum, has a potent inhibitory effect on intestinal bacterial growth, is a minimally-absorbed oral antibiotic and only plays a role in the gastrointestinal part. administration of rifaximin (550 mg, twice a day) can significantly prevent the occurrence of he compared with placebo-treated patients [275] [276] [277] ; rifaximin was equivalent to or better than lactulose and neomycin in treating patients with chronic he [278] . a study indicated that rifaximin-α in combination with lactulose was a cost-effective therapy for patients who had experienced at least two prior overt he episodes [270] , and this therapy could also improve the driving ability of patients with covert he without toxicity to the auditory nerve and renal function [274, 277] . thus, rifaximin has been recommended by the us food and drug administration (fda) for the prevention of recurrent he. the efficacy of rifaximin and relation between longterm use of rifaximin and intestinal flora in the treatment of he needs to be further investigated. however, a recent study in mice showed that rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression in mhe [277] . the study may provide a new opportunity to study intestinal flora in the treatment of he. microecologics with bifidobacterium and lactobacillus can regulate intestinal flora structure to inhibit the growth of bacteria that produce ammonia and urease. in combination with prebiotics, microecologics can reduce the production and absorption of intestinal ammonia and other toxic substances [279, 280] . in a recent openlabel study, 190 patients with cirrhosis were randomized to three groups and treated with lactulose (30-60 ml daily), probiotic capsules, or with both drugs. after a month of treatment, patients with he showed better results in the neuropsychological test, p300 auditory evoked potentials, and blood ammonia. however, there was no difference in the therapeutic effect among the three groups [281, 282] . clinicians commonly use sodium glutamate, potassium glutamate, arginine hydrochloride and potassium magnesium aspartate, but the exact efficacy is highly controversial at present and effective drug reduced ammonia is described below. (a) l-ornithine-l-aspartate lola, a dipeptide, can lower blood ammonia by promoting ammonia consumption and the synthesis of urea, glutamic acid and glutamine in brain, liver and kidney [283, 284] . ornithine, a substrate of the ornithine urea cycle, can increase activity of carbamyl phosphate synthetase and ornithine carbamyl transferase, and promote urea synthesis. n-methyl-d-aspartate (nmda) is a substrate of glutamine synthesis, and the conversion of glutamic acid to glutamine in the body can remove blood ammonia [285] . nmda is also involved in nucleic acid synthesis in liver cells and indirectly improves the metabolism of the krebs cycle process in liver cells to facilitate the repair of liver cells. clinical studies show that, compared with a placebo group, 20 g/days lola intravenously could noticeably reduce fasting blood ammonia (fnh3), postprandial blood ammonia, and improve the mental status of patients with he [286] . patients with oral lola also had improved he examination results for the digital connection test, the flapping tremor, and eeg results [287] . in addition, glycerol phenylbutyrate (gpb) can safely reduce the incidence of he as well as ammonia in patients with cirrhosis and he. the results showed that gpb had therapeutic potential in this population [288] . zinc is an important cofactor in urea cycle enzyme catalysis. a study in he patients showed that serum zinc concentration is reduced, and showed a negative correlation with the blood ammonia concentration; the serum ammonia level is much lower after zinc supplementation in patients, and he can be improved in some patients. a new study suggests that antioxidant and zinc supplementation can improve mhe in patients with liver cirrhosis [289] . oral zinc preparation can also reduce absorption of divalent cations such as manganese in the intestine; however, it has not been determined if zinc has a positive therapeutic effect on he. (c) sodium benzoate sodium benzoate can lower the blood ammonia concentration by activating the urea cycle for ammonia detoxification and promoting urinary ammonia. randomized controlled studies showed that sodium benzoate had the same efficacy as lactulose in treating patients with he. the recommended sodium benzoate dose is 5 g twice a day; nevertheless, few patients can tolerate this dose because of its high gastrointestinal side effects [232] . a recent study showed that tranilast could protect patients from thioacetamide-induced acute liver injury and alleviate he [290] . endogenous bz analogues combine with the inhibitory neurotransmitter gaba receptor to depress the action on the cns, and is one of the occurring hallmarks of he pathogenesis. a large-scale clinical study on 560 he cases showed that the improvement rate in brain function in treatment and control groups were 15% and 3%, respectively [291] . the study showed that treatment of he with receptor antagonists such as fluorine marcie is feasible. a meta-analysis which included 13 casecontrol studies of 805 patients show that fluorine marcie can noticeably improve he, but didn't show any long-term benefits or improve patient survival rate. so fluorine marcie should only be considered for he patients who had used bz. although the reduction of dopamine neurotransmitter activity is also one of the pathogenesis, the application of bromocriptine, levodopa, has been unable to bring more benefits besides partly improving symptoms of patients. oral or intravenous infusion with a bcaa-based amino acid mixture can theoretically correct an imbalance in amino acid metabolism and control false neurotransmitter formation in the brain [292] . a meta-analysis which included five studies showed that intravenous bcaa did not reduce the mortality rate of he. three studies with bcaa did not reduce the mortality rate of he; however, two larger studies (randomized controlled study about patients with liver cirrhosis in 174 cases and 622 cases, respectively) show that the application of bcaa not only reduced the occurrence of he and liver failure, but also improved the nutritional status, liver function and survival rate in patients. another study showed that bcaa could stimulate liver cell regeneration thus reducing the occurrence of liver failure. supplementation with a bcaa-rich amino acid mixture showed improved restoration of the patients' positive nitrogen balance, and increased the patient's susceptibility to protein food, improving cerebral perfusion [293] . considerable progress has been made to understand treatment of he, studies of basic and clinical research are underway using newly discovered treatment strategies, such as toll-like receptor 4 antagonists (with the ability to reduce systemic inflammation and oxidative stress) as well as non-steroidal anti-inflammatory drugs (ibuprofen) [286, 294] , nmda antagonists, anticholinesterase. however, research using gene therapy should not be ignored [295] . after tips, lola can significantly reduce the increase of venous ammonia concentration in patients with he [296] . and the positive dietary intervention can significantly reduce the incidence of he [297] . for patients with refractory he, embolization of pss is a safe and effective treatment strategy [298] . improving liver function antiviral treatment with nucleos(t)ide analogues can reduce or eliminate liver inflammation and necrosis, promote the regeneration of liver cells, and help restore the functions of hepatic metabolism and detoxification in chronic liver failure caused by hepatitis b virus. artificial liver support systems can be divided into three types including nonbiological type, biological type and mixed type. the non-biological liver support system is the most widely used type, and consists of hemodialysis, hemofiltration, plasma exchange, blood perfusion, plasma adsorption, and the molecular adsorption recirculation system (mars) [299] . the artificial liver support system can replace the partial function of the liver, remove the poison accumulated in the body, create conditions that allow for the regeneration of liver cells and provide enough time to wait for liver transplantation for patients with he. an artificial liver support system can be used to treat acute and chronic he, but patients with overt stage 2 he should be especially careful with plasma exchange. liver transplantation remains the only promising therapy for patients with an acute liver failure or endstage liver disease. liver transplantation is an effective means for all kinds of persistent and severe he; however, in patients with he there is a significant increase in mortality among patients awaiting liver transplantation [300] . recently, it has been reported that cognitive function was not fully recovered after liver transplantation in some patients with severe he [301] . the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. theoretically, for patients with serious pss, interventional therapy, surgery or permanently/temporarily and partially/totally blocking the pss can improve the patient's symptoms. the use of this therapy should be carefully weighed because it can increase the risk of gastrointestinal bleeding in case of portal hypertension. covert he has gained an increasing amount of attention in recent years. patients with covert he do not exhibit obvious signs and symptoms; however, their quality of life is reduced because of reduced operational ability or sleep disorders. without treatment, covert he will progress to overt he over time. the population with high risk should be examined and treated early, especially those engaged in potentially dangerous occupations. the following solutions can be referred to: (a) adjusting dietary structure (vegetable protein is the main intake); (b) oral administration of lactulose (15-30 ml, 2-3 times daily); (c) oral administration of rifaximin (550 mg, twice a day); (d) oral administration of lola (6 g, 3 times a day); (e) oral administration of baaa; and (f) oral administration of probiotic preparations [271, 275, 289 ]. although medical technology has made great progress and the research into he is also increasing in recent years, the pathogenesis of he is still unclear. due to a lack of specific methods, combination treatment is still the main therapy for he. it is generally believed that the onset of he may be a result of the synergistic effects of many factors. therefore, it is difficult to implement and draw convincing conclusions from randomized controlled trials with a single intervention targeting a specific pathogenesis and risk factor. ongoing issues remain, such as standardizing the research design of he treatment and evaluating the efficacy of he treatment more scientifically and objectively. some clinical studies may bring new hope for he treatment by new ongoing strategies of targeted systemic inflammation, oxidative stress, and neurosteroids. in addition, the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. it is difficult to popularize the cognitive dysfunction detection methods of latent he. so the key and difficult point is to develop new method of assessments for clinicians in the future. jia shang hepatopulmonary syndrome (hps) is a syndrome of shortness of breath and hypoxemia induced by vasodilation in the lungs of patients with a variety of acute and chronic liver disease. essentially primary liver disease, pulmonary vasodilation and arterial oxygen lack of co-triad constituted. due to abnormal increase of vasodilators、ventilation/ blood flow disproportion and pulmonary hypertension caused by liver disease, the hypoxemia (pao 2 < 9.33 kpa) (70 mmhg) is included in hps. when fluckiger reported a 37-year-old syphilis female patient as early as in 1884, he described cirrhosis, cyanosis and clubbing at the same time, while he was not aware of the intrinsic relationship between these clinical manifestations [302] . in 1935, snell reported decreased arterial oxygen saturation (sao 2 , less than 94%) with abnormal hemoglobin in 38 patients with liver parenchymal lesions and biliary obstruction, and 3 years later, he proposed that such a phenomenon was associated with decreased affinity of o 2 with hemoglobin. in 1956, rydell and hoffbauer reported the detailed clinical diagnostic and treatment process of a 17-year-old male with "juvenile cirrhosis", and found multiple arterial-venous anastomoses in the lungs during autopsy, which he thought contributed to clinical cyanosis mainly. this provided a histological basis for the patient, and people conducted a large amount of studies thereafter. in 1966, berthelot et al. injected opaque glue into the pulmonary vascular beds at the time of biopsy after the patient's death for the first time, and he found abnormal small arterial dilation in the lungs of the patient with cirrhosis, which he termed lung spider nevus. the term hepatopulmonary syndrome (hps) was first proposed by kennedy and knudson in 1977 [303] . after nearly 20 years of studies in a large number, people gradually developed a clear understanding of the mechanisms underlying its pathogenesis. in 1988, eriksson used the term functional hepatopulmonary syndrome for the first time. in 1989, the famous liver disease expert sherlock formally used this diagnostic term in his monograph hepatobiliary diseases, which has been recognized by many scholars [304] . hps can occur in patients of any age groups, and various literature reports show conflicting incidences of hps in patients with cirrhotic portal hypertension, with the average incidence of various chronic liver diseases being about 5-29%. the incidence of cirrhosis in patients is high, and 30-70% of patients can additionally develop mild arterial hypoxia and 12-28% develop arterial hypoxia. in the study by binay on indian cirrhotic populations arising from hepatitis b mainly, the incidence of this disease is relatively low (6.7%) [305] . the differences in incidence were mainly attributable to the different diagnostic criteria adopted. schenk et al. studied the incidence of hps by performing transthoracic contrast echocardiography (ttce), pulmonary function tests and blood gas analysis on 98 patients with cirrhosis patients. the results showed that the incidence of hps patients in whom alveolar-arterial partial pressure of oxygen (aapo 2 ) was used as an indicator of hypoxemia was significantly higher than those in whom arterial partial pressure of oxygen (pao 2 ) was used [306] . when arterial partial pressure of oxygen (pao 2 ) was reduced to reflect hypoxemia, hps incidence was 19% when <80 mmhg and 15% when <70 mmhg, respectively. while when increase in alveolar-arterial partial pressure of oxygen (aapo 2 ) was used to reflect hypoxemia, the incidence of hps was high, with 32% when >15 mmhg and 31% when >20 mmhg, respectively. hps is most common in cirrhosis due to various causes. pulmonary vascular abnormalities and arterial hypoxemia can occur in a variety of acute and chronic liver diseases, and this is true mainly when it comes to cirrhotic patients due to chronic liver diseases, especially cryptogenic liver cirrhosis, alcoholic cirrhosis, hepatitisinduced cirrhosis and primary biliary cirrhosis. besides, hps can also occur in chronic hepatitis, acute severe hepatitis, cholestasis, ɑ-anti-trypsin deficiency [307] , tyrosinemia, wilson disease, and non-cirrhotic portal hypertension (such as idiopathic portal hypertension and schistosomiasis cirrhosis, etc.). arterial hypoxemia can also occur in extrahepatic portal vein occlusion. the observation of these patients suggests that portal hypertension may be the main factor for the pathogenesis of hps. hps can also occur in non-cirrhotic portal hypertension, and even cirrhosis-and portal hypertension-free chronic viral hepatitis. in 2000, binay et al. found that patients with progressive liver failure with hyperdynamic circulation are most likely to suffer from hps, while they did not find the correlation with the severity of liver cirrhosis. hps is, in essence, hypoxemia due to anomaly in pulmonary vascular dilatation and arterial oxygenation when liver disease occurs. arterial hypoxemia occurs as the result of insufficient oxygenation by blood cells in the blood when blood flows through the lungs, or a proportion of blood fail to flow through the alveoli [308] . since primary heart and lung diseases have been excluded when hps occurs, the abnormal pathways that red cells may pass through include: (1) passing through the pleural and hilar bronchial vessels while not reaching the alveoli; (2) blood flows directly into the pulmonary veins due to the high pressure portal system in the mediastinum, thereby bypassing the pulmonary circulation; (3) flowing directly into the pulmonary veins through the expanded alveolar capillaries or the pulmonary-venous fistula. alveolar telangiectasia may be more important to the formation of hypoxemia, and existing study data show that the development of hps is at least associated with the systemic hyperdynamic state, portal hypertension, hepatic encephalopathy, hepatorenal syndrome and pulmonary hypertension [308] . therefore, it is believed that the main causes of hps are systemic metabolism and hemodynamic disorders, and that it is involved in the formation of systemic metabolism and hemodynamic disorders, which is of important pathophysiological significance. 1. the basic pathological change of hps is pulmonary vascular dilatation, which is manifested as: (a) dilation of anterior capillaries in a large number. (b) formation and opening of the pulmonary basilar arterial -venous communicating branches. (c) formation of pleural "spider mole", which is mainly manifested as dilation of anterior capillaries. in autopsies, it was found that the basic pathological changes in patients with liver cirrhosis and other chronic liver diseases were extensive pulmonary vascular dilatation and arteriovenous communicating branches. some people found the pathological changes through vascular shaping, with pleural vasodilation at the basal aspect of the lungs or the formation of subpleural spider nevus. domestic professor gu changhai summarized these pathologic changes in 1997 as arterial dilation within the pulmonary acinus in a pattern of inhomogeneous distribution, thin-walled blood vessels, 60-80 μm in diameter, in the lower lobes of the whole lungs, extensive dilation of pulmonary vascular beds adjacent to the alveolar gas at the anterior capillary level, and significantly expanded pulmonary artery branches and pulmonary capillaries up to 160 μm in diameter. electron microscopy showed thickened pulmonary capillaries, pulmonary arterial walls and the basal layers of small veins. 2. factors that affect the dilation of blood vessels: the mechanisms underlying pulmonary vascular dilatation have not yet fully elucidated, and the possible influencing factors include: (a) increased activity of vascular dilators various acute and chronic liver diseases, liver cell failure and metabolic disorders, particularly reduced inactivation of vasoactive substances which can enter directly into the systemic circulation through abnormal anastomotic collateral vessels, result in disorder of the systemic hemodynamics and increased contents of vasodilators in the blood circulation. just as visceral congestion in patients with portal hypertension, they can act on the intrapulmonary vessels, causing pulmonary vascular dilatation and pulmonary congestion. substances that cause vasodilation include glucagon, prostaglandin, vasoactive intestinal peptide, nitric oxide, angiotensin, bradykinin and endotoxin, etc. (b) reduced vasoconstrictors or decreased sensitivity of intrapulmonary vascular beds to the endogenous vasoconstrictors, such as norepinephrine, endothelin, atrial natriuretic peptide, vasopressin, serotonin and tyrosine, etc. the contents of the substances are not absolutely reduced because maybe their sensitivity is reduced. when chronic liver disease occurs, the anterior communicating branches of the originally closed non-functional capillaries may be opened, and a disorder occurs in the hypoxic pulmonary vascular systolic dysfunction which should have been normal, and it is only 75% of the normal state [309] . (c) neurological factors cirrhotic patients show sympathetic nerve hyperactivity, but after the formation of portal hypertension, their sympathetic nerve function may be damaged, which play an important role. animals with portal hypertension often show abnormal pressure responses and reduced sensitivity of blood vessels to norepinephrine, resulting in increased cardiac output, and dilated pulmonary vascular volumes. besides, hemodynamics within the lungs is also a manifestation of the body's hyperdynamics. (d) decreased reactivity of intrapulmonary blood vessels to hypoxia recent inert gas dispersion tests show that cirrhotic patients with over two spider nevus are manifested as not only liver damage, but also decreased systemic intrapulmonary vascular resistance, decreased reactivity of blood vessels to hypoxia and dilated pulmonary vessels. however, it was also found using pulmonary angiography that in spite of the dilated vessels at the ending of arteries, the responses of vessels to oxygen were almost normal, which did not support this view. (e) intrahepatic angiogenesis or dysplasia may also be one of the factors for the formation of hps. to date, the mechanisms underlying pulmonary vascular dilatation caused by hps is still unclear. however, long-term administration of intrapulmonary vasoactive substances can cause significantly increased intracellular cyclic adenosine monophosphate (camp) and/or cyclic guanosine monophosphate (cgmp), resulting in hypoxic pulmonary vasomotor dysfunction and pulmonary artery dilatation, which may be an important cause of this disease and also pulmonary manifestations of systemic hyperdynamic circulation. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, some of the blood around the capillaries in contact with the alveoli can still undergo exchanges with gases, while the central blood, due to the increased diffusion distance from the alveoli, leads to insufficient gas exchange, resulting in insufficient arterial oxygenation and thus a series of hypoxemic manifestations. to date, the pathogenesis underlying the pathogenesis of hps has not yet been elucidated. in view of the above pathophysiological changes and current studies, it is believed that the disease may be caused by insufficient ventilation, diffusion disorder, ventilation/blood flow imbalance and decreased oxygenated hemoglobin affinity, or the above factors in combination. under normal circumstances, insufficient ventilation due to a variety of reasons causes insufficient oxygen inhaled into the alveoli and reduced blood oxygen exchanges, which can result in hypoxemia [310] , such as chronic bronchitis, foreign bodies in trachea, atelectasis and respiratory muscular paralysis, etc. and the presence of insufficient ventilation in patients with chronic liver disease and cirrhosis or not is still controversial. in 1982, fujiwara studied the lung function in 22 patients with decompensated liver cirrhosis and reported that vital capacity (vc), functional residual capacity (frc) and respiratory reserve volume (evr) in the patients were significantly reduced, that r/t was mildly increased, and that there was no changes in 1 s forced expiratory volume (fev1). therefore, it was believed that mechanical compression and insufficient ventilation due to pulmonary interstitial edema in patients with liver cirrhosis was the main reason for impaired lung function. subsequently, edison et al. studied the pulmonary function of 21 patients with decompensated liver cirrhosis, and found that their vc, maximum ventilation volume (mvv), frc, total lung volume, and r/t were significantly reduced, and they believed that patients with cirrhosis had obvious obstructive and limited insufficient ventilation, which were mainly caused by compression of lung tissue due to increased abdominal pressure, elevated diaphragm and increased chest volume and pressure when patients had additional ascites, and atelectasis [306] . however, decreased fev1 resulted from compression of small trachea due to pulmonary interstitial edema and vasodilation, and early closure of expiration. theoretically, all of the above factors can lead to insufficient ventilation, one of the factors resulting in this disease. this was also substantiated by significantly increased arterial partial pressure of oxygen and decreased co 2 partial pressure in cirrhotic patients with pleural effusion after pleural effusion extraction and recovery from atelectasis. however, there are also some people who do not think that hypoxemia results from insufficient ventilation, but because cirrhotic patients are not complicated by high concentrations of co 2 when their arterial partial pressure of oxygen is decreased [311] . this is likely because when patients have hypoxemia, compensation of hyperventilation causes arterial blood co 2 partial pressure not to increase, and results in decreased paco 2 or even respiratory alkalosis. besides, in some patients without decompensated liver cirrhosis, arterial hypoxemia can also occur. even it has been found that the lung function tests in patients with decompensated liver cirrhosis are normal. therefore, the majority of scholars currently believe that insufficient ventilation is not the main cause of hypoxemia in cirrhotic patients. for patients with hps, the inert gas exclusion technique should be performed to prove that there is a disorder in the diffusion of oxygen, which is determined by the basic pathological changes of hps -pulmonary vasodilatation. pulmonary angiography can show small spider-like to obviously cavernous diffuse vasodilation within the lungs. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, the diffusion distance of the blood flow in central blood vessels and the alveoli is increased, preventing the gases in the alveoli from entering the pulmonary capillaries, thereby affecting the gas exchanges. studies have shown that hypoxemia often occurs in patients with cirrhosis or aggravates during exercises, and it is believed that diffusion disorder or limitation of oxygen occurs in patients. in fact, factors that affect o 2 diffusion do occur in patients with cirrhosis, but they are still not sufficient to explain the apparent hypoxemia. although vascular dilatation occurs at arterial endings in patients with hps, their arterial partial pressure of oxygen can be reduced while inhaling air and increased when they are given oxygen inhalation, which further proves that although diffusion disorder does exist and it plays a role in the formation of this disease, the role is not important. to engage in gas exchanges is the most important biological function of lung tissues, and this gas exchange must be completed when there is an appropriate ventilation/blood flow ratio. under the normal circumstance (normal adult resting state), the most appropriate ventilation/blood flow ratio physiologically is 0.8. changes in the ratio due to any cause can affect the gas exchange, and the imbalanced ventilation/blood flow ratio in hps patients with hypoxemia is mainly because of pulmonary vascular dilatation and arteriovenous shunt [312] . 1. intravascular vascular dilatation: pulmonary vascular dilatation has been confirmed pathologically and by angiography. dilated blood vessels in the lungs leads to gas diffusion disorder. besides, since the oxygen molecules in the air can not be diffused to the central dilated blood for the gas exchange, causing decreased ventilation/blood flow ratio and pulmonary arterial partial pressure of oxygen. this decreased ventilation/blood flow, together with increased amount of reactive cardiac output, shortens the duration of blood that flows through the capillary network and insufficient oxygenation [312] . excessive ventilation can in part enhance patients' pao 2 . if the alveolar oxygen partial pressure is increased at this time, some oxygen molecules can reach the central areas of dilated blood vessels, increasing the arterial partial pressure of oxygen. therefore, it is called the diffusion -perfusion disorder or pulmonary arteriovenous functional shunt rather than the true lung shunt. 2. arterial -venous shunt: intrapulmonary vascular fistula and pleural spider nevus can occur in cirrhosis of the liver and allow the pulmonary arterial blood to circumvent gas exchange and directly flow into the pulmonary vein so that patients may develop hypoxemia. this hypoxemia can not be corrected by oxygen inhalation and represents the true pulmonary shunt, which has been confirmed by pulmonary histopathology, angiography, transthoracic echocardiography and other examinations. it is now believed that pulmonary vascular casting is still the most direct evidence for determining the arterialvenous shunt. this intrapulmonary arterial -venous shunt is the main cause of abnormal ventilation/blood flow ratio and insufficient gas exchange. although pleural spider nevus can also cause arterial -venous shunt, it generally does not suffice to cause significant hypoxemia due to the small amount of shunt. in addition, studies in recent years also show that portal-pulmonary vein shunt in a small amount occurs in some patients with cirrhosis, in whom blood flow circumvents the alveolar gas exchange and enters directly the systemic circulation. this can also cause ventilation/blood flow abnormalities, causing insufficient gas exchange 3. airway closure: in 1971, ruff et al. proved that cirrhotic patients had significantly increased closed volume (cv) and total amount of closed gas (cc) and increased gases trapped in the lower fields of the lungs, resulting in an extremely low ratio of ventilation/blood flow, and they believed these were due to reduced airway ventilation [302, 313] . in 1984, furukawa et al. measured the lung function of 105 patients with liver cirrhosis and did not find abnormalities; however, most patients had flow -volume abnormalities and significantly increased cv, suggesting the closed the airway in advance and decreased ratio of ventilation/blood flow, which might important causes of hypoxemia. 4. decreased affinity of oxygen with hemoglobin: some reports showed that 15 patients with cirrhosis (mostly alcoholic cirrhosis) patients had mild systemic vascular or pulmonary vascular dilatation, normal pao 2 , mild hypocapnia, mild right shift of the oxygenated hemoglobin dissociation curve, normal amount of carbon monoxide diffusion [313] , and mild imbalance of the ventilation/pao 2 blood flow, indicating that the right shift of the oxygen dissociation curve due to decreased affinity of oxygen with hemoglobin in the patients. this was possibly caused by the increased concentration of 2,3-diphosphate glyceride in red blood cells, which, however, is not an important factor in the occurrence of hypoxemia [302] . in summary, hypoxemia can result from many factors, while none of the factors can completely explain the pathogenesis underlying the disease. since the basic pathological changes in patients with hps are intrapulmonary vascular dilatation and opening of arterial -venous communicating branches, together with recent findings, it is suggested that the diffusion disorder of the alveoli and pulmonary capillaries and ventilation/blood flow imbalance may coexist, and are the main cause of hypoxemia in this disease. other factors may aggravate hypoxia and are secondary factors. therefore, it is believed that the disease occurs as result of the above factors. the pathological features of hps are dilation of capillaries in the anterior aspect of the lungs and telangiectasia. autopsies show arterial-venous short circuit within the lungs, vasodilation and thickened pulmonary muscles [314] . at the same time, arterial hypoxemia is common in liver diseases, often attributable to a variety of factors (such as ascites, hepatic pleural effusion, and copd in patients with alcoholism); it shows unique pathophysiological characteristics under specific circumstances of hps. its prominent features are dilation of micro-arteries in the anterior aspect of pulmonary capillaries and true capillaries (the normal diameter of these vessels is 8 to 15 μm, which can reach 15-100 μm when patients rest), with the number of dilated vessels increased macroscopically. some patients show arterial-venous communicating between the pleura and lungs, vascular anastomosis in the liver and the lungs, and thickened walls of small veins and capillaries. pulmonary vascular dilatation is promoted, and mixed venous blood quickly or directly enter the pulmonary veins through the anastomosis in the lungs, leading to oxygenation defects. increased nitric oxide (no) is a key cause for pulmonary vasodilation, and whether other mediators, such as heme oxygenase-derived carbon monoxide, are causes of pulmonary vasodilatation are not yet confirmed. abnormal arterial oxygenation seriously affects the survival of patients, and is an important indicator that determines the timing and risk of liver transplantation as well as an important basis for the grading of severity of hps. causes of deaths associated with hps are often multifactorial and are associated with basic liver diseases, and there are few cases of respiratory failure due to severe hypoxemia. hps is a triad composed by intrapulmonary vascular dilatation and insufficient arterial oxygenation due to primary liver disease, and it is mainly clinically manifested as primary liver disease and pulmonary lesions. hps can occur in various liver diseases, mostly in chronic liver disease, especially cirrhosis caused by various causes, such as cryptogenic cirrhosis, alcoholic cirrhosis, liver cirrhosis, viral cirrhosis, postnecrotic cirrhosis and biliary liver cirrhosis, etc. the most common clinical manifestations include liver palms [314] , spider nevus, jaundice, ascites, hepatosplenomegaly, gastrointestinal bleeding and abnormal liver function, etc., while they are not significantly correlated with hps. some patients with clinically stable liver disease may also develop the clinical manifestation of progressive pulmonary insufficiency. since hps patients have no primary cardiopulmonary diseases, most (80-90%) patients gradually develop respiratory manifestations on the basis of various liver diseases, such as cyanosis, dyspnea, clubbing, orthodeoxidation and platypnea, etc. among them, progressive dyspnea is the most common lung symptoms of hps. binay et al. believed that cyanosis was the only reliable clinical sign, and that platypnea and orthostatic hypoxia are the most characteristic manifestations. pulmonary examinations generally show no obvious positive signs [315] . a small number of patients (about 16-20%) can present complaining dyspnea on exertion in the absence of clinical manifestations of a variety of liver diseases, to which attention should be paid clinically so as to prevent misdiagnosis. the domestic researchers gao zhi et al. reported that 2 patients presented to hospitals with cyanosis, palpitation after exercises and shortness of breath; meanwhile, they found that the patients had clinical manifestations of liver cirrhosis [308] (such as liver palms, spider nevus, hepatosplenomegaly and ascites), which were conducive to the diagnosis of this disease. if liver disease patients have other lung diseases (such as chronic bronchitis, emphysema and pneumonia, and pleural effusion, etc.), then significant respiratory symptoms may occur. therefore, differential diagnosis should be made. data show that the duration of initial dyspnea to the conformed diagnosis in patients with hps average 2-7 years; that is to say, about 18% of patients already have dyspnea at the time confirmed diagnosis. 1. orthodeoxidation: pao 2 is decreased by >10% when patients switch from the supine position to the standing position. 2. platypnea: when patients switch from the supine position to the standing position, they have palpation, chest tightness and shortness of breath, and when patients resume the supine position, the above symptoms are improved [313] . krowka reported that about 80-90% of patients with hps had the above two manifestations because vascular dilatation in the patients was mainly distributed in the middle and low lung fields. when patients switch from the supine position to the standing position, the blood flow in the middle and lower lobes of the lungs is increased under the action of gravity, aggravating hypoxemia [315] . although the two manifestations are not unique to hps, they suggest the significant abnormality in the patients' pulmonary vascular system. if patients with a variety of liver diseases present with the above two manifestations, further examinations are needed for confirmation. patients may present with liver palms, hepatosplenomegaly, spider nevus and ascites; patients show palpitation, chest tightness, shortness of breath when switching from the supine position to the standing position due to hypoxemia. schenk et al. defined the values of pao 2 for the diagnosis of hps, thinking that pao 2 < 70 mmhg suggested a high possibility of hps, and that for pao 2 < 65 mmhg, the diagnosis of hps could be made [306] . pulmonary function tests mainly showed significantly decreased vc, frc, mvv and fev1, but sometimes the total lung volume and fev1 were normal. chest x-ray, ct scan and transthoracic contrast echocardiography (ttce) hps patients are mostly normal on chest radiography or show diffuse small millet shadows predominantly in both lower lobes of the lungs, nodular shadows in both lower lung interstitium, dilated pulmonary arterial trunks, and thickened pulmonary markings, whereas these manifestations have no specific values to the diagnosis of this disease. ct scan shows certain diagnostic values in that it demonstrates distal vasodilation and even pleural blood vessels, and can suggest the presence of hps. arteriovenous communicating occurs in hps patients due to pulmonary vascular dilation, indicating that subclinical pulmonary vascular dilatation and abnormal gas exchanges occur in cirrhotic patients with normal pao 2 [306] . contrast echocardiography: when hps is suspected, transthoracic echocardiography can be used as a preliminary screening to determine whether the intrapulmonary vascular dilation occurs or not. the microbubble contrast material in the right atrium, after intravenous injection of dioxane isotonic saline, will develop images in the left atrium through the dilated vascular beds after 3-6 cardiac cycles, while the microbubbles cannot pass through the normal capillaries (normal capillaries are <8-15 μm in diameter). approximately 40% of patients with cirrhosis had positive changes on contrast echocardiography, while only a small proportion of patients are in line with the diagnosis of hps due to the influence of dilated blood vessels. if contrast echocardiography is positive for liver cirrhosis or portal hypertension patients with hypoxemia and cardiopulmonary diseases in them can be ruled out, then the diagnosis of hps is established. this means is used to confirm the diagnosis of intrapulmonary vasodilatation. the pulmonary vascular abnormalities in hps patients are as follows: (1) diffuse spider nevus images. patients of this type have severe hypoxemia and erectile hypoxia and respond well to inhalation of 100% oxygen; (2) cavernous or spotted arterial dilatation mainly seen in the basal aspect of the lungs. patients during this period respond poorly to 100% oxygen; and (3) intermittent local arterial malformation or communicating branches, isolated earthworm-like or bulk images. in addition to severe hypoxemia and erect hypoxia, patients of this this type respond extremely poorly to the inhalation of 100% oxygen. when hypoxemia is caused by hps and cardiopulmonary diseases concurrently, 99cm tc-maa scan can determine hypoxemia resulting from hps more likely. radiolabeled albumin 99cm tc, which is administered through intravenous injection, is about 20 pm in diameter. when pulmonary vascular shunt occurs, a proportion of the polymerized albumin passes through the lungs and enters the systemic circulation, the intake of albumin by other organs can be simultaneously determined by scintigraphy [308] . therefore, the amount of shunt can be calculated. a study showed that 99 cm tc-maa scan was positive for hps patients with pa02 < 60 mm hg, while the scan was negative for chronic obstructive pulmonary disease (copd) patients with the same degree of hypoxemia, a result indicative of the good specificity of this means. compared with contrast echocardiography, 99cm tc-maa scan, in spite of its low sensitivity, can be used for the diagnosis of hps in patients with copd. pathological examinations are the most reliable means for the diagnosis of hps, whose basic pathological change is pulmonary vasodilation manifested as diffuse anterior capillary dilation or discontinuous formation of arteriovenous branches [312] . in addition, pulmonary perfusion scan and right cardiac catheterization are also valuable for the diagnosis of hps to a certain extent. there is no unified standard for hps diagnosis to date. diagnosis should be based on clinical manifestations plus imaging evidence of pulmonary angiography. (c) the domestic scholars gao zhi et al. thought in 1998 that the diagnosis of this disease should be based on the following manifestations in patients, hepatosplenomegaly, ascites, liver palms, spider nevus, dyspnea on exertion, hypoxia while breathing in the supine and orthostatic positions, increased mesenchyma in the basal aspects of the lungs and vascular markings on chest radiography, patchy or nodular shadows, dilated basilar pulmonary vessels and increased pulmonary vascular branches on ct, severe hypoxemia or not on blood gas analysis, increase in alveolar -arterial oxygen gradient by ≥20 kpa (150 mmhg), and 82% diffusion disorder on pulmonary function tests [316] . in addition, shunt-related examinations should be performed, such as 99cm tc-maa scanning, contrast-enhanced two-dimensional echocardiography and pulmonary angiography, etc., while the last one does not show the same sensitivity as that of the former two because the small blood vessels in the lungs may not develop on angiography. most hps patients have a slow onset of the disease, are difficult to treat, and have a poor long-term prognosis, with a mortality of more than 40% after 3 years. therefore, early diagnosis of the disease and its differential diagnosis are vital to improving the prognosis of patients. first of all, the previous liver and lung diseases in the patients should be ruled out, such as chronic obstructive pulmonary emphysema, pulmonary infection, interstitial pneumonia and silicosis, etc. at the same time, cirrhosis with pulmonary hypertension, infections secondary to pleural effusion, interstitial pulmonary edema, atelectasis and hyperventilation syndrome, etc. need to be ruled out. hps should be differentiated mainly from the following diseases: 1. liver cirrhosis following pulmonary heart disease: this is mainly because pulmonary diseases result in cardiac insufficiency and thus increased pulmonary venous pressure. repeated or long-term existence of liver congestion can lead to central venous hypertrophy and lobular central connective tissue hyperplasia, and further progression of the lesion will lead to the formation of liver cirrhosis following pulmonary heart disease. patients with pulmonary cirrhosis often have a long history of chronic lung disease and signs of cardiac insufficiency, such as edema of lower extremities, palpitation, shortness of breath and other symptoms. this patient has no history of chronic lung disease or edema of lower extremities, making him inconsistent with liver cirrhosis following pulmonary heart disease. 2. left heart insufficiency: both hps and left heart insufficiency can cause severe dyspnea and hypoxemia. a history of liver disease or evidence of chronic liver damage and decreased po 2 can be found in patients with hps, especially such characteristics as orthostatic hypoxemia and intrapulmonary vascular dilatation. patients with left heart insufficiency have a history of heart disease, orthopnoea, pink frothy sputum and moist rales in the lungs, etc. this patient is inconsistent with such manifestations. 3. primary pulmonary hypertension: after inhalation of pure oxygen, hypoxemia in most of patients with hps will be significantly alleviated. the effects of oxygen are poor in patients with hypoxemia [317] , and hps is manifested as orthostatic hypoxemia. the characteristics of hemodynamics of patients with hps are hyperdynamics and normal or decreased pulmonary artery pressure and pulmonary vascular resistance, while those in hypoxemic patients are increased. 4. others: ductus arteriosus, eisenmenger syndrome and pulmonary embolism, etc., need to be differentiated from this disease, and comprehensive judgments should be provided based on other clinical data of the medical history. hps patients can also have the above-mentioned diseases, and careful and meticulous examinations are needed for differentiation. because hps is developed on the basis of original liver disease, the frequency of its occurrence and its severity are mostly associated with the liver cell function of patients, while there are also hps patients in whom chronic liver disease is relatively stable and liver functions are normal. besides, pleural effusion, ascites and infections secondary to pulmonary edema after liver function decompensation can aggravate patients' respiratory function injury. therefore, under the current circumstances in which there are no effective measures for hps, active and effective treatment of primary liver diseases is the basis for the treatment of hps. therapy of primary diseases, including correction of hypoproteinemia, elimination of pleural effusion, improvement of liver function and treatment of complications, etc., can improve tissue oxygenation and improve arterial oxygen saturation. on this basis, the following treatment can be given. oxygen therapy also helps the differential diagnosis of pulmonary shunt: if pao 2 is resumed after oxygen inhalation, then the diagnosis of intrapulmonary vascular dilatation (ipvd) can be made; for patients with partial improvement, pulmonary anatomical shunt and functional shunt may coexist; for patients in whom the oxygen therapy proves inefficacious, pulmonary arteriovenous fistula is a possible diagnosis. it is now believed that once the diagnosis is established, treatment should be given as soon as possible. in the early stage of correcting hypoxemia in patients with mild conditions, even in patients in whom the critical value of hypoxemia (pao 2 , 8-9 kpa (60-67.5 mmhg) is reached and who have ascites, the hemoglobin saturation may still be less than 85% when patients are in activities or even sleep. that is to say, nasal catheter oxygen inhalation at 2-3 l/min is needed so as to improve hypoxemia [318] . with the development of the disease, oxygen flow needs to be gradually increased, and intratrachea oxygen supply can be offered when necessary. during the late stage, patients can receive pressurized oxygen through a ventilator or a hyperbaric oxygen chamber. for patients whose conditions are severe, the efficacy of oxygen therapy alone is not obvious. 2. vasoactive drugs vasoactive drugs for the treatment of patients with hps are most studied; however, since its pathogenesis has not been clarified to date and primary liver disease is difficult to reverse, it is hard to define the clinical efficacy of these drugs. the commonly used drugs include: used aerosolized ephedrine hydrochloride for the treatment of 12 patients with hps, and the preliminary efficacy was significant. the mechanisms were that ephedrine could excite the pulmonary vascular α receptor, resulting in contracted bronchial mucosa and pulmonary capillaries and alleviated bronchial edema, so that the dilated blood vessels within the lungs were contracted and intrapulmonary shunt was reduced. meanwhile, the bronchial β2 receptors were excited and the bronchi were dilated so as to improve the ventilation/blood flow ratio and relieve hypoxia. further studies are merited. (f) others: there have been reports on sympathomimetic drugs (isoproterenol) and β-blockers (propranolol), etc. that improve the symptoms of hps. theoretically, vascular endothelin, estrogen suppressor (tamoxifen) and so on can relieve the spider nevus and pulmonary vascular dilatation in patients with liver cirrhosis and improve their respiratory symptoms, while further studies are needed. no is most studied currently, and there are reports indicating that no synthesis inhibitors can increase pulmonary vascular resistance. alexander et al. used no for the treatment of severe hypoxemia in patients after liver transplantation, and obtained good results. durand et al. also reported that an hps patient was cured by inhaling no, while its mechanisms and clinical efficacy needed to be further confirmed. 3. pulmonary embolism it is generally considered that pulmonary vascular dilatation can vanish after liver transplantation in hps patients who are normal on pulmonary angiography or who have cavernous vessels on imaging [310] ; for patients who show diffuse pulmonary vascular dilation features on pulmonary angiography, embolization is usually not adopted since patients' lesions are extensive and the efficacy is poor; for patients with isolated and severe pulmonary vascular dilation or arterial-venous communicating branches, local pulmonary embolism therapy can yield a satisfactory effect. 4. liver transplantation it is currently considered that liver transplantation is still a possible fundamental measure for the treatment for hps. in the past, it was believed that serious hypoxemia was an absolute contraindication against liver transplantation, while recent studies show that liver transplantation is preferred for patients who have good alveolar gas diffusion function, who can respond well to pure oxygen inhalation and who can undergone oxygenation safely during anesthesia. recent reports further prove that hypoxemia can be cured after liver transplantation [308] . through literature review and case reports, krowka et al. believed that progressive hypoxemia in hps could be used as an indication of liver transplantation. temporary hypoxemia following liver transplantation can be adjusted by using no and taking the head-down supine position and the alternate lateral decubitus position. and for hps patients who fail to respond to the inhalation of pure oxygen, who have direct pulmonary arterial communicating branches on pulmonary angiography and who have severe clinical hypoxia, liver transplantation cannot improve their hypoxic status, has limited efficacy, or even increases intraoperative and postoperative risks. therefore, liver transplantation should not be performed on them. tips was an effective method for the treatment of hps, and its effects of improving symptoms, enhancing oxygenation and reducing intrapulmonary shunt could last up to 4 months. riegler et al. performed tips on an hps patient with diffuse intravascular dilatation who was not suitable for vascular embolization, and the results showed significantly increased pao 2 and significantly improved hypoxemia. however, coley et al. also reported that a patient failed to respond to tips, and therefore, its exact effects remain to be studied. 6. other treatment options one patient with hps was once treated with garlic, and 18 months later, his oxygenation was significantly improved and his symptoms were relieved. there are also patients who receive plasma replacement therapy, which has limited effects on the oxygenation of patients with hps [308] . to sum up, no effective treatment options are currently available for hps. since the basic cause of hps is liver cell failure, the usual cause of patients' deaths is not lung failure, mostly complications such as gastrointestinal bleeding, renal failure, hepatic encephalopathy and sepsis. therefore, we consider that the therapy of primary liver disease is particularly important. oxygen inhalation alone can be given in the early stage of hypoxemia, or conservative treatment can be provided if additional drugs are effective. liver transplantation is the best solution whenever possible. it is generally accepted that liver transplantation is the most promising regimen with confirmed efficacy. if oxygen inhalation is less satisfactory and patients are diagnosed with local intrapulmonary vascular dilatation or arterial -venous fistula by such means as pulmonary angiography, pulmonary embolism should be carried out as soon as possible. for patients with additional obvious portal hypertension, tips treatment can also be given. the interval from chronic liver disease and cirrhosis in patients to the confirmed hps due to such respiratory symptoms as anoxic dyspnea is usually several years or even more than 10 years [average interval, (4.8 ± 2.5) years], and a small number of patients can develop such a disease acutely in the short term. besides, signs of chronic liver disease can be traced in patients complaining breathing difficulties. once hps is established, obvious hypoxemia has occurred already. it confers a poor prognosis, and most patients die within 2-3 years often due to other complications of liver disease [312, 315] . if patients' oxygenation is satisfactory and they have undergone liver transplantation, or with the improvement in liver function, their hypoxemia can be resolved or improved of its own volition with good prognosis. if patients' oxygenation deteriorates severely and they have a very poor prognosis, most of them will die in the short term. hps often progresses slowly. although it is not a direct cause of death in patients with cirrhosis, it can significantly aggravate the disease. therefore, cirrhotic patients, especially those with positive liver palms and spider nevus as well as patients with portal hypertension, should be careful of the possibility of hps. timely detection and symptomatic treatment (such as low flow oxygen inhalation) can improve the prognosis of patients. active and effective treatment of primary liver disease forms the basis for the prevention of this disease. education of common sense should be given to patients with liver diseases so as to avoid factors inducing hps in their life. for patients with liver disease, mild hps should be found as soon as possible and appropriate treatment should be given. jia-quan huang and dong xu lipopolysaccharide (lps) is a constituent of bacteria cell wall which plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as nitrous oxide, cytokines, superoxide anions, and lipid mediators. despite the recent advances in antibiotic treatment and hemodynamic monitoring, septic shock still remains a serious disorder that is associated with a high mortality rate, to more comprehensive definition of the mechanisms that underlie innate immunity against bacterial pathogens, lps has been extensively studied [319] . the pathophysiological consequences of bacterial sepsis are contributed by the dysregulation of these same mechanisms. before we can hope to design effective anti-sepsis therapies, greater insight into the nature of host interactions with lps is extremely essential. the gram-negative bacterial envelope is composed of two bacterial membranes, outer and inner membrane. the outer membrane consists of the following substances, like lipopolysaccharide (lps), several kinds of outer membrane proteins, lipid a and metal ions. for most gram-negative bacteria, lps is a major component in the outer monolayer of the outer membrane which works like a tight shield. the shield is composed by unique molecules, such as polysaccharide, or long chain of sugar, and lipid a. during the process to evoke the signaling events of lps, lipid a plays a pivotal role. the entire lipid component of lps molecule, however, is required for optimal activity [320] . the basic principles of lps bioactivity are nowadays well understood. endotoxins do not elicit their toxic effects -as we might suspect and as it is known for many proteinous exotoxins which can kill host cells or inhibit cellular functions. rather, lps requires the active response of host cells. according to present knowledge we get, lps interacts with various host cell types through lipid a, those cells include mononuclear cells, thrombocytes, endothelial and smooth muscle cells, and polymorphonuclear granulocytes, among which macrophages/monocytes are of particular importance. through the lps-induced activation, macrophages produce many substances, like bioactive lipids, reactive oxygen species, and in particular, cytokines such as tumor necrosis factor a (tnf), interleukin-1, il-6, il-8, and il-10. it appears that when low levels of mediators are produced, beneficial effects (e.g., induction of resistance to infection, adjuvant activity) are elicited and when high levels of mediators reach the circulation that detrimental effects (e.g., high fever, hypotension, irreversible shock) are induced. however, when the host organism is in a hyperreactive state lps, low mediator concentrations may also become harmful. the hyperreactivity to endotoxin may be caused by exo-toxins, chronic infection, and by growing tumors, and interferon-γ. to function properly, organism requires an immune system that must detect pathogens, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. the immune system can be classified into humoral immunity versus cell-mediated immunity or the innate immune system versus the adaptive immune system. when microbes invade organism, the innate response is usually triggered by pattern recognition receptors, which recognize components that are conserved among broad groups of microorganisms, or when injured, damaged, or stressed cells send out alarm signals, many of which (but not all) are recognized by the same receptors as those that recognize pathogens. the innate immune system defenses are nonspecific, meaning that the system responds to pathogens in a generic way. this system does not confer long-lasting immunity against a pathogen. in most organisms, the innate immune system is the dominant system of host defense [321] . they activate innate immune responses by identifying some conserved non-self molecules, so as to protect the host from infection,. bacterial lipopolysaccharide (lps), an endotoxin which is found on the bacterial cell membrane, is considered to be the prototypical pamp. lps is specifically recognized by toll-like receptor (tlr) 4, a recognition receptor of the innate immune system. the interaction of the lipid a moiety of lps with macrophages appears to be especially important because subsequent cellular activation results in the release of systemically active pro-inflammatory molecules, which in turn mediate systemic toxicity. lps has extreme potential in macrophages activated at concentrations of lps as low as 1 pg/ml [322] . host-defense peptides (hdps) could be a possible alternative solution since they possess the antimicrobial, antiseptic, and immunomodulatory properties [323] . endotoxins lipopolysaccharide is released not only from dead gram-negative bacterial, but also from the growing ones. endotoxins are very stable molecules, which are resisted to extreme temperatures and ph values in comparison to proteins. endotoxins are shed largely during cell death as well as growth and division. they are highly heat-stable and are not destroyed under regular sterilizing conditions. endotoxin can be inactivated through exposed at temperature of 250 ° c for more than 30 min or 180 ° c for more than 3 h. acids or alkalis of at least 0.1 m strength can also be used to destroy endotoxin in laboratory scale [324] . gut microbiota is composed of strict anaerobes, facultative anaerobes and aerobes. recent reports suggest the existence of over 35,000 bacterial species in the human gut microbiota. an important characteristic of gut microbes is their heterogeneity [325] . the composition and the frequency of the microbiome changes with the different segments of the elementary tract. the composition is influenced by the environment, consumed diet and host factors. endotoxin to surrounding tissues and organs or blood shift that shift pathway include: (1) via the portal vein, liver into the systemic circulation; (2) through the intestinal tract into the lymphatic system lymphatic; (3) through the intestinal wall into the peritoneal cavity and then absorbed into the bloodstream. under physiological conditions, although a small amount of toxins continued to parenteral shifted via the portal vein into the liver, but it does not cause endotoxemia; mild in gram-negative bacilli infections, although bacteria continue to release to the tissue or blood endotoxin, but it does not give rise to a strong inflammatory response, the above are dependent on the presence of an effective mechanism within the body to remove toxins and detoxification. the liver is the main organ of clearance of endotoxin, and the spleen, also removes toxins. molecules in lps removed include cationic antimicrobial peptides (cationic antimicrobial peptides, cap), acyloxy acyl hydrolase (acyloxyacyl hydrolase, aoah) lipoprotein binding protein and anti-lps antibodies are important endotoxin clearance methods [326, 327] . liver blood endotoxin clearance, primarily through kupffer cells, hepatocytes internal toxin endocytosis achieved, but the specific metabolic process is not entirely clear. mediated by kupffer cells engulf toxins may be scavenger receptor, it may be a molecular weight of 119,000 and 83,000 protein; mediate phagocytosis liver toxin structure may be the lectin-like receptor (lectin-like receptor). endotoxin receptor hepatocyte sinusoidal plasma membrane on the surface: after one to one binding, is taken up within the liver cells endocytosis way to microtubule-dependent vesicular transport through the liver cells, transported to the liver cells bile duct surface to exocytosis into the bile duct, and then discharged into the biliary system through the intestine [328] . splenic macrophages containing approximately 15% of the body to settle within the organization, and macrophages are important endotoxin removal cells. when endotoxin intravenously into the body, except gathering in the liver, a lot of endotoxin can be quickly gathered and taken up into macrophages in the spleen, the spleen and therefore equally important endotoxin removal organs. in addition to its clear role in the performance of its direct effect, but more importantly, spleen macrophages is the precursor cells of kupffer cells in the liver, having a very big impact on removing toxins within the liver [329] . mechanisms for removing toxins in the intestine are related to the intestinal villus tip epithelial cells. under normal circumstances, injected into the intestinal, endotoxin in intestine does not enter intestinal epithelial cells, but after intravenous injection of endotoxin, endotoxin may enter intestinal epithelial cells inside. therefore, endotoxin receptor may identify ways by endotoxin and (or) simple diffusion way into the intestinal epithelial cells. endotoxin way into the intestinal epithelial cells intravenously may have two: (1) displaced from the lamina propria macrophages to intestinal epithelial cells basolateral; and enter intestinal epithelial cells from the side; (2) including lower toxin, intestinal lamina propria macrophages, intestinal epithelial cells release large amounts of no and oxygen free, resulting in intestinal lamina propria microvascular injury, increased permeability, extravasation of endotoxin and ultimately displaced into the outer intestinal epithelial cells. villus tip epithelial cells within a stronger uptake of toxins, thus endotoxin can be started from the crypt, moving along the intestinal villi, and finally to the top of the inner hair cells of the intestinal epithelium. uptake of endotoxin villus tip epithelial cell loss, while the endotoxin into the intestine, which constitutes one of the effective clearance mechanisms of endotoxin [327, 330] . plasma lipoproteins in endotoxin detoxification mechanisms play an important role, in which the lipopolysaccharide binding protein (lipopolysaccharide binding protein, lbp) and high-density lipoprotein (high density lipoprotein, hdl) play a major role. endotoxin into the bloodstream within minutes there were half white blood circulation due to binding to the edge of the pool or the pool is cleared, the remaining residual endotoxin and rapidly bound to plasma lipoprotein is inactivated. in plasma, lipoproteins and endotoxin when hdl plays a major role in its binding of endotoxin to reach more than 50% of the total, hdl, and thus research endotoxin important [331] . cationic antimicrobial peptides are an ancient ingredients in the natural evolution of the immune system, including bactericidal/permeability-increasing protein (bactericidal/permeability-increasing protein, bpi), cathelicidin, lactoferrin, defensins and other substances, with not only the activity against gram-negative bacteria, but also the ability to combine internal toxins. cationic antimicrobial peptides are mainly in regular contact with the pathogen site mammalian skin, digestive tract, respiratory tract, and inherently express or express induced by pathogens and their products in the blood, secretions and neutrophil granules. cationic antimicrobial peptides have two types of three-dimensional structure; one is a α-helix, having such a molecular structure include cathelicidin and lactoferrin; the other is β-fold, including mammals α and β-defensins, etc. [332] aoah aoah is a glycoprotein produced by white blood cells with weight of 5.2-60,000, the large subunit of 50,000 and small subunits of 1.4 million to 20,000, the large and small subunits connected by covalent disulfide bond. aoah as a lipase with hydrolysis for toxin, can selectively hydrolyze the secondary acyl chain on lipid a acyl groups acyloxy. when hydrolyzing endotoxins, both the large and small subunits of aoah play an important role, and both are indispensable. in addition to directly destroying toxin, the deacylated lps after the hydrolysis of endotoxin by aoah is also involved in aoah's detoxification mechanism on endotoxin; the material can accumulate and inhibit endotoxin-induced inflammatory response in the cell. however, due to a limited number of secretion by local infiltration of leukocytes, the internal detoxification of toxins is also limited [333] . when the body respond with endotoxin, one trigger inflammation, on the other hand can be cleared to produce or activate, specific polysaccharide inactivate toxins, including antimicrobial-specific polysaccharide antibody and anti-core polysaccharide antibody. after two antibodies binding with endotoxin, and then with the fc receptors on the cell membrane, inner source of the toxin-mediated, so that the endotoxin inactivated intracellularly. anti-endotoxin antibodies can interfere with toxin within lbp binding, thus preventing lbp endotoxin transport [334] . in the body's defense system, the shift from the inhibition of intestinal endotoxin ingredients include intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, the normal intestinal flora and liver hepatocytes and kupffer cells, in which intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, hepatocytes and kupffer cells play a direct inhibitory effect, while the normal flora plays an indirect inhibition. the intestine is huge "endotoxin library", a special anatomical location determines the intestinal mucosa must be an effective defense barrier. immunological barrier intestinal barrier formed by the epithelial cells of mechanical barrier and secretory iga (siga) and the like components [335] . intestinal epithelial cells and tight junction formation mucosal mechanical barrier, is a significant barrier in the intestine endotoxin translocation defense to maintain its integrity is it to play an important role in defense guarantee. in severe trauma, burns, infection, considerable loss of body fluids, hypovolemia, cause the body ischemia and hypoxia. in order to maintain blood pressure, to ensure that the blood supply to the heart brain and other vital organs, compensatory splanchnic vascular contraction, including gastrointestinal ischemia and hypoxia longer time than other organs, even after shock patients after resuscitation to restore normal hemodynamics, stomach intestinal still in a state of shock occult. therefore, when the intestinal microvascular perfusion recovery, intestinal ischemic/reperfusion injury, epithelial cells produce large amounts of reactive oxygen species and other media, resulting in intestinal epithelial cell apoptosis, destruction of tight junctions between cells, thus rapidly increasing intestinal permeability mechanical barrier function weakens, thus contributing to the intestine of the endotoxin absorbed through the intestinal wall to parenteral tissue displacement [336] . intestinal mucosal intestinal immunology barrier is a defense of the invasion of pathogens and endotoxin important line of defense, siga plays an important role in intestinal mucosal immunity. siga is an important component of the protection of the intestinal mucosa, both to prevent bacteria in the intestine mucosal surface colonization, but also in endotoxin. studies have found that e. coli o157 infection suffered intestinal mucosa, the anti-endotoxin core polysaccharide-specific siga secretion, in convalescent patients has been particularly evident, suggesting siga endotoxin to prevent the transfer of the body has a protective effect. in addition, studies suggest that nitric oxide (nitrogen monoxide, no) preventing endotoxin translocation in intestinal mucosal barrier oxide formed locally. under physiological conditions, nitric oxide synthase (inducible nitric synthase, inos) expression only in the respiratory epithelium, the pregnant uterus and ileal mucosa and a few other parts. induced by endotoxin including but under normal colonic epithelial cells also express inos and catalytic synthesis of no, are formed in the local oxidation barrier to prevent bacterial translocation colon, thus effectively preventing bacterial translocation, also indirectly prevents endotoxin shift [337] . under physiological conditions, intestinal flora forms a relatively balanced microecosystem. flora distribution in the intestine has certain rules: deep close to the intestinal mucosal surface, parasitic anaerobic bifidobacteria and lactobacilli, these anaerobic bacteria are sugar coated, relatively stable, known as membrane flora; middle class bacteria, streptococcus digest, veillonella and excellent bacilli; the surface of e. coli and enterococci, can swim in the intestine, known as cavity flora. the antagonism between the layers flora, mutual cooperation, to maintain a dynamic equilibrium, in which the film anaerobic flora is a very important body's natural defense barrier that can prevent opportunistic pathogens such as e. coli colonization in the mucosa, but also can inhibit the overgrowth of opportunistic pathogens. intestinal flora micro-ecosystem is a very sensitive system, in severe stress or long-term systemic administration of large doses of broad-spectrum antibiotics, etc., the film significantly reduced the number of anaerobic bacteria, e. coli and other bacteria thrive conditions and continuous release of endotoxin to the intestine, since the film flora defense decreased, these opportunistic pathogens to colonize the intestinal mucosa, resulting in intestinal mucosal barrier damage, followed by the occurrence of intestinal bacteria, endotoxin translocation [338] . under normal circumstances, the liver is one of the major barriers preventing endotoxin translocation, via the portal vein into the liver hepatocytes and a small amount of the toxin can be kupffer cell depletion. in conditions such as stress, not only liver cell dysfunction, so the ability to reduce endotoxin detoxification and collaterals between the portal vein and the vena cava, causing an overflow of endotoxin from the liver into the systemic circulation. endotoxin absorbed into the bloodstream, which in turn may increase the intestinal epithelial cells of the intestinal microvascular endothelial cell damage and, in a vicious cycle [339] . when the body's defense system to produce responses in endotoxin, the innate immune system plays a major role. pathogens can be identified conserved receptors called pattern recognition molecules (pathogen-associated molecular patterns, pamps), including endotoxins of gram-negative bacilli, peptidoglycan grampositive cocci, lta and other cell wall composition and gram-negative bacteria, gram-positive bacteria such as dna. although a variety of pattern recognition molecules of different chemical structures, but they have similar characteristics: (1) characteristic structure in which different types of pathogens in a relatively constant conserved; produced by a pathogen, the host body without these molecules; survival or disease-causing pathogen is generally the essential, such as mutations, death or loss of a pathogen will pathogenicity. natural immune system to recognize the receptor molecule called pattern recognition receptors (pattern-recognition receptors, prrs), including cd14, toll-like receptor family (toll-like receptor, tlrs) and scavenger receptors. but in recognition of toxins, some differences exist between the different kinds of cells [340] . macrophages in addition to expressing cd14, tlrs and scavenger receptors and other associated endotoxin receptors on the cell surface, but in the cytoplasm also express the protein molecules nod1 recognizing toxins. cd14, tlrs are key receptors that mediate endotoxin within macrophage activation; and scavenger receptor has relationship with macrophage clearing and inactivating toxins [341] . kupffer cell is the main cell that clears the endotoxin in the liver. under physiological conditions, although there is still a small amount of bacteria and endotoxin via the portal vein into the liver, but kupffer cells will clear. kupffer cells are the most resident macrophages in the liver and are the largest number of resident cells in tissues. there is a theoretical speculation that if kupffer cells and on is very sensitive to endotoxin as other macrophages, the cell will be in constant activation, but in fact when kupffer cell engulfs, removes endotoxin, its itself is not activated by endotoxin, which suggests that in the treatment of endotoxin, kupffer cells have different mechanisms with other macrophages: kupffer cells treats endotoxin mainly depending on its phagocytosis. in the absence of serum, the phagocytic effects of kupffer cell on endotoxin can play a normal; and with the appropriate increase in endotoxin concentration, the phagocytic activity of kupffer cell was enhanced. the effect has something to do with phosphorylation events of two protein tyrosine residue individually weighting 11.8 million and 8.3 million. cd14 is the main receptor that mediates endotoxin activating kupffer cell, scavenger receptor is kupffer cells' important defense of receptor mediated kupffer cell to remove and inactivate endotoxin. there are four stages of the activation of kupffer cells, of which cd14 is the characteristic marker of cellular activation and function change. (1) the stationary phase; the performance of less number of kupffer cells, small shape, a number in the hepatic sinusoids, cd14 staining negative; (2) reaction period: for the local kupffer cells stimulate hyperplasia and systemic mononuclear macrophage intrahepatic accumulation; (3) pre excitation period: kupffer cell phenotype occurred transformation period, expressed cd14 cell membrane receptor, kupffer cell functional changes; (4) the activation period: the performance of nuclear transcription factor nf kb activation, cellular secretion cytokines [342] . the cd14 (cd14 membrane-bound, mcd14) and tlr4 endotoxin were activated by the neutrophil surface to activate the neutrophils by binding with the receptor. in addition to the expression of the high affinity endotoxin receptor cd14, the surface of the neutrophils also expressed the low affinity endotoxin receptor l-and the activated cells were activated by the receptor. in addition, the integrin is considered to be a low affinity endotoxin receptor for the surface of the neutrophils [343] . it is generally believed that the expression of mcd14 was not on the surface of endothelial cells and serum soluble cd14 (soluble cd14 (scd14) is mediated endothelial cell recognition of lps molecules, and tlr4 is involved in lps induced endothelial cell activation. lbp was transported to scd14 by endotoxin, and tlr4 was activated by lps/scd14 and activated endothelial cells in the endothelial cell membrane. scd14 in addition to mediated endothelial cell activation and also mediated by endothelial removal of endotoxin and lps/scd14/lbp form trimers and binding to endothelial cells, following the lps/scd14 endogenise, thus the removal of endotoxin [344] . the epithelial cells of the intestinal mucosa were consistently associated with the bacterial and its products, and the bacteria and its products could stimulate other types of cells and induce inflammatory response, but did not induce intestinal epithelial cell defense, this feature for colonic epithelial cells is particularly important, because if can react to the normal intestinal flora in intestinal epithelial cells, it will cause adverse effects on the body. but this does not mean that intestinal epithelial cells are immune cells, when suffered pathogens and their products invasion, intestinal epithelial cells produce normal response. description: intestinal epithelial cells with normal differentiation of natural bacteria and pathogenic ability, and the recognition system of subcellular localization. there are different endotoxin recognition mechanisms in the intestinal epithelium and the myeloid cells [327] . uncontrolled inflammatory responses (uncontrolled inflammatory response) has a relationship with infection, bacteremia, septicemia, sepsis, systemic inflammatory response comprehensive sign (systemic inflammatory response syndrome (sirs), compensatory anti-inflammatory response syndrome (compensatory antiinflammatory response syndrome, cars) and other related terms used for a long time, but also has an essential difference. out of control including inflammatory reaction syndrome (msas anti-inflammatory response syndrome, mars), a dynamic process of sirs and cars and the mixed antagonistic response syndrome, at present clinical many diseases occurrence and development are closely related. uncontrolled inflammation is a common pathological phenomenon in clinic, which is the important mechanism of the development of the complication after trauma. lps is one of the main factors that induce the uncontrolled inflammatory reaction in the most common. lps receptor on the monocyte/macrophage surface is the he initial factor for the body to recognize and start inflammatory reaction, also is one of the key links for the induction of uncontrolled inflammatory response. the concept of uncontrolled inflammatory response refers to inflammatory disorders then resulting in multiple organ dysfunction syndrome (multiple organ dysfunction syndrome. mods), emphasizes the importance of the of balance inflammatory/anti-inflammatory mechanism in the body, changes the limitations that in the past we only attached importance of the pathogenic effects of inflammatory factors. it is believed that the response of the body to the inflammatory factor is the dominant factor in the development of the whole body inflammatory reaction and mods. this concept is more focused than the previous focus on the dynamic changes of the whole process of inflammation. this can be divided into two types of mods: one is the early stage after the injury, that is, the speed hair style. the main blame is a strong inflammatory reaction induced by proinflammatory factors, and the other is a late phase of the disease, which is "late style", mainly due to the immune paralysis or worse immune disorders caused by cars or mars. the inflammatory is actually a kind of medium disease mainly caused by the chain reaction of cytokine. endotoxin is thought to be one of the most important predisposing factors in the chain reaction and can be referred for chain reaction "trigger" (trigger). endotoxin induced inflammation mechanism is mainly mediated by pamps that can induce cytokines such as il-1, tnf alpha and other active molecules synthesis, the formation of the cytokine network, has a very important role in the occurrence and development of infection. the excessive activation of cytokines can cause septic shock, and is the leading cause of death in patients with bacterial infections. accordingly, prrs plays an important role in innate immunity and inflammation, and it can distinguish the pathogens from self organization through prrs organism, which is characteristic of immune response [345] . inflammatory response syndrome systemic (sirs) is a systemic inflammatory response caused by any pathogenic factor to the body. the concept is first proposed by coris in 1985. 1991 august american college of chest physicians and critical care medicine to present the diagnostic standard of sirs, think to have the following each of the two or more than two, sirs can be established: (1) temperature > 38 deg c or < 36 deg c; ii heart rate 90 beats/min; (3), the breathing frequency > 20 times per minute or arterial blood carbon dioxide into pressure (paco 2 ) < 4.27kpa 32 mmhg; (4) peripheral white blood cell count >12 × 109/l or 4 × 109/l < or immature myeloid cells >10%. what should be paid attention to is that sirs is a common athophysiological state of body with severe inflammatory reactions, and should be differentiated from some abnormal factors such as leukemia or cause increase or reduction of white cells after chemotherapy. although the naming of sirs has been generally concerned, but some scholars have raised objection to the concept, for example sirs has following problems: the sensitivity and specificity of the diagnostic criteria is poor, has the same meaning with the "critical"; can not understand the pathophysiology of the original disease; is difficult to guide clinical trials and practice. the production of sirs can be divided into two cases, the sirs caused by the infection and the non infectious sirs. from the point of view of the clinical development process, sirs can be followed by injury immediately aroused, then known as the "single phase velocity hairstyle; also to start local, and later developed into a systemic sirs, namely after the initial shock is brief period of stability, later gradually intensified when sirs is called" dual phase delayed onset. either of the factors or the clinical development process, the systemic inflammation of the control of the uncontrolled, and ultimately can lead to mods [346] . the intestine is the biggest bacterial and endotoxin warehouse in the body. in severe trauma, systemic infection, intestinal ischemia and liver disease, there may be the occurrence of endotoxin. the main source is due to intestinal gram-negative bacteria in the excessive growth and reproduction, or due to increased intestinal permeability lps entry into the portal vein increased. if hepatic kupffer cell phagocytic function is low, the amount of endotoxin over the liver ability to remove endotoxin can "flood" (spill over) into the body of the loop and the endotoxemia formated. because of the endotoxin from the gut, so it is called intestinal endotoxemia (intestinal endotoxemia, ietm) [347] . hepatitis b patients are often accompanied with ietm. its formation mechanism is: the production and absorption of intestinal endotoxin increased. there is a large number of gram negative bacteria in the body's normal intestinal, so endotoxin in intestinal contents is very high, but the intestinal mucosal epithelial cells have stronger resistance to toxins so that endotoxin is not easy to run through the intestinal mucosa into the blood, even a small amount of endotoxin breaking through the intestinal mucosal barrier into the portal vein, will be swallowed up by the kupffer cells in the liver. severe hepatitis b when the intestinal flora disturbance, endotoxin increased, increased intestinal hyperemia, edema and the permeability of the intestinal mucosa; endotoxin itself can damage the mitochondria and lysosome of intestinal epithelial cells, leading to epithelial cell autolysis; endotoxin can cause intestinal microvascular contraction of the intestinal mucosa, reduce blood, intestinal ischemia, hypoxia, cause the intestinal mucosal barrier function decreased, increased the absorption of endotoxin; severe hepatitis, due to intrahepatic bile acid and bilirubin deposition in kupffer cell phagocytosis was inhibited, resulting in the removal of endotoxin in the endotoxin decreased; through the door body circulation circuit into the systemic circulation, resulting in blood within liver cells to escape kupffer toxin the phagocytosis and clearance, which aggravate endotoxemia; the endotoxin can also pass through the celiac lymphatic system into systemic circulation by thoracic duct. in addition, severe hepatitis patients with sepsis, spontaneous bacterial peritonitis, etc., in the release of endotoxin, so the formation of the endotoxin is exogenous [348] . in viral hepatitis and other basic diseases complicated with ietm and liver function failure are closely related and endotoxin can directly cause arterial vasoconstriction, the organ ischemia; endotoxin can activating endogenous clotting system, coupled with kupffer cell dysfunction, decrease delimination of blood coagulation or fiber soluble substances, easily lead to dic, so as to damage to multiple organs. endotoxin activated phospholipase a2 mediated membrane phospholipid degradation and lipid peroxidation, which is an important part of liver cell damage. nolan has pointed out that the effect of kupffer cell dysfunction induced by intestinal endotoxemia on liver and body, far more than the direct action the endotoxin, and production and release of inflammatory mediators and factors from kupffer cells activated by endotoxin are closely related. the occurrence of ietm affect hepatic energy metabolism, resulting in liver cell damage and necrosis, also caused hepatic microcirculatory disturbance, performing liver hemorrhagic necrosis. on the basis of severe hepatitis, it can accelerate liver function failure [349] . hepatic cellular jaundice the acute and chronic liver function is often accompanied by intrahepatic cholestasis jaundice, and ietm plays an important role in the occurrence of intrahepatic cholestasis.. endotoxin involves in liver cell damage mainly through activation of phospholipase a2, and inhibits the activity of na + − k + − atpase on liver canalicular membrane to make the bile excretion disorder, and then to cause intrahepatic cholestasis in the liver cells. endotoxin can start the peroxidation of liver parenchymal cells mitochondrial membrane lipid so that an increase in the content of oxygen free radical in blood, resulting in the disorder of energy generation, atp was reduced, so that the active uptake, metabolism and secretion of bile acid by liver parenchymal are short of energy, resulting in cholestasis [350] . the liver disease with ietm often causes the coagulation dysfunction, the serious person appears the different degree bleeding, in particular the severe liver disease patient may concurrent dic, endangers the life. violi found that, when liver dysfunction in patients with ietm, the expression of tissue factors on the surface of macrophages and endothelial cell factor induced by endotoxin increased, promoting the synthesis of tumor necrosis factor (tumor necrosis, factor, tnf) to increase, and thrombin generation increasing, activation of coagulation system in about 70% of patients, followed by hyperfibrinolysis, suggesting that ietm in liver dysfunction can be used as the warning signal as the activation of coagulation and fibrinolysis system and activate, and with the increased hepatic lesions, plasminogen activation decreases with endotoxin levels increased, thus plasminogen may decline with endotoxin induced by chronic consumption of dic on the microstructure of ii related factors, new blood can not correct. in fact, before variceal rupture bleeding, patients with liver function severely damaged already have the gastrointestinal mucosa extensive ischemia and erosion, which is the potential causes of bleeding, ietm in the process. and gastric h + at this time can occur abnormal reverse diffusion and stimulate mast cells to release histamine, may lead to mucosal blood vessel dilation and permeability enhanced. as a result, hemorrhage, edema; histamine and directly stimulate the secretion of gastric acid, so that an increase in the number of h + and reverse diffusion, lesions persisted, form a vicious circle [351] . it is important for the formation of ascites in ascites due to the obstruction of the hepatic vascular outflow tract obstruction. the initial vascular response to endotoxin was the rapid obstruction of the hepatic venous outflow tract and increased the portal pressure which may be related to endotoxin induced swelling of kupffer cells, liver cells with microvilli swelling, platelet aggregation and fibrin deposition effect, while others think that is endotoxin of blood vessels of the liver has a direct effect. ietm continuous damage to the liver cells, resulting in albumin synthesis and of hormones such as aldosterone de live function obstacles, thus affecting the renal function, and led to the emergence of the refractory ascites plays an important role in the process [352] . patients with severe liver disease always are complicated with the functional renal failure, hepatorenal syndrome (hrs). the patients with severe liver disease can be associated with the pre-renal azotemia and acute renal tubular necrosis, and there is a certain relationship with ietm. the clinical studies showed that the levels of no 3 --no 2 and endotoxin in serum of patients with liver cirrhosis were significantly higher. at the same time, plasma renin activity, aldosterone and vasopressin levels increased and urinary sodium excretion decreased. the mechanism about ietm induced by hrs is not clear, may be related to the following factors: leukotrienes (leukotrienes, lts) lts can lead to renal vasoconstriction, increased renal vascular resistance, reduced renal blood flow and renal blood redistribution, decreased glomerular filtration rate induced by hrs, in ietm lts generation and release increased obviously. in addition, liver dysfunction, liver's uptake, inactivation and excretory function of lts decline, causing blood concentration increased; the thromboxane a2 (thromboxane a2 txa2)/i2 (prostaglandin i2, prostaglandin pgi2) can contract renal arterioles, decrease glomerular filtration rate, while pgi2 and pge2 (prostaglandin e2, pg e2) is caused by on the role of txa2. in patients with severe hepatitis with ietm, elevated systemic levels of pgi2, reducing the renal vascular resistance, leading to renal vascular resistance txa2, reduce the renal blood flow and glomerular filtration rate, promote the formation of hrs; third, nitric oxide, nitrogen monoxide) nono can through vasodilatation of the systemic, resulting in effective circulating blood volume reduction and evoked hrs; endothelial endothelin (et) et caused renal cortical blood priming hrs; and platelet activating factor (growth factor (paf) endotoxin and platelet activating factor (paf) can lead to a decrease in cirrhotic rats cardiac ejection fraction, reducing blood flow to the kidney, and paf antagonist can improve hemodynamics changes [353] . the mechanism of endotoxin induced hepatic encephalopathy is not clear. we have known that lps can increase the permeability of blood brain barrier, promote intestinal toxic substances through the blood brain barrier (bbb), damage mitochondrial oxidative metabolism in brain cells, reduce oxygen utilization in patients with liver cirrhosis and disrupt energy metabolism of brain cells, induce brain edema. the clinical symptoms of chronic severe hepatitis with endotoxemia included in addition to fatigue, anorexia, tiresome of the oil, nausea, vomiting, yellow skin and sclera and, performance of endotoxemia. endotoxin can cause the release of histamine, 5-hydroxytryptamine (5-ht), prostaglandin, bradykinin, resulting in micro circulation expansion, venous blood volume reduction, decreased blood pressure, inadequate tissue perfusion, hypoxia and acidosis, and main symptoms and signs are: fever, elevated white blood cell count, bleeding tendency, heart failure, renal dysfunction, hepatic injury, nervous system symptoms and shock [354] . improve liver function this is the basic treatment of ietm. liver function improved can strengthen mononuclear phagocyte system function to help the endotoxin removal. it can also decrease portal vein pressure to relieve intestinal congestion, edema, hypoxia, improve the intestinal microenvironment, reduce production and lymph reflux, and lower door shunt. these all contribute to the prevention and treatment of ietm. clean the gut saline is available as enema if severe liver disease, which helps reduce intestinal endotoxin generation and absorption. decompensated cirrhosis is often accompanied by small intestinal bacterial overgrowth and intestinal flora disturbance. thus, the promotion of intestinal flora back to normal state help prevent and treat intestinal endotoxemia. a variety of bifidobacterium, lactobacillus can be selected. a synthetic disaccharide, it is not digested and absorbed in the small intestine, but can be broken down into lactic acid, acetic acid and other small molecules by the bacteria into the colon. such acidification of the intestine reduces the generation and absorption of endotoxin, and promotes the growth of intestinal bacteria, stimulates bowel movements so as to increases tool frequency and so on. in addition, the lactulose may have internal direct inactivation of toxins, prevents activation of macrophages to release cytokines. oral absorption of antibiotics can effectively suppress the generation of intestinal endotoxemia. patients with liver cirrhosis taking oral polymyxin e or neomycin, the level of plasma lps and no 2 -/no 3 -horizontal declines in synchronization. polymyxin b has an internal direct antitoxin effect [355] . it play a role by reducing intestinal absorption of toxins, inactivating toxins and inhibiting those lps-induced media by monocyte macrophages. the new anti-endotoxin therapy including interrupt endotoxin synthesis, binding or neutralizing its activity, preventing its interaction with the host effector cells, or interfering with toxin-mediated signal transduction pathways. therapeutic formulations include endotoxin analogs, antibodies, subunit vaccines, polymyxin combination column, recombinant human protein, small molecule inhibitors of endotoxin synthesis and intracellular signal transduction. bacteriophage producing a piece of short nucleotide sequence which plays the role of an antisense rna, blocking the synthesis of bacterial lps synthase. current clinical studies carried out an experiments in cloning of human anti-endotoxin lipid a light and heavy chain variable region. it laid the foundation for the next screening and expression that recombination between dna of antibodies and phage's succeeded. the clone45 is one kind of anti-polymyxin b (polymyxin b) monoclonal antibody of the igm class. it can play the role of anti-endotoxin shock by imitating the surface antigen structure of lipid a so as to substitute receptor antagonist of lipid a and lps blocking the causative link that the endotoxin induces inflammatory mediators [356] . isolating antibodies having a high affinity of various g-bacteria to prepare a chimeric monoclonal igg1 antibody sdz219-800 which have a therapeutic effect on the human endotoxemia. anti-endotoxin core glycolipid monoclonal antibody (antimonoclonal antibody r595) can prevent and treat the metabolic disorders in peritoneal infection with mods; it plays a significant role in conditioning in high catabolism, and can significantly improve metabolic disorders under the condition of abdominal infection associated with mods. bactericidal/permeability-increasing protein (bpi) bpi is a human endogenous protein, found primarily in neutrophils primary particles. its molecular amino-terminal and carboxy-terminal appear v-shaped structure planar symmetry. many amino acids to form a hydrophobic capsule hold lps's lipid a. it was reported that bpi has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin [357] . reconstructing hdl (high density lipoprotein, hdl) hdl can be used as an endogenous lps scavenging system, binding of bacterial endotoxin with high affinity to form a stable hdl-lps. lps-hdl complexation may contribute to a reduction in endotoxic activities in vivo by preventing lps (lipid a) from generating important transmembrane signals after binding to cells [358] . e5531 is the first generation lipid a analogue, which is derived from the lipid a structure from the endotoxin of rhodobacter capsulatus. it can block lps in cell culture without any endotoxin-like activity. e5531 can protect mice from lethal doses of lps, and viable e. coli infections in combination with antibiotics. in human healthy volunteers who are exposed to intravenous lps. e5531 also blocks the endotoxin response [359] . an anti-cd14 antibody cd14 has a very important role in monocyte-macrophage cell signaling. since epitopes of lps on the cell membrane at the binding site is the same material with soluble cd14, so we can develop a monoclonal antibody interfering with lps binding to cd14 and blocking to pass activation signals from immune effector cell [360] . tyrosine kinase and mitogen-activated protein kinase are involved in lps cellular signal transduction. build anti-endotoxin (lps) single-chain antibody gene and attempt to make it express in e, coli. the scfv gene was successfully constructed and gst scfv fusion protein highly expressed in e. coli was obtained. glucocorticoids, including the synthetic glucocorticoid dexamethasone, are recognized for their anti-inflammatory properties and have the ability to inhibit the production of proinflammatory cytokines such as tnf-α. in intestinal ischemia-reperfusion methylprednisolone pretreatment can prevent endotoxemia. combined lps and dexamethasone treatment at 120 h significantly changed tnf-α [361] . this points that glucocorticosteroids added before or during stimulation of macrophages can prevent tnf release, after which the administration would be invalid. in fact, it is very difficult to use corticosteroids before tnf release. current clinical anti -tnf antibodies and tnf antagonist use exists, and before many scholars have obtained a more satisfactory results of blocking or neutralizing excessive tnf with anti-tnf. although the clinical symptoms improved, yet the survival rate is not higher than expected. the effect of anti--tnf clinical application needs further evaluation. the first proposed concept is sequential organ failure, based on which multiple organ failure (multiple system organ failure, msof or mof) is put forward, and in 1980 diagnostic criteria is developed, but it reflected the end-stage, denied reversibility, ignore the dynamic development from organ dysfunction to failure. therefore, the us accp/sccm proposed to replace the concept with mods. mods emphasized an early phase of organ dysfunction before overt failure occurred. it is defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention." [362] organ dysfunction may be relative, or can be absolute; with extension of time, mods can increase or reverse. thus, the term. mods was coined to indicate the wide range of severity and the dynamic nature of this disorder, which contributes to early diagnosis and treatment of patients and is more in line with clinical practice. there are two relatively distinct (although not mutually exclusive) pathways by which mods can develop: in primary mods, there is a direct insult to the organ that becomes dysfunctional. examples of such direct insults include gastric aspiration in the lungs or rhabdomyolysis in the kidney. this direct insult causes: an inflammatory response that is localized, at least in the beginning, to the affected organ. secondary mods is a consequence of trauma or infection in one part of the system that results in the systemic inflammatory response and dysfunction of organs elsewhere [363] . secondary mods means not directly caused by damage, but to experience the "second strike", the first blow can make the immune system in a preactivated state under which inflammation lost control and significant sirs appears, then the following second strike can quickly cause multiple distant organ dysfunction and easily form sepsis with the basis of sirs/cars. this type of mods often develops as the following model: the original cause → stress → immune wpw → sirs/cars → infection → sepsis → mods → mof. the process of severe hepatitis, can cause intestinal damage and massive release of endotoxins into the blood, leading to intestinal endotoxemia (ietm). endotoxin is a powerful trigger for complement activation, and then these complement can activate "cascade effect" (cascade) to release oxygen free radicals, prostaglandins, endorphins, paf, cytokines and other inflammatory mediators to cause cytotoxicity, the error of microcirculation and tissue metabolism, eventually leading to the occurrence of mods. in this case, we emphasize mods originated in continuous, uncontrolled inflammation and factors causing systemic inflammatory response can be induced mods, including bacteria, fungi, parasites, viruses, toxins and other infectious agents. it is a debate of long-century problems that whether endotoxin has effects on hearts. the late 1970s a large number of experiments prove endotoxins has a role of heart. it enables reduction of coronary blood flow, decrease of total coronary vascular resistance, and have meanings in heart failure. studies have shown that endotoxin can damage myocardial mitochondria, muscle paddle net, muscle membrane, contractile proteins etc., leading to cell membrane system damage, energy metabolism. past research in cardiac dysfunction under endotoxin shock did not attract enough attention, for they think that the heart is the final failure among all organs so that clinical treatment value is little. now people's awareness has completely changed that heart dysfunction can occur early in sepsis or septic shock. therefore, early identification and prevention of the occurrence and development of cardiac dysfunction may have some clinical value for treatment of septic shock and other serious complications [364] . clinical gram-negative bacteria sepsis often complicated by adult respiratory distress syndrome, or server development of mods. in this pathological process, the high biological activity of endotoxin plays an important role. endotoxins often involving the lungs firstly, and the pathogenesis of non-injury may be associated with the direct damage on endothelial cell by endotoxin through complement pathway and induction of cytokines [365] . the liver is the major site of endotoxin on clearing and detoxifying and the place where clinical gram-negative bacteria sepsis often can be complicated, and it is also the primary organ suffering attacks by toxins. it is generally believed that toxins in the liver circulating mainly from the gut and liver dysfunction is closely related to the formation of endotoxemia. histological examination revealed the inner toxins damage the liver cells, showing sinusoidal congestion, dieldrin expansion chamber, kupffer cell swelling, endoplasmic reticulum、mitochondria swelling, crest destroy and lysosomal activation etc. [366] . the liver is the body's largest metabolic organ, and many cases of acute liver failure often involve other organ complications, which has become an important factor in determining the prognosis. endotoxins may cause the reduction of liver nutritional blood flow, mitochondrial oxygen metabolism, interfering with sugar metabolic pathways in liver leading to metabolic disorders. various damaging factors (such as gastrointestinal disorders, ischemia, immunocompromised, dysbiosis) promote absorb of intestinal bacteria and toxins and displacement via the portal vein, the lymphatic system into the systemic circulation. on the one hand these infectious agents can directly damage liver cells, or mediate hepatic injury whether by kupffer cell; on the other hand it can induce systemic inflammatory response by the monocyte-macrophage cells to release the media, both leading to organ perfusion disorder, affecting protein synthesis and energy metabolism, eventually resulting in severe sepsis, mods and even death. the effect of endotoxin on kidneys is not clear yet. in the early stage, it can affect the kidneys, decreased its blood flow renal via vasoconstriction. when endotoxemia is complicated by renal failure, the mechanism of glomerular filtration rate decreasing is unclear. the pathophysiology of aki in sepsis is complex and multi-factorial and includes intrarenal hemodynamic changes, endothelial dysfunction, infiltration of inflammatory cells in the renal parenchyma, intraglomerular thrombosis, and obstruction of tubules with necrotic cells and debris [367] . the relationship between endotoxin and dic is quite complicated. dic is considered to be an important incentive of mods, especially patients with severe sepsis with dic having a highly possibility of developing mods, what's more, the prognosis is very poor, and the mechanism is multifaceted. endotoxin can start the endogenous coagulation system directly or via activating factor xii (hageman factor) by damaged endothelial cells, also can act on the monocyte-macrophage cells, stimulate the release of tissue factor to trigger the extrinsic coagulation pathway [368] . in clinical practice it has been noted that serious infections is very possible to be complicated by gastrointestinal failure. the intestine is the biggest reservoir of bacteria in the body and leakage of bacteria or microbial products, notably lps, from the lumen of the gut into the systemic compartment, leads to initiation or amplification of a deleterious inflammatory response and mods [369] . after endotoxins challenging the gastrointestinal mucosa, it initially shows mucosal telangiectasia, interstitial edema and hemorrhage. microcirculation leading to damage of lysosomal and release of proteases in the cell, cell degeneration and necrosis. in addition, mucosal cellular energy metabolism decrease, h + reverse diffusion, prostaglandins and bradykinin further aggravate mucosal damage. at the same time destroy of the gastric mucosal barrier also make a lot of bacteria and endotoxins pass through the gastrointestinal mucosa, migrate to the blood circulation, the lymphatic system and the abdominal cavity etc., leading to systemic multi-system organ damage. clinical characteristics of mods 1. organ failure usually do not result directly from the primary injury. there is a certain time interval from the primary injury to organ failure. 2. not all of infection have bacteriological evidence, and more than 30% of patients and autopsy found no infected lesions. thus, to identify and treat the infection may not be able to improve the patient's survival. 3. mods may have perfectly healthy organ involved, and it is ferocious and rapidly progresses. once happened, it is difficult to depress in the event almost, so often with a high mortality rate. 4. in pathology, mods lacks features, the affected organ only showing acute inflammation, such as inflammatory cell infiltration and so on, and these changes are very inconsistent with severe clinical manifestations, and once restored, patients do not have any clinical sequelae. 5. mods is closely with shock and infection. shock, infection, injury (including trauma and surgery, etc.) are the three main causes of mods. 6. generally the later period of shock will typesetting idc and mods, and the order of occurrence of mods usually is the lungs, liver, kidney, gastrointestinal tract, finally the heart. the characteristic clinical manifestations 1. instability of circulation due to a variety of inflammatory mediators have effects on the cardiovascular system, the circulation is most likely involved. almost all cases, at least in the course of the early and middle will be in highpower type of cycle of "high ranked low resistance". cardiac output up to 10 l/ min or more and low peripheral resistance cause shock and need vasopressors to maintain blood pressure. 2. high metabolic systemic infection and mods are usually accompanied by severe malnutrition. its metabolic mode has three salient features: (1) persistent high metabolism, metabolic rate up to 1.5 times more than normal; (2) abnormalities of energy pathway. in starvation, the body obtain energy mainly through the decomposition of. however, with systemic infection, the body will get energy by breaking down proteins while the use of sugar is limited and fat utilization may increase early, fall later; (3) poor response to exogenous nutrient, supplement of exogenous nutrition can not effectively prevent itself consumption, which suggests that a high metabolism itself has a "mandatory" also known as "autophagy metabolism." high metabolic may have serious consequences. first, protein malnutrition result from it will cause serious damage to the structure and function of the enzyme system of organs; secondly, imbalance of branched-chain amino acids and aromatic amino acid which makes the latter formate into a pseudo-neurotransmitter, then further lead to dysfunction of nerve. 3. hypoxia in tissue cells at present many scholars believe that the high metabolic and circulatory disorders often cause oxygen supply and oxygen demand does not match, so that the tissues of bodies are in a hypoxic state, mainly clinically manifesting "oxygen supply dependency" and "lactic acidosis. ". currently mods still lacks an unified diagnostic criteria, and any one of the diagnostic criteria of mods is difficult to reflect the entire contents of organ dysfunction, so in clinical practice we can select one according to our own specific situation. 1. the main contents from national critical care medicine conference standard in 1995 are: (1) respiratory failure: r > 28/min; pao 2 < 6.7 kpa; pco 2 > 5.89 kpa; pao 2 /fio 2 ≤ 26.7 (200 mmhg); p (aa) do 2 (fio 2 1.0) > 26.7 kpa (200 mmhg); x-ray of chest shows alveolar consolidation ≥1/2 lung (which have more than three or three); (2) renal failure: except prerenal factors, little or no urine, serum creatinine, increased blood urea and nitrogen levels, exceeding more than twice the normal value; (3) heart failure: systolic blood pressure < 80 mmhg (10.7 kpa), sustained more than 1 h; ci <2.6 l/(min · m 2 ); ventricular tachycardia; ventricular fibrillation; degree atrioventricular block; resuscitation after cardiac arrest (with which three or more); (4) liver failure: total bilirubin>34 μmol/l; liver enzymes increased more than 2 times compared with the normal; prothrombin time > 20s; with or without hepatic encephalopathy; (5) dic: platelets 100 × 10 9 /l; prothrombin time and partial thromboplastin time prolong 1.5 times, and fibrin degradation products increase; systemic hemorrhage; (6) brain failure: glasgow score below 8 means coma, and less than 3 points means brain death. 2. the sooner the primary diseases or the primary risk factors are eliminated or controlled, the greater the possibility of organ recovery is. 1. to effectively rescue and debride as soon as possible, prevent infection, prevent ischemia-reperfusion injury, use a variety of supportive care; 2. to reduce stress response, mitigate and shorten high metabolism and the magnitude and duration of glucocorticoid receptor; 3. to pay attention to the patient's breathing and circulation, as soon as possible to correct hypovolemia and hypoxia; 4. 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tubular dysfunction in men lipopolysaccharide precipitates hepatic encephalopathy and increases blood-brain barrier permeability in mice with acute liver failure early use of polymyxin b hemoperfusion in abdominal septic shock: the euphas randomized controlled trial polymyxins as novel and safe mucosal adjuvants to induce humoral immune responses in mice neutrophil phagocytosis and killing: normal function and microbial evasion. the neutrophil modulation of endotoxic activity of lipopolysaccharide by high-density lipoprotein toll-like receptor 4 modulation as a strategy to treat sepsis cd14 controls the lps-induced endocytosis of toll-like receptor 4 macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and antiinflammatory agents: key role for tnf-α the accp-sccm consensus conference on sepsis and organ failure sepsis and the heart endotoxin and lung injury the role of intestinal endotoxin in liver injury: a long and evolving history pathophysiology of septic acute kidney injury: what do we really know? sepsis, thrombosis and organ dysfunction bile high-mobility group box 1 contributes to gut barrier dysfunction in experimental endotoxemia key: cord-022555-a7ie82fs authors: nan title: digestive system, liver, and abdominal cavity date: 2011-12-05 journal: the cat doi: 10.1016/b978-1-4377-0660-4.00023-5 sha: doc_id: 22555 cord_uid: a7ie82fs nan these complex pathways highlight the need to consider the whole cat and not just the cat's gastrointestinal vomiting can be defined as the ejection of part or all of the contents of the stomach and/or upper intestine through the mouth, usually in a series of involuntary spasmodic movements. the disturbances in gastrointestinal (gi) motility are coordinated with respiratory and abdominal muscle contractions and mediated by the central nervous system (cns). vomiting begins with retching, a series of brief negative intrathoracic pressure pulses that coincide with positive abdominal contractions. these pressure changes occur as a result of repeated herniations of the abdominal esophagus and cardiac portion of the stomach into the esophagus. during retching, food freely moves back and forth in the esophagus, which is now dilated because of the ingesta. ultimately, the diaphragm rapidly moves cranially, resulting in positive intrathoracic pressure that leads to expulsion of these contents. 12 vomiting is such an active process that it seems to involve the whole cat, and so it is little wonder that it concerns owners so much. since vomiting is mediated by the cns with input and influence from just about anywhere in the body, it is important to summarize this physiology so it can be appreciated when managing clinical cases. vomiting results from stimulation of the "vomiting center," which is located in the brainstem; there are four main pathways that stimulate the vomiting center, 12 and these are summarized below and in figure 23 (though not all older cats have grown out of this habit). some extragastrointestinal problems, such as hyperthyroidism and renal disease are more likely to occur in older cats. most texts and references instruct clinicians to distinguish between vomiting and regurgitation, with the latter noted as being quite passive. 3, 11, 12 in practice, it can be hard to make this distinction, because it is the author's experience that cats with esophageal disease can have quite forceful, spasmodic movements when ejecting ingesta by regurgitation-although it is also possible for regurgitation to be a passive process. given that the physiology of vomiting, as described above, results in ingesta being forced to and then evacuated from the esophagus, it is hardly surprising that it can resemble regurgitation. fortunately, regurgitation and esophageal disease do vary from vomiting in other ways! vomiting 3. blood and urine testing 4. imaging (radiography, ultrasonography) 5. biopsy samples 6. treat and manage underlying problem the decision to proceed to steps 4 and 5 is based on the assumption that the prior steps have narrowed down the underlying cause as gastrointestinal, pancreatic, or hepatic in origin. the important aspects of the clinical history are given in chronic kidney disease. the author has found that some cats with dental disease can gorge their food, resulting in vomiting; so, paying attention to the state of the teeth and gums is important. of course, some cats have multiple problems, and correction of dental disease may not resolve vomiting if there is another process. in the examination, it is also important to note consequences of both the underlying process and the vomiting itself; these include the demeanor of the cat, hydration status, and abdominal pain. the physical examination findings, together with the clinical history, help determine the next appropriate steps. well cats that are not continually vomiting and are appropriately hydrated, with no other specific signs, may be treated as outpatients by fasting them for 24 hours, then returning to food with a bland diet, such as plain cooked chicken or commercial, low-residue prescription diets designed for this purpose. follow-up is important to ensure signs do not progress. cats with nonspecific signs may require supportive care with subcutaneous or intravenous fluids and perhaps analgesia (with opioids). if clinical signs do not resolve, the pursuit of a specific diagnosis should be attempted. the practitioner must ask the following important questions: • are ancillary tests appropriate? • is supportive care necessary? • are any medications required? routine serum/plasma biochemistries, hematology, urinalysis, and total thyroxine (t 4 ) (for older cats) testing is not only important to distinguish primary from secondary gastrointestinal disease but to look for consequences of vomiting that may need to be addressed, such as hydration status and electrolyte abnormalities. careful interpretations should be made. severe azotemia, even with hyperphosphatemia, can occur as a result of primary gastrointestinal disease, and the distinction from renal disease usually requires an assessment of urine specific gravity. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity (fpli) tests are useful markers of intestinal and pancreatic disease, 7, 8, 9, 10 but it is important to note that they mostly do not give a precise diagnosis. more detail about the utility of these tests is noted below in the section approach to the cat with diarrhea. is usually preceded by the cat licking its lips, salivating, or making attempts to swallow. regurgitated ingesta is often in a tubelike structure and if undigested can be covered with frothy saliva. partially digested food suggests vomitus, and the presence of bile or digested blood confirms this. it is important to determine if the cat vomits regularly. many owners have seen their cats vomit on a regular basis with no evidence of the cat being unwell, and this is noted frequently in the veterinary literature. 3, 12 hairballs can cause gastric irritation, and it may be that eating quickly also stimulates the peripheral sensory receptors that contribute to vomiting. if a cat does vomit regularly, it is important to assess if the cat is presenting for a change in the vomiting pattern (e.g., frequency or timing in relation to eating) and if the cat is unwell in any way, such as anorexia or weight loss. the pattern of vomiting is important in all cases, because cats presenting with acute gastritis usually have a sudden onset of frequent vomiting compared with those with chronic disease processes that may vomit every few days. the timing in relation to eating can be helpful, because the stomach should empty by 6 to 8 hours after a meal; so, vomiting longer than 8 hours after a meal can suggest motility or retention disorders. the description of the vomitus can be helpful. if bile is present, the pylorus is not obstructed; the presence of blood (digested or fresh) indicates ulceration. hair in the vomitus can indicate hairball gastritis, and the possibility of trichobezoar obstruction should be considered. access to foreign bodies or toxins is an important aspect of the clinical history. has the cat been seen playing with an insect, mouse, or other prey? are there any medications unaccounted for (e.g., a dropped aspirin tablet)? are lilies present in the house? vomiting is the major sign of gastric disease, but given the number of potential organ systems that can be involved, a thorough physical examination should be undertaken. because linear foreign bodies are a common cause of vomiting, all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of string caught there. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see . a thorough examination may reveal specific signs, such as a palpable thyroid nodule and tachycardia in the case of hyperthyroidism or palpably small kidneys with and analgesia, many cats recover uneventfully. one survey assessed that 83% of cats undergoing exploratory laparotomy survived the hospitalization, and although complications occurred in 26% of cats, these were more likely to be associated with the underlying disease process and not surgery or anesthesia. 4 laparoscopy is not readily available in all veterinary clinics. this alternative is less invasive and allows exploration of the abdomen but not as thoroughly as with laparotomy. organs are usually exteriorized for biopsy. there is the possibility of anesthetic complications associated with insufflating the abdomen. endoscopy is the least invasive procedure and is the only alternative that allows examination of the intestinal lumen. this option limits the parts of the gastrointestinal tract that can be biopsied; it does not allow examination or sampling of any other part of the gastrointestinal tract and does not enable full-thickness biopsy samples. one study found that, of cats investigated for gastrointestinal disease, 9 of 33 cats (27%) had no pathology recognized proximal to the jejunum (i.e., the effective length of diagnostic endoscopes would have precluded diagnosis), and other organs were affected in 9 of 10 cats with inflammatory bowel diseases and 7 of 8 cats with intestinal small cell lymphoma. 1 careful case selection for endoscopy from survey ultrasonography can reduce the number of missed diagnoses from endoscopy, but the possibility still remains. the quality of endoscopically obtained biopsy samples varies greatly with the skill of the endoscopist. it has been stated that "it is exceedingly easy to take inadequate tissue samples with a flexible endoscope." 5 in an assessment of endoscopically obtained biopsy samples, two laboratories were compared, one that received samples from any practitioner and the other that received samples only from practitioners trained to take, mount, and submit endoscopy samples. all slides were reviewed by three pathologists who found that, of samples from the first laboratory, 15% of the slides were considered inadequate for diagnosis, 71% were considered questionable, and only 14% were adequate. by comparison, in the second laboratory (with samples from experienced practitioners) 0% of slides were inadequate, 21% were questionable, and 79% were considered adequate for diagnosis. 13 in the case of distinguishing between lymphocytic intestinal infiltrates (commonly known as inflammatory bowel disease) and lymphocytic neoplasia (small cell lymphoma), endoscopically obtained samples can give an incorrect diagnosis. 2 many of these problems can be minimized with experienced operators and careful case selection from prior ultrasonography. radiography is most useful for identifying foreign bodies or signs of intestinal obstruction from other causes. the major findings are noted below in the section intestinal obstruction. contrast radiography can aid the diagnosis for both discrete and linear foreign bodies but should be used with caution, because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium irritates the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. 6 ultrasonography is a useful diagnostic adjunct and helps to detect and characterize localized thickening of the stomach or intestinal wall, lymphadenopathy, radiolucent foreign bodies, and changes in the size and echogenicity of the pancreas, liver, kidneys, or spleen. abdominal effusions can be assessed and sampled. ultrasound-guided fine-needle aspiration can be used to sample masses, bile, or peritoneal fluid. it should be recognized that in most cases of gastrointestinal disease, imaging will not give a definitive diagnosis and biopsy will be required, usually using either endoscopy or laparotomy. ultrasonography can be a considered as a means to "survey the field," assessing • the nature of the underlying disease, such as • thickened intestines with or without discrete layers • lymph node involvement • other organ involvement • the location of disease, for example, • diffuse or focal • proximal duodenum (reachable by endoscope) versus distal ileum these factors may be used to assess the appropriateness of endoscopy versus laparotomy to obtain diagnostic samples. most commonly used antiemetics all control vomiting by different mechanisms and include mirtazapine, metoclopramide, dolasetron/ondansetron, maropitant, and the phenothiazines (tables 23-2 and 23-3) . metoclopramide functions both as an antiemetic and prokinetic in cats, while cisapride functions solely as a prokinetic. mirtazapine, a piperazinoazepine, antagonizes the presynaptic alpha 2 -adrenergic receptor, increasing noradrenergic and serotonergic neurotransmission; the primary mechanism targeted for its use is as an antidepressant in humans. mirtazapine is also a potent antagonist of the postsynaptic serotonergic receptors (5-ht 2 and 5-ht 3 ) and histamine h 1 receptors. because of its antiserotonergic and antihistaminic effects, mirtazapine is used as an entiemetic and appetite stimulant in cats. anorexia is a common clinical problem in ill cats, and in some anorexic or partially anorexic cats the use of an appetite stimulant as adjunctive therapy to nutritional support (i.e. feeding tubes) may be of clinical benefit. prior to the development of mirtazapine, cyproheptadine was used as an appetite stimulant in cats, with variable clinical results. recently, the pharmacokinetics and pharmacodynamics of mirtazapine have been reported in cats. in a group of healthy cats, mirtazapine was found to be an effective appetite stimulant, with a shorter half-life than that reported in humans. the recommended oral dose is 1.88 mg/cat every 48 hours. 55a in humans, age and kidney and liver dysfunction affect mirtazapine metabolism (hepatic cyp 450 enzymes) and clearance (excreted in urine and feces), suggesting that dose adjustment may be necessary. 69a side effects reported in cats treated with mirtazapine include behavior changes (vocalization and interaction), tremors, muscle twitching, and hyperactivity. 9a,55a metoclopramide is both an antiemetic and prokinetic drug that acts peripherally on the gastrointestinal tract and centrally within the central nervous system (cns). at low doses metoclopramide inhibits dopaminergic (d 2 ) transmission, and at higher doses it inhibits serotonergic 5-ht 3 receptors in the chemoreceptor trigger zone (crtz). 15, 23 metoclopramide also acts peripherally as a prokinetic at the level of the gastrointestinal smooth muscle of the stomach and duodenum, triggering gastric emptying and duodenal contractions. multiple mechanisms mediate metoclopramide's prokinetic activity, including augmentation of acetylcholine release and increased smooth muscle sensitivity to cholinergic neurotransmission, which may in part be because of antagonism of dopamine, but more recently, serotonergic 5ht 4 receptor activation has been suggested. 23, 56 metoclopramide has been reported to increase the lower esophageal sphincter tone in humans, 20 although in cats metoclopramide's affect on the lower esophageal sphincter is reported to be weak. 32 adverse central nervous system, extrapyramidal signs occur secondary to dopamine (d 2 ) antagonism, including excitement and behavior changes. extrapyramidal signs are most often seen at the higher doses needed to block 5-ht 3 receptors. because of metoclopramide's prokinetic properties, an intestinal obstruction should be ruled out prior to its use. dopamine is a less important neurotransmitter in the chemoreceptor trigger zone of cats than alpha 2adre nergic and 5-ht 3 -serotonergic receptors, suggesting that d 2 -dopaminergic antagonist may be a less effective antiemetic in cats. clinically metoclopramide commonly controls vomiting in cats, although this clinical response may be secondary to 5-ht 3 antagonism and/ or its prokinetic effects. 32, 44 extrapolated from the short elimination half-life of metoclopramide in dogs (90 minutes), frequent be clinically significant side effects. phenothiazines have the potential to lower the seizure threshold; their use is not recommended in patients with a known seizure history. other cns-associated side effects linked to d 2 antagonism occur at higher doses and produce extrapyramidal signs, including rigidity, tremors, weakness, and restlessness. antagonism of the histaminergic receptors carries the risk of sedation. because of the need for frequent dosing (0.2 to 0.4 mg/ kg subcutaneously every 8 hours) and the risk of hypotension and sedation, the clinical use of phenothiazine antiemetics is limited to hospitalized patients with refractory vomiting and should be avoided in patients who are dehydrated or hypotensive. cisapride is a serotonergic 5-ht 4 agonist that increases propulsive gastrointestinal motility from the lower esophageal sphincter to the colon. cisapride binds serotonergic 5-ht 4 receptors in the myenteric plexus, increasing the release of acetylcholine in gastrointestinal smooth muscle. in dogs cisapride has greater prokinetic activity in the stomach relative to metoclopramide. 29 cisapride has no direct antiemetic effect, although it is indicated in a vomiting cat with colonic dysmotility secondary to megacolon. colonic distention can trigger the vomiting reflex in cats. cisapride induces colonic smooth muscle contractions in cats with megacolon that is dependent on the influx of extracellular calcium and is only partially cholinergic dependent. 30 other potential indications include refractory generalized ileus or gastroesophageal reflux. dosage recommendations based on the pharmacokinetics in healthy cats is 1.5 mg/kg orally every 12 hours. 41 prior to the use of cisapride, an intestinal obstruction should be ruled out because of its strong prokinetic effects. side effects reported in humans are cramping and diarrhea. potentially life-threatening side effects include qt prolongation and ventricular arrhythmias, the primary concern in humans that led to cisapride's removal from the market in the united states. 47 in cats qt prolongation associated with cisapride administration requires 20 times the therapeutic dose. 37 because of the risk of prolongation of the qt interval and ventricular arrhythmias, the concurrent use of cisapride and dolasetron is not recommended. 14 other potential drug interactions associated with cisapride include concurrent therapy with azole antifungals (ketoconazole and itraconazole), because of their inhibition of hepatic cyp3a isoenzyme system and the inhibition of cisapride metabolism. 47 diet trials are commonly used in cats with idiopathic gastrointestinal signs or in cats with suspected or known intermittent dosing or delivery by a constant rate infusion (cri) is necessary. empirical dosing in cats is 0.2 to 0.4 mg/kg subcutaneously or orally every 8 hours or 1 to 2 mg/kg/day as a cri. approximately 25% of metoclopramide is excreted in the urine, thus dose reduction is recommended in cats with underlying renal azotemia. 42 dolasetron and ondansetron are selective serotonin antagonists that inhibit central and peripheral 5-ht 3 receptors. their main antiemetic effect is through antagonism of the peripheral 5-ht 3 receptors in the gastrointestinal tract. in cats 5-ht 3 antagonism of the crzt is also likely important in the antiemetic effect of dolasetron and ondansetron. dolasetron and ondansetron were originally used for vomiting secondary to chemotherapy because of their superior clinical efficacy. the clinical use of dolasetron and ondansetron in cats has not been associated with reported side effects, and experimental studies report minimal toxicity in animals at doses 30 times the antiemetic dose. 15 side effects reported in humans include headaches, elevated liver enzymes, rare hypersensitivity reactions, prolongation of the qt interval, and arrhythmias. 14, 24 dolasetron is commonly used for parenteral administration and ondansetron for oral administration, dictated primarily based on the tablet sizes available and cost. recommended dosing of dolasetron is 0.5 to 1 mg/kg intravenously every 24 hours and ondansetron 0.5 mg/ kg orally every 12 hours. maropitant is a neurokinin-1 (nk-1) receptor antagonist, blocking the binding of substance p to the nk-1 receptors located in the emetic center, crtz, and the enteric plexus. 55 in cats maropitant has been reported to be efficacious in treating xylazine-induced vomiting and motion sickness. 31 recommended dosing in cats is 1 mg/ kg intravenously, subcutaneously or orally every 24 hours for up to 5 days. 31 maropitant is reported to be well tolerated in cats. prochlorperazine and chlorpromazine are considered broad-spectrum antiemetics by antagonism of d 2dopaminergic, histaminergic (h 1 and h 2 ), and cholinergic (muscarinic) receptors within the crtz and, at high doses, the alpha-adrenergic receptors (alpha 1 and alpha 2 ) within the vomiting center. in cats alpha 2 -receptors play a key role in emesis (recall xylazine is the emetic of choice in cats), suggesting cats may be more sensitive to the antiemetic effects of the phenothiazines. prochlorperazine and chlorpromazine produce an antiemetic effect at relatively low doses, thus avoiding profound sedation; although, because of antagonism of the alpha-receptors, vasodilation and hypotension can hyperacidity alone is not considered a common cause for vomiting in cats, but famotidine is effective in treating vomiting in cats associated with gastric ulcers or gastritis. recommended dosage in cats is 0.5 mg/kg every 12 to 24 hours. ranitidine is also a competitive inhibitor of the h 2 receptor associated with gastric parietal cells. in addition, ranitidine increases lower esophageal sphincter tone and functions as a prokinetic agent (increasing gastric emptying and stimulating intestinal motility, including colonic motility), because of its anticholinesterase food hypersensitivities. dietary strategies used to control vomiting in cats focus on either a highly digestible diet or an elimination (novel protein/carbohydrate or hydrolyzed protein) diet. 72 the empirical use of elimination diets in cats is reported to be relatively successful, with approximately 50% of cats with idiopathic gastrointestinal signs responsive to a novel protein/carbohydrate diets within 2 to 3 days. 28 interestingly, traditional diet trials are recommended for a minimum of 8 to 12 weeks, but in this group of diet-responsive cats with chronic gastrointestinal disease, clinical improvement was reported within days. 28 thus if a cat is going to be diet responsive, clinical improvement to a diet trial should be noted relatively early. highly digestible diets enable more effective absorption and assimilation of nutrients in the face of a compromised digestive tract. these diets contain highly digestible proteins and carbohydrates, moderate to low fat, soluble fiber but low concentrations of insoluble fiber, and are supplemented with omega-3 fatty acids. these diets are recommended when food allergy or intolerance is suspected. these diets contain a single highly digestible novel carbohydrate source and novel protein source. alternatively, diets formulated with hydrolyzed proteins can be used as an alternative to novel protein/carbohydrate diets. see tables 23-4 and 23-5 for information on gastrointestinal ulcers. famotidine has no direct antiemetic effect but is a competitive inhibitor of the histamine (h 2 ) receptors associated with the gastric parietal cells. the h 2 -receptor is the dominant receptor involved in gastric acid secretion. h 2receptor antagonism is reported to result in a 70% to 90% reduction in acid production. 13 famotidine is more effective at suppressing gastric acid secretion relative to ranitidine. famotidine is well tolerated, although, with chronic therapy, there is the potential for hypoacidity and gastric bacterial overgrowth. in humans dose reduction is recommended in association with renal dysfunction. 21 famotidine is not an inhibitor of the hepatic microsomal cytochrome p-450 enzyme system, therefore significant drug interactions are not anticipated. activity. 40, 54 significant drug interactions associated with hepatic microsomal cytochrome p-450 enzyme system inhibition are not a clinical concern with ranitidine. 46 an adverse effect to be aware of in cats treated with ranitidine is transient hypotension associated with ranitidine administered as an iv bolus. 19 in humans dose reduction is recommended in patients with renal azotemia. 39 ranitidine is effective in decreasing gastric acid in cats. 22 ranitidine would be a logical choice in a cat with gastrointestinal ulceration and/or atony. the reported dosage recommendation for ranitidine in cats is 3.5 mg/ kg orally every 12 hours or 2.5 mg/kg intravenous every 12 hours. 19 omeprazole omeprazole is a proton pump inhibitor that targets the h + /k + atpase pump on the luminal surface of partial cells. omeprazole is effective at suppressing parietal cell acid secretion, and its effects persist for ≈24 hours after drug withdrawal because of drug accumulation in the parietal cell (by ion trapping). indications for omeprazole therapy are for the treatment and prevention of nonsteroidal antiinflammatory drug (nsaid)-induced ulcers. 9 omeprazole is enteric coated to prevent its degradation by gastric acid; therefore oral formulations should not be crushed. based on human studies, omeprazole is a hepatic microsomal cytochrome p-450 enzyme inhibitor with known drug interactions with diazepam. 2 the extent of clinically significant drug interactions in cats has yet to be studied. omeprazole is reported to be effective in reducing gastric acid secretion in cats. 22 the recommended empirical dosage in cats is 0.5 to 1 mg/kg orally once daily. long-term use in humans 33 and dogs 11 is associated with gastric polyps and parietal cell hyperplasia, respectively, but the effect of long-term use in cats is currently unknown. sucralfate is a disaccharide complexed with aluminum that dissociates to sucrose octasulfate and aluminum hydroxide upon exposure to gastric acid. the sucrose octasulfate spontaneously polymerizes, producing a viscous material capable of binding ulcerative lesions in the gastric mucosa. once bound to the exposed mucosa, it prevents back diffusion of h + , inactivates pepsin, absorbs bile acids, and increases mucosal prostaglandin synthesis, collectively supporting ulcer healing. sucralfate is not systemically absorbed but does prevent the absorption of drugs capable of chelating with aluminum, including fluoroquinolones, tetracyclines, and digoxin. if sucralfate is indicated in a cat being treated concurrently with fluoroquinolones, tetracyclines, or digoxin, the recommendation is to administer the other drug 2 hours prior to the administration of sucralfate to optimize drug absorption. clinical indications for the use of sucralfate in cats are for the treatment of gastric ulcers and esophagitis. 36 dosage recommendation in cats is 250 mg orally every 12 hours. sucralfate can be crushed, suspended in water, and administered as slurry. diet trials are used in some cats with diarrhea if the underlying cause is from known or suspected food hypersensitivities. dietary management includes either a highly digestible diet, an elimination (novel protein/ carbohydrate or hydrolyzed protein) diet (see above for both), or a diet high in fiber. 72 high-fiber diets contain a mixture of both soluble and insoluble fiber that can be beneficial in patients with signs of large bowel diarrhea. insoluble fiber, such as cellulose, functions to increase the bulk of the stool, bind fluid, and regulate intestinal motility. soluble fiber, including fruit and vegetable pectins and beet pulp, functions as a source of butyric acid that can be used by the colonic mucosa and decreases proinflammatory cytokines. 69, 72 cobalamin cobalamin (vitamin b 12 ) is an essential vitamin needed by a number of different enzymes, including key enzymes involved in methionine metabolism and the conversion of methylfolate to tetrahydrofolate needed for dna synthesis. cobalamin and folate are intimately linked, and hypocobalaminemia can lead to a functional deficiency of folate. 57 ingested cobalamin requires intrinsic factor binding for enterocyte absorption at the level of the ileum. hypocobalaminemia is commonly associated with distal small intestine diseases in cats, including inflammatory bowel disease. in addition, low cobalamin has a negative impact on enterocyte function; therefore in many cats with intestinal disease and hypocobalaminemia, cobalamin supplementation is necessary for resolution of clinical signs. 60, 64 quantification of serum cobalamin levels is recommended in cats with clinical signs of small bowel diarrhea, ones suspected to have an infiltrative disease of the small intestine (inflammatory bowel disease or gastrointestinal lymphoma), or ones with pancreatic dysfunction. when hypocobalaminemia is identified, supplementation is recommended mannanoligosaccharides, inulin, chicory, and lactosucrose. 72 reports on the use of prebiotics in cats are limited to their use in healthy cats; healthy cats fed fructooligosaccharides were reported to have a trend toward an increase in fecal concentrations of lactobacilli and a decrease in concentration of c. perfringens and e. coli relative to the controls. 65 to date no reports are available on the use of prebiotics in cats with gastrointestinal disease. probiotics and prebiotics potentially have a supportive role in the treatment of gastrointestinal disease in cats. the important clinical consideration in the use of probiotics as an adjunctive therapy is to ensure the use of live nonpathogenic microorganisms that have been documented to colonize the intestinal tract of cats. gastrointestinal flora co-evolve with their host. gastrointestinal microorganism colonization varies among species and within each individual animal. the distribution of fecal microflora for a given individual is considered unique but stable over time. 68 antimicrobial and antiparasitic therapies for the treatment of feline diarrhea are indicated based on the specific diagnosis of infectious diarrhea, bacterial enteritis, or as adjunctive therapy for inflammatory bowel disease. infectious pathogens more commonly associated with feline diarrhea include bacterial enteropathies (clostridium, campylobacter), protozoal enteropathies (tritrichomonas foetus, giardia spp.), and helminthic enteropathies associated with ascarids, hookworms, whipworms, and tapeworms. only the more common anthelminthic, antimicrobial, and antiprotozoal therapies are discussed below (tables 23-6 and 23-7) . more information about antimicrobials and antiparasitics is found under specific infections in the discussions of infectious enteritis and gastrointestinal parasites. fenbendazole is an anthelmintic used to treat common helminth infections, including ascarids, hookworms, whipworms, and a single species of tapeworm, taenia pisiformis. giardia spp. are also considered susceptible to fenbendazole. fenbendazole binds beta-tubulin subunits of microtubules, interfering with their polymerization. side effects include vomiting and diarrhea, although both are considered rare. fenbendazole is not approved for use in cats in north america but is commonly used clinically, and an empirical dosage of 50 mg/kg (250 µg/cat every 7 days) while the underlying cause of cat's malabsorption is being investigated and at initiation of targeted therapy. probiotics probiotics are ingested live microorganisms intended to benefit the host, specifically to support the microflora environment of the gastrointestinal tract as well as to provide an overall benefit to the body's immune function by immunomodulation. 8, 18, 51 probiotics chemically modify ingesta and intestinal mucus, as well as affect immune cells, enterocytes, and goblet cells within the intestinal mucosa through direct receptor interactions and indirectly through the action of cytokines. the microorganisms commonly used are nonpathogenic bacteria and yeast that have a vital role in gastrointestinal health, including lactobacillus spp., enterococcus faecium, bifidobacterium spp., and saccharomyces spp. for example, lactobacilli synthesize b vitamins, digestive enzymes, and folate coenzymes. 63 clinical indications for the use of probiotics are diverse, including primary gastrointestinal disease, chronic renal disease, and pancreatitis. 71 the rational use of probiotics in the treatment of gastrointestinal diseases include their ability to modulate gastrointestinal flora, minimize colonization by pathogenic bacteria, and decrease the likelihood of bacterial translocation. 17 in healthy cats, lactobacillus acidophilus is reported to reduce fecal clostridium counts. 45 when lactobacillus acidophilus was used adjunctively with antimicrobial therapy, fecal shedding of campylobacter was reduced in cats with campylobacter-induced diarrhea relative to cats treated with antimicrobials alone. 3 specifically, in cats with gastrointestinal disease, available research supports the probiotic enterococcus faecium as clinically beneficial in resolving diarrhea in kittens. 16 relative to the control group, the kittens treated with probiotics had increased fecal bifidobacteria and blood iga concentrations and decreased fecal counts of clostridium perfringens. prebiotics are dietary supplements used to select for the more beneficial enteric flora, support gastrointestinal function, and prevent the overgrowth of pathogenic bacteria, including salmonella, escherichia coli, clostridium, or campylobacter. for a food additive to be considered a prebiotic, it must be nondigestible by the gastrointestinal tract (resistant to gastric acidity, gastrointestinal hydrolysis and absorption), yet fermentable by gastrointestinal microflora to short-chain fatty acids to stimulate the growth of "good" intestinal bacterial. 72 prebiotics include nondigestible oligosaccharidescommonly, oligofructose, fructo-oligosaccharides, pyrantel pamoate is a nicotinic anthelmintic used primarily for the treatment of ascarids, but its spectrum of activity also includes hookworms and the stomach worm, physaloptera spp. pyrantel is toxic to susceptible parasites through its selective action on their nicotinic acetylcholine receptors, resulting in depolarization and spastic paralysis. pyrantel is not approved for use in cats but is considered safe in cats and is commonly used clinically. the dosage recommendation in cats is 5 mg/ kg orally once, repeat in 3 weeks, and finally repeated in 3 months. metronidazole is a nitroimidazole antibiotic with an anaerobic antibacterial spectrum with antiprotozoal activity against giardia spp. in an anaerobic environment, metronidazole is converted to unstable intermediates (nitroso free radicals) that disrupt bacterial dna synthesis. immunomodulatory properties capable of inhibiting cell-mediated immunity have been described for metronidazole, although its immunomodulatory properties are reported at dosages well beyond what is recommended for clinical use, 62 raising questions about the clinical use of metronidazole as an adjunctive therapy for treating inflammatory bowel disease. 34, 43 resistance to metronidazole is considered rare. 43 the most common adverse reaction is gastrointestinal upset, including inappetence, anorexia, nausea, and vomiting. profuse salivation can occur in cats after oral administration of metronidazole base (formulation used in standard tablets), which has lead to the use of metronidazole benzoate (a compounded formulation not approved by the food and drug administration) in some cats because of its better oral palatability. 61 at high doses (>200 mg/ kg/day) benzoic acid is reported to be neurotoxic in cats, but with appropriate clinical dosing of metronidazole benzoate benzoic acid toxicity is unlikely. 6 dose-related metronidazole toxicity in cats results in cerebellovestibular ataxia secondary to gamma-aminobutyric acid (gaba) inhibition at dosages greater than or equal to 58 mg/kg/day 12, 52 ; clinical signs include nystagmus, head tilt, ataxia, seizures, and obtundation. in cats with inflammatory bowel disease, the dosage recommendation for the metronidazole base is 10 to 15 mg/kg/day. metronidazole benzoate contains approximately 60% metronidazole base by weight, translating to an empirical dosage of 20 mg/kg/day of metronidazole benzoate (equivalent to 12.4 mg/kg/day of metronidazole base). 61 little is known about the safety of chronic metronidazole use in cats, but oral metronidazole has been reported to disrupt dna within feline peripheral mononuclear cells following 7 days of therapy. 61 this metronidazole-induced genotoxicity is reversible and is no longer detected 6 days after antibiotic therapy is discontinued. ronidazole is a nitroimidazole antibiotic (similar to metronidazole) and available as a powder-on-feed antibiotic. ronidazole is not approved for use in cats but has immunosuppressive therapies used in cats with inflammatory bowel disease include glucocorticoids, cyclosporine, and chlorambucil (tables 23-8 and 23-9 ). more information on the treatment of inflammatory bowel disease is found elsewhere in this chapter. glucocorticoids are considered first-line therapy in the treatment of cats with inflammatory bowel disease. glucocorticoids bind their intracellular glucocorticoid receptors, modifying the expression of genes with glucocorticoid response elements. immunomodulation is achieved through inhibition of cytokine release and response, including decreasing leukocyte phagocytosis, chemotaxis, and antigen expression. the more common side effects in cats include gastrointestinal ulceration, opportunistic infections (e.g., urinary tract infections), pancreatitis, and diabetes mellitus. cats are less susceptible to iatrogenic hyperadrenocorticism than dogs. initial therapy is usually with oral prednisone or prednisolone. prednisone is a prodrug that is metabolized to its active form prednisolone. cats are reported to be less efficient in the conversion of prednisone to prednisolone 27 ; therefore prednisolone may be preferred in cats, especially in cats refractory to prednisone therapy. been used off-label to effectively treat tritrichomoniasis in naturally and experimentally infected cats (30 mg/kg orally every 12 hours for 14 days). 25 t. foetus reduces nitroimidazoles to their nitroso free radicals. ronidazole has been reported to have better in vitro and 10-fold higher in vivo activity against t. foetus relative to metronidazole. 25, 35, 49 ronidazole resistance is beginning to be reported in t. foetus isolates from cats with diarrhea. 26 side effects include hepatoxicity and neurotoxicity. neurotoxicity is associated with high doses and has been reported in cats. 59 the use of ronidazole is recommended only for confirmed cases of t. foetus, and dosing should not exceed 30 mg/kg once daily in cats, especially in cats at risk for neurotoxicity. ronidazole is not registered for human or veterinary use in the united states; therefore its use in cats requires owner informed consent and client education of the potential human hazards. immunosuppressive therapies are considered the standard of care for cats with gastrointestinal biopsies consistent with inflammatory bowel disease (lymphoplasmacytic or eosinophilic inflammation). the common alternative forms of glucocorticoids can be considered in specific patient populations. in patients with severe malabsorption, injectable dexamethasone may provide improved bioavailability and clinical response. also dexamethasone maybe preferred in patients with a history of heart failure, fluid retention, or hypertension because of its lack of mineralocorticoid activity relative to prednisone/prednisolone. dexamethasone's potency is 4 to 10 times that of prednisolone; therefore a dose reduction is necessary when prescribing dexamethasone (the dexamethasone dose is one seventh that of prednisolone). 4, 10 budesonide is an oral, locally active, highpotency glucocorticoid that is formulated to be released in the distal gastrointestinal tract (based on the ph differential between the proximal and distal small intestine), where it is absorbed and is locally immunomodulating at the level of the enterocyte. the amount of systemically absorbed budesonide is minimized, because 80% to 90% of the budesonide absorbed from the gastrointestinal tract undergoes first-pass metabolism in the liver. some systemic absorption does occur, as evidenced by a blunted adrenocorticotropic hormone (acth) stimulation test in dogs treated with budesonide at 3 mg/m 2 for 30 days. 66, 70 the use of budesonide in cats remains anecdotal, with a suggestive empirical dose of 0.5 to 1 mg/cat/day. initial glucocorticoid therapy for cats with inflammatory bowel disease consists of antiinflammatory (0.5 to 1 mg/kg/day) to immunosuppressive (2 to 4 mg/kg/ day) dosages, with dosages based on the potency of prednisone/prednisolone. the goal of therapy is to achieve clinical remission and slowly taper the dose of glucocorticoids to the lowest dose that will control the cat's clinical signs. 67 some cats may be completely weaned off therapy, while others require long-term lowdose therapy. the tapering of therapy should be slow, with a 25% to 50% dose reduction every 3 to 4 weeks. cyclosporine is considered a second-tier immunosuppressive drug used to treat inflammatory bowel disease in cats. use of cyclosporine in the treatment of diarrhea associated with inflammatory bowel disease in cats is extrapolated from its use in dogs to treat glucocorticoid refractory inflammatory bowel diarrhea. 1 cyclosporine suppresses t-lymphocyte-mediated inflammation in the gastrointestinal tract secondary to suppression of inflammatory cytokines. specifically, cyclosporine attenuates t-lymphocyte activation and proliferation through the inhibition of interleukin-2 (il-2) production. side effects of cyclosporine in cats include dose-dependent inappetence and vomiting, which may occur at the onset of therapy and are generally responsive to dose reduction. other less common side effects reported in cats are opportunistic infections, including toxoplasmosis 5 and hepatoxicity. the microemulsion formulation of cyclosporine has higher oral bioavailability and less variable pharmacokinetics. 58 a suggested initial dosage of cyclosporine is 4 mg/kg every 12 or 24 hours. serum cyclosporine levels can be used to monitor for excessive trough plasma concentration (>400 ng/ml) as determined using a highperformance liquid chromotography (hplc) analytical method. 53 chlorambucil is a slow-acting nitrogen mustard that alkylates and effectively cross links dna, leading to altered protein production. the immunosuppressive effects of chlorambucil are the result of its cytotoxic effect on lymphocytes, similar to other nitrogen mustards. bone marrow suppression is considered mild to moderate and is rapidly reversible. neurotoxicity and myoclonus has been reported in a cat accidently overdosed with chlorambucil. 7 chlorambucil is used as a second-tier drug in cats to treat immune-mediated disorders, in part because of ease of administration and its low risk of myelosuppression. for the treatment of inflammatory bowel disease, the recommended dosing in cats is 2 mg/cat every 48 hours in cats greater than 4 kg and 2 mg/cat every 72 hours in cats less than 4 kg. 50 chlorambucil is commonly used in combination with glucocorticoids in the treatment of immune-mediated diseases, including inflammatory bowel disease, 48 overlooked. awareness about feline esophageal diseases is low, the clinical signs are often not specific, and imaging beyond survey radiographs may be required for diagnosis. the esophagus is composed of four layers (from inner to outer): mucosa, submucosa, muscularis, and adventitia (there is no serosal layer). in the dog, the muscle layer is entirely composed of skeletal muscle, but in cats, the distal third of the esophagus is composed of smooth muscle. the upper esophageal sphincter prevents reflux of esophageal contents into the pharynx and minimizes aerophagia. the lower esophageal sphincter prevents gastroesophageal reflux and relaxes during swallowing to allow food and fluid to enter the stomach. clinical signs of esophageal disease include drooling, dysphagia, pain on swallowing (odynophagia), and, most classically, regurgitation. weight loss may occur secondary to inadequate food intake when disease is severe or chronic. other clinical signs, such as anorexia, cough, dyspnea, and fever, may occur if complications such as aspiration pneumonia or esophageal perforation occur. regurgitation is passive expulsion of food or fluid from the esophagus. the food is undigested and often accompanied by mucus and saliva. mucosal erosions may produce frank blood in the regurgitated material. regurgitation must be differentiated from vomiting (table 23 esophageal disease is uncommon in the cat when compared with dogs, but it is also likely that problems such as esophagitis and esophageal strictures are often salivation, retching, and abdominal contractions. the vomitus consists of partially digested food from the stomach and/or intestines and may be mixed with bilestained fluid. some cats will have both vomiting and regurgitation. expectoration may also be confused with vomiting or regurgitation. expectoration is associated with coughing, but cats that cough excessively may also stimulate vomition so that a careful history is needed to characterize the clinical signs correctly. coughing may also occur in cats that have aspirated as a result of regurgitation. drooling, dysphagia, and odynophagia are most commonly seen with conditions of the oropharynx and/or proximal esophagus. odynophagia is most commonly associated with esophagitis and foreign bodies. dysphagia and regurgitation together most commonly indicate oral or pharyngeal dysfunction; if regurgitation is not accompanied by dysphagia, esophageal dysfunction is likely. 55 regurgitation in cats with esophageal disease is caused by obstruction or muscular dysfunction. causes of obstruction include vascular ring anomaly, foreign object, stricture, and neoplasia. causes of muscular dysfunction include congenital disease, esophagitis, myopathies, neuropathies, and dysautonomia. regurgitation may occur immediately after eating if the lesion is in the proximal esophagus. however, a dilated esophagus provides a reservoir for food and fluid so that regurgitation may not be associated in time with eating. young cats with signs of esophageal disease should be suspected of congenital defects, such as vascular ring anomaly, or a foreign body. adult cats with esophageal disease may have a recent history of general anesthesia, administration of certain oral medications, or ingestion of irritant chemicals. acute onset of clinical signs may suggest a foreign body, while chronic, slowly worsening signs may indicate a stricture or tumor. all cats suspected of esophageal disease should have a minimum database as part of the diagnostic plan (complete blood cell count, serum chemistries, urinalysis, and other tests as indicated by age or concurrent diseases, such as serum total t 4 and blood pressure measurement). an important part of diagnosis is observation of the cat while eating food, to localize the location of the dysfunction. if the cat is unwilling to eat while in the veterinary clinic, the owner can make a video of the cat eating at home for the clinician to view. the general diagnostic approach to regurgitation in cats is found in figure 23 -3. plain and contrast radiography and endoscopy are important diagnostic tools for esophageal disease. fluoroscopy is valuable for the diagnosis of motility disorders, but availability is limited to universities and referral centers because of the cost of equipment. ultrasonography is limited to evaluation of * references 1, 8, 11, 15, 38, 43. the cervical esophagus and a small segment of abdominal esophagus between the cardia of the stomach and the diaphragm. the entire esophagus should be evaluated with cervical and thoracic radiographs. thoracic radiographs may also show evidence of complications such as aspiration pneumonia or esophageal perforation. the normal esophagus is not visualized on plain radiographs, but may be seen if food or fluid are retained or a foreign body or mass is present. radiographic contrast agents useful for esophagrams in cats include liquid or paste barium. a water-soluble iodinated contrast agent (e.g., iohexol, gastrografin) is preferred if there is any risk the esophagus is perforated, because these agents are less irritating and more rapidly reabsorbed. esophagrams are most useful for diagnosis of luminal obstructions, extraluminal compression, mucosal irregularities, and possibly alterations in motility. dilute liquid barium can be administered with a syringe or it may be mixed with canned food, especially if a motility disorder or stricture is suspected. multiple lateral radiographs are taken rapidly, starting within 10 seconds of swallowing the contrast agent. contrast is rapidly cleared from the normal esophagus by peristalsis. if the contrast in the esophagus terminates abruptly, an obstruction is likely. if the contrast is retained throughout the esophagus, muscular dysfunction is suspected. some conditions, such as esophagitis, are difficult to diagnose radiographically, because contrast agents may or may not adhere to ulcerated mucosa. flexible endoscopy is a noninvasive diagnostic tool for esophageal disorders and is often used if plain and contrast radiographs have failed to establish a diagnosis. it is most sensitive for diagnosis of masses, ulcers, perforations, and obstructions. in addition, it is often possible to retrieve foreign bodies using endoscopy as well as to assist with dilatation of strictures or placement of gastrostomy feeding tubes if required. biopsy of the esophageal mucosa is more difficult than biopsy of gastric or intestinal mucosa and is not commonly performed with the exception of mass lesions. esophagitis may result from various causes of inflammation, such as contact irritation from foreign bodies (including trichobezoars lodged in the esophagus), chemical irritants or caustic medications, gastroesophageal reflux, persistent vomiting, hiatal hernia, or general anesthesia. inflammation disrupts the esophageal mucosa and exposes the submucosa. an important part of the treatment plan is identification and treatment of the underlying cause. clinical signs include dysphagia, regurgitation, salivation, and repeated swallowing, although signs may be absent in cats with mild esophagitis. cats with odynophagia may repeatedly extend the head and neck while swallowing. if the esophagitis or underlying disease is severe, weight loss and dehydration may occur secondary to anorexia. if the submucosa and muscularis are damaged, strictures may form as a result of the production of fibrous connective tissue and compromise the esophageal lumen. 54 neoplasia is an important cause of esophageal stricture in humans, but not in cats. most cases have single strictures, but multiple strictures are possible. in two studies, the mean stricture diameter was reported as 5 mm. 26, 32 most strictures are less than 1 cm in length. clinical signs associated with strictures appear 5 to 14 days after the esophageal injury and may be present for weeks before definitive treatment is pursued. regurgitation typically occurs immediately after eating, although if the stricture is long standing, a pouch may form cranial to the lesion where food accumulates. survey radiographs may be normal in cats with esophagitis and strictures, but are useful to rule out other causes for the clinical signs, such as a foreign body, or to detect related problems, such as aspiration pneumonia. in some patients, dilation of the esophagus with fluid or air may be seen. 45 a contrast esophagram may disclose irregularities of the mucosa in cats with severe esophagitis. segmental dilation may occur with severe inflammation. strictures may be diagnosed with an esophagram (figure 23-4) ; however, in some cases, it may be difficult to differentiate a stricture from intramural thickening (e.g., because of neoplasia). endoscopy is useful for diagnosis of esophagitis; findings include mucosal erythema, hemorrhage, and erosions or ulcerations. if gastroesophageal reflux is present, the lesions will be most severe in the distal esophagus, and the lower esophageal sphincter may be dilated. endoscopy is often used for definitive diagnosis of esophageal stricture as well as to visualize the lesion during treatment by bougienage or balloon catheter dilation. strictures appear as a ring of white fibrous tissue that narrows the esophageal lumen. if endoscopy is performed after a barium esophagram, 24 hours should be allowed to elapse between the procedures or the barium will obscure visualization with the endoscope. 19 general anesthesia is an important cause of esophagitis (sometimes leading to stricture formation) in cats, probably because gastroesophageal reflux appears to occur commonly in anesthetized cats.* for example, in a series of seven cats with benign esophageal stricture, recent anesthesia for ovariohysterectomy was the suspected cause in five cases. 1 clinical signs appeared up to 21 days after anesthesia. abnormal esophageal tissue was performed in two cases. the authors noted that the esophageal mucosa may appear grossly normal, but submucosal inflammation may be found on histopathologic examination of biopsies. a consequence of chronic severe gerd in humans is the development of metaplastic columnar epithelium (barrett esophagus) that replaces the normal squamous epithelium. one case series reported on barrett-like esophagus in three cats. 23 two cases were associated with hiatal hernia and one with cardial incompetence. drug-induced esophageal damage and stricture formation is well known in humans and cats (see . in humans over 70 drugs have been implicated, and most are antibacterials or nsaids. 30 implicated drugs in the cat include tetracycline, doxycycline, and clindamycin in tablet or capsule form administered without a food or water bolus. 4, 17, 32, 36, 37 clinical signs (dysphagia, regurgitation, salivation, anorexia) appear 3 to 16 days after drug treatment is started. strictures commonly form in the midcervical esophagus or over the heart base in the thoracic esophagus. doxycycline hyclate is most commonly associated with esophageal strictures in cats, and the principle reason for its irritating properties is an acidic ph. the monohydrate salt of doxycycline is less irritating and is marketed as tablets and a palatable paste licensed for use in dogs and cats in some countries. 48 in humans esophageal ulceration after doxycycline therapy is more common than stricture formation. although the development of strictures in cats would appear to be uncommon, it seems possible the incidence of esophagitis is underestimated, because the clinical signs (e.g., odynophagia, chest pain) may go unrecognized. esophageal transit studies of normal cats have shown that the passage time of dry-swallowed tablets and capsules is often prolonged (longer than 30 seconds). 20, 53 complete entrapment (retention for more than 4 minutes) in the midcervical region occurs commonly. however, a small bolus of food or water is sufficient to ensure immediate passage of the medication into the stomach. 20, 53 the risk of esophageal retention can also be lessened by coating a tablet or capsule with butter or a gel dietary supplement (nutri-cal; vétoquinol, fort worth, tex.). 21 one study determined that tablets or capsules administered using a one-step pill gun with flavored liquid (flavorx pill glide; flavorx, columbia, md.) or a pill delivery treat (greenies pill pockets; nutro products, franklin, tenn.) ensured an average transit time of 60 seconds or less. 6 delayed esophageal transit of medications allows tablets and capsules to disintegrate within the esophagus, exposing the mucosa to irritating chemicals. cats may be at risk of delayed esophageal transit, because they do not typically drink water with medication, and they do not have an upright posture. in addition, medications are often given to sick or dehydrated patients many preanesthetic drugs and induction agents reduce lower esophageal sphincter pressure. 27, 28 other predisposing factors may be intraabdominal surgery and a head-down position on the surgery table. reflux fluid with a ph less than 4 is likely to cause esophageal mucosal damage, as is prolonged contact time. esophageal defense mechanisms include clearance of the reflux fluid by peristalsis and neutralization of the acidic ph by the bicarbonate present in saliva. in a study of 40 kittens less than 15 weeks of age, risk of gastroesophageal reflux during anesthesia was evaluated with use of a laryngeal airway mask versus endotracheal intubation. 47 gastroesophageal reflux was observed in 50% of kittens with use of the laryngeal airway mask but more importantly in 22% of kittens with endotracheal intubation. the reflux episodes occurred shortly after anesthesia induction. in a study of 50 cats anesthetized with thiopentone or propofol, gastroesophageal reflux occurred in 14%. 16 reflux also occurred shortly after anesthesia was induced and lasted for a mean of 23 minutes. it is unknown why esophageal strictures form only in a small number of cats that experience gastroesophageal reflux during anesthesia. gastroesophageal reflux disease (gerd) is a commonly reported cause of esophagitis in humans, but it is rarely reported in cats when not associated with general anesthesia. 24, 33 the true incidence is unknown, and diagnosis may be hampered by scant knowledge about the clinical presentation and diagnosis. clinical signs and diagnostic procedures are as for other causes of esophagitis. in one case series of three cats, diagnosis of gerd was based on clinical signs, contrast radiography, and endoscopic findings. 24 biopsy and histopathology of obtained from a compounding pharmacy in most countries and can only be given orally. h 2 -receptor antagonists are competitive inhibitors that block parietal h 2 receptors and decrease the amount of gastric acid produced. proton pump inhibitors are noncompetitive inhibitors that act on the h + /k + atpase enzyme system at the secretory surface of gastric parietal cells. they are considered superior for decreasing gastric acid secretion and are therefore the first choice, despite their greater cost. 45 a drawback of proton pump inhibitors is that they must be administered orally. sucralfate may be beneficial for reflux esophagitis, because it binds to mucosal erosions in an acid environment and provides a protective barrier. it is given as oral slurry, ideally separate from meals or other medications. antibiotics are not commonly recommended unless aspiration pneumonia is present or the eroded mucosa is at risk of bacterial infection in a patient with severe disease or a compromised immune system. corticosteroids are often recommended for cats with esophagitis to reduce esophageal inflammation and impair the formation of fibrous connective tissue. however, the benefit of corticosteroids in cats with esophagitis has not been investigated and administration must be weighed against potential adverse effects, especially in patients with aspiration pneumonia. treatment of esophageal stricture typically requires dilation with either bougienage or a balloon catheter; both are used with endoscopic visualization under general anesthesia. appropriate analgesia should be provided, because dilating the stricture is painful. it does not appear that placement of a gastrostomy feeding tube is specifically required to recover from dilation procedures, although a tube may be placed in some anorexic cats to ensure nutritional intake and administer oral medications. a bougie is a long, narrow, oblong, mechanical dilator available in various sizes (typically 9-to 12-mm sizes are used in cats) that is gently passed through the stricture, usually over a guide wire. established criteria for selection of bougie diameter and dilation end points are not available. in one study, the initial bougie chosen was approximately the same size as the estimated diameter of the stricture, or no more than 2 mm larger. 8 once the first bougie is passed, subsequent bougies of increasing diameter are employed. two to four bougies of increasing size may be passed in a single session, with the goal of dilating the stricture without causing esophageal tear or perforation. determining when dilation should be stopped is a matter of clinical judgment. the procedure may be repeated as needed to maintain improvement; the total number of procedures required is variable. in one retrospective case series of eight cats treated with bougienage, the median number of procedures was 4.5, and a good outcome was achieved in 75% of the cases. 8 in some cases, the endoscope tip itself has been used for that may be at greater risk of esophageal retention of medication. all oral medication given to cats in tablet or capsule form should be followed with food or a liquid. mild esophagitis will resolve on its own, especially if an underlying cause can be removed or treated. frequent meals of canned food should be provided. cats with moderate to severe esophagitis will require medical therapy, and those with difficulty eating or weight loss may also require gastrostomy tube feeding. esophagostomy or pharyngostomy feeding tubes should be avoided in these patients. treatment is provided to control inflammation and promote healing while reducing gastric acid secretion and increasing lower esophageal sphincter tone. the length of medical treatment will vary from about one week to several weeks, depending on the underlying cause and severity of disease. medications indicated for esophagitis include prokinetics, h 2 -receptor antagonists, proton pump inhibitors, and sucralfate (table 23-11) . prokinetic drugs enhance gastric emptying and increase lower esophageal sphincter tone. metoclopramide also has antiemetic effects, which may be beneficial in patients with chronic vomiting. it can be administered by the subcutaneous (sc) route, an advantage in a vomiting or regurgitating patient. cisapride may be more effective at enhancing both gastric emptying and lower esophageal sphincter tone, but it must be stent placement has recently been described in cats with esophageal strictures with variable results. a 1-year-old cat presented with a 4-week history of dysphagia and regurgitation caused by a single cervical esophageal stricture after treatment with oral clindamycin. 18 guided balloon dilation was performed 6 times over a period of 3 weeks, but stricture formation always recurred. a self-expanding metal stent was placed using endoscopy and fluoroscopy after another dilation procedure. the cat did well eating a canned diet from an elevated position for 10 months, but by 12 months, the cat was no longer able to eat even liquid food and was euthanized. on necropsy, the stent had migrated and was obstructed by swallowed hair. in another case, a biodegradable self-expanding stent was used to successfully treat an 11-year-old cat that presented with a stricture in the cervical esophagus after anesthesia for dentistry. 3 balloon dilation was performed twice, but regurgitation recurred 5 days after the last procedure. the stricture was dilated a third time with a balloon catheter, and a tubular selfexpanding polydioxanone stent was placed with fluoroscopic guidance. the life span of the stent was estimated to be 10 to 12 weeks, sufficient time to allow healing of the esophagus. foreign bodies are less commonly found in the esophagus of the cat than in other gastrointestinal locations. reported foreign bodies include string, needles, fish hooks, and bones. trichobezoars may cause obstruction when they become lodged in the esophagus during vomiting ( figure 23 -5). recurrent esophageal trichobezoars have been infrequently reported in the literature. 12, 51 it is not known if an esophageal motility disorder is the underlying cause for recurrent obstructions. in one case, an esophageal diverticulum developed in association with recurrent trichobezoars. 12 treatment for recurrent trichobezoars includes prokinetic drug therapy (e.g., cisapride), moderate to high-fiber diets, and shaving of long-haired cats. common areas for foreign bodies to lodge include the thoracic inlet, the heart base, and the esophageal hiatus in the diaphragm. 5 obstruction of the esophageal lumen may be complete or partial. clinical signs include acute onset of gagging, salivation, repeated swallowing, dysphagia, and regurgitation. however, chronic esophageal foreign bodies have been reported in cats with dysphagia, intermittent regurgitation, and weight loss over a period of weeks or months. 2 bougienage when bougies or balloon catheters were not available. balloon catheter dilation has become a popular method in recent years. 26, 32, 38 although some clinicians feel this is a safer procedure than bougienage, there is no data in the literature to support this assumption. the catheter can be placed through the endoscope biopsy channel, alongside the endoscope, or with the aid of a preplaced guide wire. as for bougienage, established criteria for selection of balloon diameter and dilation end points are not available, and the clinician's best judgment must be used. various balloon sizes are available; in one study, the size was selected so that the inflated diameter was 4 mm larger than the stricture diameter. 32 the balloon is passed into the stricture with endoscopic guidance. it is then inflated to a predetermined pressure for 1 to 2 minutes to stretch the stricture, usually with saline, but contrast agents may also be used if fluoroscopy is used. as for bougienage, some cases may require more than one dilation procedure (typically two to four). cuffed endotracheal tubes are not appropriate substitutes for balloon catheters. regardless of the method used, after the dilation procedure, the endoscope should be used to look for other strictures and should be passed into the stomach to look for potential causes, such as causes of chronic vomiting. after treatment, medical management to decrease ongoing gastroesophageal reflux, resolve inflammation, and prevent further stricture formation should be instituted (as described previously). most cats are able to eat the day following the dilation procedure. corticosteroid treatment after dilation is controversial, and no controlled studies in animals are available. antibiotics are not routinely recommended. the prognosis for cats undergoing esophageal dilation is generally good based on the ability to eat canned food with minimal episodes of regurgitation. however, published studies show 10% to 30% of cats died or were euthanized despite multiple episodes of dilation, and up to 30% could only be fed liquid diets. 1, 8, 32, 38 even among cats with good outcomes, a return to a dry kibble diet may not be possible. the dilation technique employed may be dictated by the clinician's experience, the equipment available, and the cost. potential complications of both methods include esophageal tear or perforation, hemorrhage, infection, and aspiration. esophageal tears or perforations may lead to pneumothorax or pneumomediastinum. repeated stricture formation is also possible, leaving only less desirable treatment options, such as long-term percutaneous gastrostomy tube feeding or surgery. esophageal surgery is generally avoided whenever possible, because it is difficult and invasive (requiring a thoracotomy), with risk of serious complications, such as failure of anastomosis, necrosis, and stricture formation. closure of incisions in the esophagus is following uncomplicated foreign body removal, the esophagus should be carefully inspected for lesions and bleeding before the endoscope is withdrawn. food and water should be withheld for 24 to 48 hours. supportive care includes fluid therapy and analgesia; a gastrostomy feeding tube may be required in selected cases for nutritional support. broad-spectrum antibiotics are administered to control bacterial infection and therapy for esophagitis should be instituted as described previously. careful follow-up should include evaluation for stricture formation. if an esophageal perforation has occurred, conservative management may be sufficient if the defect is small. a broad-spectrum antibiotic should be administered along with other supportive care, such as fluid therapy and analgesia. feeding through a gastrostomy tube for several days is recommended as well as close monitoring for complications such as pleuritis. large perforations require thoracotomy for surgical repair. megaesophagus is a diffuse hypomotility disorder that may be classified as congenital versus acquired or idiopathic versus secondary to other diseases. it is uncommon in cats compared with dogs. at least two dog breeds have been identified with heritable congenital megaesophagus. a heritable form of megaesophagus has been suggested for cats, particularly for siamese cats, although no detailed studies have been performed. 13, 29 it is often frustrating to determine the underlying cause of acquired megaesophagus. megaesophagus may be a manifestation of neuromuscular diseases, such as dysautonomia or myasthenia gravis (see chapter 27) . megaesophagus may also develop secondary to esophagitis from chronic vomiting or gerd. 24, 43 other uncommon causes of megaesophagus are found in the literature. one case report describes a young cat with megaesophagus secondary to a large nasopharyngeal polyp that extended into the cervical esophagus. 10 megaesophagus resolved once the polyp was removed. in another report, a young cat with diaphragmatic hernia was diagnosed with megaesophagus and gastric dilation. 31 megaesophagus resolved with medical treatment and surgical correction of the diaphragmatic defect. clinical signs are typically those of esophageal dysfunction; regurgitation is the most consistently found sign. regurgitation may not be closely related in time to eating if the esophagus is markedly distended and holds food. cats with long-standing disease may suffer from weight loss or secondary rhinitis. the appetite is typically normal or increased. additional signs may occur if systemic neuromuscular disease is present. aspiration pneumonia may cause fever, dyspnea, and cough. two case reports describe cats with idiopathic cough, mucopurulent nasal discharge, and fever may be found if aspiration has occurred. trauma to the esophagus may cause esophagitis and even esophageal stricture. perforation of the esophagus by the foreign body may lead to pneumothorax, pneumomediastinum, or pyothorax with signs of depression, anorexia, fever, and dyspnea. if the perforation occurs in the cervical esophagus, swelling, cellulitis, and drainage of serous or purulent material may be noted. many foreign bodies are readily diagnosed with survey radiographs, especially if they are radiopaque. other radiographic findings include an esophagus dilated with fluid or air. radiolucent objects may be detected with an esophagram. care must be taken when performing esophagrams on cats that may have an obstruction, because aspiration is a concern. if abnormalities that could be consistent with an esophageal perforation (e.g., periesophageal gas or fluid, pleural effusion) are detected on survey radiographs, an aqueous iodine contrast solution should be used. removal of esophageal foreign bodies should be performed as soon as possible to minimize esophageal trauma and pressure necrosis. endoscopy can be used to confirm the diagnosis and often to remove the object. both rigid and flexible endoscopes may be used along with accessories such as various forceps and foley catheters. care should be taken to remove the object as atraumatically as possible, especially if the object is sharp or pointed. if the object is in the caudal esophagus and it cannot be grasped and removed, an attempt should be made to gently push it into the stomach, where it can be retrieved using laparotomy and gastrotomy. if esophageal perforation has occurred, esophagotomy is recommended and is described elsewhere. 5, 14 removal of fish hooks may require a combination of surgery and endoscopy. 5, 39 a surgical approach to the esophagus is made, but the esophagus is not incised; rather, the portion of the hook protruding through the esophagus is cut and removed, and the endoscope is used to retrieve the remainder. clinical sign is regurgitation, and most patients are underweight. a distended cervical esophagus may be palpated, and secondary aspiration pneumonia may occur. a history of regurgitation since weaning is very suggestive of a vascular ring anomaly, but other causes of regurgitation must be ruled out. survey radiographs show a dilated esophagus cranial to the heart, while the caudal esophagus is usually normal. the bulge of the aortic arch normally seen on a ventrodorsal radiographic view is absent. an esophagram is used to confirm the location of the obstruction and the severity of disease. definitive treatment is surgical repair of the vascular defect (i.e., ligation and transection of the ligamentosum arteriosum). some patients will require nutritional support through gastrostomy tube feeding and treatment for aspiration pneumonia before surgery. early diagnosis and surgical intervention brings the best prognosis for return of normal esophageal function. some affected cats are left with residual esophageal hypomotility, which is managed as for idiopathic megaesophagus. esophageal neoplasia is rare in the cat as in the dog. although parasitic granulomas caused by spirocerca lupi are associated with esophageal neoplasia in dogs, this parasite does not infect cats. both primary and metastatic esophageal tumors can occur in the cat. squamous cell carcinoma is the most common primary esophageal tumor in cats and is often found in the caudal two thirds of the esophagus. 7, 22, 25, 46 affected cats are middle aged or older. clinical signs are typically those associated with esophageal obstruction, such as regurgitation, dysphagia, odynophagia, and salivation. patients with advanced disease may suffer anorexia, depression, and weight loss. on physical examination, an esophageal mass may or may not be palpable. survey and contrast radiographs reveal esophageal dilation, a soft tissue mass, or periesophageal lesions that displace the esophagus. computed tomography is useful to identify periesophageal or intraluminal masses. definitive diagnosis is made with endoscopy and biopsy. mucosal biopsies are difficult to obtain, because the esophageal mucosa is tough; exfoliative cytology may also be helpful. treatment is rarely undertaken, because disease is often advanced at the time of diagnosis, and many patients have complications such as aspiration pneumonia. palliation may be attempted with chemotherapy or radiation, although data on efficacy is unavailable. in general, squamous cell carcinomas in other anatomic locations respond poorly to treatment. surgical resection may be attempted if anastomosis can be accomplished without excessive tension. megaesophagus and chronic vomiting associated with intermittent gastroesophageal intussusception. 35, 50 survey and contrast radiographs may identify a dilated esophagus (figure 23-6), but contrast fluoroscopy is the diagnostic tool of choice when available, because it allows for assessment of peristalsis. care must be taken with contrast studies because of the risk of aspiration. treatment of megaesophagus is largely symptomatic and supportive unless an underlying disorder can be identified and treated. frequent small meals are offered with the cat feeding in an upright position. the upright position should be maintained for at least 10 minutes after eating to allow for gravity-assisted passage of food into the stomach. this is best accomplished by having the owner hold the cat over their shoulder so that the esophagus is in a vertical position. 44 different types of diets should be offered to determine which is best for the individual patient; calorically dense diets may be beneficial for patients with weight loss. prokinetic drugs, such as cisapride, stimulate smooth muscle, but since most of the esophagus is skeletal muscle, the efficacy of such drugs is questionable for treatment of megaesophagus. prokinetic drugs also increase lower esophageal sphincter tone and may increase esophageal transit time, neither of which is desirable in patients with megaesophagus. vascular ring anomalies are congenital malformations of the great vessels that entrap the thoracic esophagus and cause obstruction. the most commonly reported anomaly is persistent right aortic arch. the esophagus is entrapped by the aorta on the right, the ligamentum arteriosum and the pulmonary trunk on the left, and the heart base ventrally. other vascular anomalies are rarely described in cats, such as a double aortic arch described in a siamese cat. 56 onset of clinical signs occurs around the time of weaning to solid food so that most affected cats are presented at less than 6 months of age. the most common surgery. 34 surgery is the treatment of choice for large defects, especially in young cats with congenital disease or cats that have failed medical management. various reconstructive surgical techniques have been described. 14 disorders of the hiatus are rare in cats. hiatal hernia is protrusion of the distal esophagus and stomach through the esophageal hiatus of the diaphragm into the thoracic cavity; the protrusion may be intermittent ("sliding") or persistent. other organs are occasionally involved, such as the omentum. 40 this is distinct from a gastroesophageal intussusception where the stomach is prolapsed into the lumen of the distal esophagus. 35, 49 both congenital and traumatic hiatal hernias have been described in cats. 9, 23, 41, 42, 52 congenital hernias appear to be more common than acquired hernias, and affected cats typically present with clinical signs before 1 year of age. it is suspected that increased inspiratory effort associated with upper airway obstruction, such as a nasopharyngeal polyp, may also lead to development of hiatal hernia. 23 hiatal herniation reduces lower esophageal sphincter pressure. clinical signs associated with hiatal hernia, such as intermittent vomiting and regurgitation, may be because of reflux esophagitis, hypomotility, or obstruction. large hernias and secondary aspiration pneumonia may be associated with respiratory distress. survey radiographs may reveal a gas-filled soft tissue density in the caudal dorsal mediastinum. an esophagram will show the gastroesophageal junction and gastric rugae cranial to the diaphragm (figure 23-7) . both fluoroscopy and endoscopy may be useful for diagnosis but are not typically necessary. the prognosis for cats with hiatal hernia is considered to be good. a trial of medical management (as for reflux esophagitis) for 1 month has been recommended before the stomach is a frequent site for gastrointestinal problems in cats, and the most common gastric problems are described in this chapter. some conditions such as gastric dilatation-volvulus are often reported in dogs but rarely reported in cats. in one report of three feline cases, all were associated with diaphragmatic hernia. 15 gastric parasites, the diagnostic approach to the vomiting cat, the gastric emptying time of normal cats is shorter than that of other mammals. in one study, the gastric emptying half-time for solid food in normal cats was 1.4 to 3.6 hours. 53 this implies prolonged fasting (longer than 8 hours) in preparation for anesthesia and surgery is unnecessary. the main clinical sign of gastric disease is vomiting, but it is important to note that vomiting is also associated with many nongastric problems, including concurrent intestinal disease, such as enteritis or colitis. vomiting patients therefore require a thorough physical examination and diagnostic plan to determine the cause. vomiting must be distinguished from regurgitation, which is primarily associated with esophageal disease (see table 23 -10). vomitus often contains food, hair, refluxed bile, and therapeutics for vomiting are covered elsewhere in this chapter. the anatomy of the feline stomach is similar to that of other mammals having a simple glandular stomach. most of the stomach is situated on the left side of the abdominal cavity. it has five regions, starting from the lower esophageal sphincter: cardia, fundus, body, antrum, and pylorus ( figure 23-8) . the pylorus of the cat is unique compared with other species in that it is narrow and has high resistance in order to maintain a tight seal ( figure 23 involve a wide variety of objects, including linear objects (e.g., dental floss, thread with or without a needle, tinsel, string). the owner may or may not be aware of the ingestion. ingestion of multiple foreign bodies may be seen in cats with pica ( figure 23-12 ). in one case report, a young domestic shorthair cat required gastrotomy for removal of 32 copper pennies. 43 some patients require multiple surgeries, because of repeated foreign body ingestion. 22 in such cases, a behavioral diagnosis should be sought and treatment instituted (see chapter 13) . trichobezoars (large masses of hair) also represent a type of foreign object. both long-and shorthaired cats may be affected. hair is normally ingested during grooming and is eliminated in vomitus and feces. cats lack the strong peristaltic contractions ("housekeeper" contractions) that clear the stomach of undigested contents normally found in other species. this may explain why cats seem to be susceptible to gastric trichobezoars. gastric motility dysfunction is suspected to cause repeated gastric trichobezoars in some cats. intestinal 3, 22 and esophageal 14, 59 obstruction with trichobezoars has also been documented. traditional treatments for cats with recurrent trichobezoars include regular grooming, shaving the hair coat of long-haired cats, flea control, or blood. fresh blood may appear as large or small clots. older blood clots have a brown "coffee ground" appearance. gastric bleeding may also cause melena. other clinical signs may be associated with gastric disease, such as anorexia, weight loss, pain, lethargy, bloating, and nausea. gastritis may be acute or chronic in nature, and this distinction may be useful in assessing the potential cause. for example, cats with acute gastritis may be suspected of foreign body or plant ingestion, drug or toxin exposure (see chapter 31), or dietary indiscretion. cats with chronic gastritis may be suspected of parasitism, helicobacter spp. infection, or dietary intolerance or hypersensitivity (see chapter 17) . chronic lymphocytic plasmacytic gastritis of unknown etiology is also a common cause of chronic vomiting. whenever possible, a specific underlying cause should be sought and treated. patients with sudden onset of vomiting may have an obvious cause in the history (e.g., dietary indiscretion), but in many cases, the cause is not apparent. abdominal radiographs should be taken if foreign body ingestion is possible, especially in a young cat. if the patient is systemically well, further diagnostic testing may be postponed pending response to therapy. treatment for uncomplicated acute gastritis is symptomatic and supportive. clinical signs are expected to resolve in 24 to 48 hours; if signs persist, re-evaluation and further investigation is warranted. subcutaneous fluid therapy using an isotonic balanced electrolyte solution may be used to correct mild fluid deficits (<5%). oral intake of fluids and food should be discontinued for up to 24 hours. a highly digestible diet, either commercial or homemade, is introduced with a gradual transition back to the normal diet over the next several days. antiemetic therapy may be indicated for acute uncomplicated gastritis if the vomiting is frequent or the cat has signs of nausea (see table 23 -3). protectants, such as kaolin and pectin, are difficult to administer to cats and are without proven efficacy. bismuth subsalicylate is controversial; it is considered contraindicated by some experts, because of the cat's sensitivity to salicylates, 39 yet is commonly used in clinical practice. cats ingest foreign bodies less commonly than dogs. in one study of 208 cases of gastrointestinal foreign body ingestion, only 12% were in cats. 22 foreign body ingestion is most likely to be seen in young cats and may a b taken just before surgery to ensure the object has not moved further down the gastrointestinal tract. postoperative management after gastrotomy includes maintenance of hydration and electrolyte balance. hypokalemia is common with anorexia and vomiting and should be treated by supplementation of iv fluids with 20 to 40 meq/l potassium chloride (not to exceed 0.5 meq/kg/hour). refractory vomiting should be treated with an antiemetic. a highly digestible diet can be introduced the day after surgery. in general, the prognosis for recovery is good. in one study, 88% of cats with gastrointestinal foreign bodies survived to discharge. 22 those cats that did not survive had linear foreign bodies of long-standing duration with subsequent peritonitis. helicobacter are spiral or curved gram-negative bacteria that inhabit the glands, parietal cells, and mucus of the gastric antrum and fundus. helicobacter contain large amounts of urease, which alters the ph in the vicinity of the bacteria and allows for colonization of the acidic environment of the stomach. in the early 1980s, the discovery of the association of helicobacter pylori with gastric disease (gastritis, peptic ulcers, and neoplasia) in humans revolutionized treatment of those diseases. since then, helicobacter spp. have been associated with gastric disease in various veterinary species, including cats and dogs. several helicobacter spp. (e.g., h. heilmannii, h. bizzozeronii, h. felis) have been identified in cats, some of which have the potential to infect humans, although transmission is thought to be rare. 19, 48 the prevalence of helicobacter infection in cats varies geographically and may be very high (>40%) in some locations. 1, 27, 37, 47, 58 the importance of helicobacter as a cause of gastric disease is cats is unclear; the bacteria may be found in the stomach of both clinically normal cats and cats with gastritis. the prevalence of helicobacter infection is not higher in cats with gastritis compared with normal cats. 61 determination of the role of helicobacter is also hampered by the paucity of controlled clinical trials that evaluate eradication of gastritis and clinical signs in infected cats. an immune response to infection characterized by gastric lymphoid hyperplasia is common, although the local immune response in cats is generally less severe than the response in humans infected with h. pylori. to date gastrointestinal ulcers have not been associated with helicobacter infection in cats. recent studies have suggested a possible association between helicobacter infection and gastric lymphoma in cats, although more research is needed to confirm the association and understand the pathogenesis. 7, 32 helicobacter spp. may be commensal in most cats, and perhaps loss of tolerance explains the development of gastritis in some individuals. 49 another possibility is that the inflammatory response is normally well managed and disease may treatment of underlying dermatologic disorders, and administration of semisolid petroleum laxatives. more recently, commercial diets have been formulated for control of trichobezoars. cats with recurrent trichobezoars causing illness and suspected motility disorders may benefit from treatment with prokinetic drugs such as cisapride. clinical signs of gastric foreign bodies are variable but typically involve intermittent or persistent vomiting because of gastric outflow obstruction, distention, and mucosal irritation. gastric obstruction may be partial or total. patients with complete obstruction will present with more dramatic signs, including anorexia and depression. the base of the tongue should always be examined, because linear foreign bodies are sometimes anchored either in this location, or they may be lodged in the pylorus, causing intestinal plication. gastric foreign bodies may also be asymptomatic and found incidentally. 5 physical examination may be unremarkable or may reveal dehydration or abdominal pain. if the stomach is markedly distended, the foreign body may be palpable in some patients. survey radiographs are always indicated when foreign body ingestion is suspected. radiopaque foreign bodies may be readily diagnosed, although some, along with radiolucent objects, will require a contrast study for diagnosis ( figure 23-13) . barium is commonly used as a contrast agent, although if gastric perforation is suspected, an aqueous iodinated agent is preferred. ultrasonography is also useful for detection of gastrointestinal foreign bodies. 56 removal of some foreign bodies can be attempted endoscopically, particularly if the object does not have sharp edges and is not too large. successful removal of fish hooks, particularly single-barb hooks, using endoscopy has been described. 35 otherwise, foreign objects are best removed using gastrotomy through a ventral midline laparotomy. a radiograph should always be to know when treatment should be attempted. one expert recommends treating only patients with clinical signs of gastritis that have biopsy-confirmed helicobacter infection with a treatment regimen of amoxicillin (20 mg/kg, every 12 hours, po), clarithromycin (7.5 mg/ kg, every 12 hours, po) and metronidazole (10 mg/kg, every 12 hours, po) for 14 days. 49 a common dilemma would be determining the treatment of choice for patients with lymphoplasmacytic inflammation of the stomach and small intestine and confirmed helicobacter infection. are such patients best treated for inflammatory bowel disease, helicobacter infection, or both? currently, guidelines for determining the best treatment approach are lacking. also, few studies on the efficacy of combination therapy have been conducted in cats. long-term eradication of infection may be difficult, and histopathologic resolution of gastritis may not be possible, which raises the question of whether helicobacter is the true underlying cause. 38, 41 in one study, two cats with clinical gastritis and helicobacter infection were treated with oral metronidazole, amoxicillin, and bismuth subsalicylate for 3 weeks and were also fed a commercial elimination diet. 25 posttreatment gastric biopsies were obtained a mean of 7 weeks after the cessation of treatment. resolution of clinical signs occurred rapidly, and clearance of helicobacter spp. was achieved at that time point, but gastric inflammation persisted in post-treatment biopsies. in another study, 13 cats with asymptomatic helicobacter infection were treated with oral omeprazole, amoxicillin, metronidazole, and clarithromycin for 14 days. 26 treatment failed to eradicate infection in 4 of the cats based on molecular analysis of post-treatment gastric biopsies. it is unclear if treatment failure is because of recrudescence or reinfection. the reader is referred to excellent reviews of helicobacter in cats for more information. 27, 38, 48 chronic gastritis chronic gastritis is common in cats with chronic intermittent vomiting. ollulanus tricuspis is a worm that infects the stomach of cats, causing chronic gastritis, and it is difficult to diagnose (see below, gastrointestinal parasites). the worm is occasionally found on histologic examination of gastric biopsy samples. 9 it is reasonable to treat empirically (fenbendazole 10 mg/kg, once daily, po × 2 days) for this parasite when the cause of gastritis is not apparent. 49 the frequency of vomiting in cats with chronic gastritis is highly variable, ranging from once or twice per week (and not necessarily every week) to more than once daily. most patients are otherwise well, although other clinical signs (inappetence, anorexia, depression, or weight loss) are possible depending on disease severity. results of routine laboratory testing are typically normal but may show neutrophilic leukocytosis, result when there is an abnormality of the immunoregulatory system. 21 the most commonly used methods for diagnosis of helicobacter infection in cats are based on gastric specimens obtained during endoscopy (or laparotomy): exfoliative cytology, histopathologic examination of biopsy specimens, and rapid urease testing of biopsy specimens. 28 however, it is important to note that even when helicobacter organisms are identified, the infection may not be the cause of the patient's clinical signs, and other causes of vomiting should always be evaluated. exfoliative cytology is the least expensive and most easily performed diagnostic test. in one study, it was also the most sensitive diagnostic method when compared with urease testing and histologic examination. 20 brush cytology samples gathered during endoscopy are airdried on microscope slides and stained with wright's stain. the slide is examined at 100× magnification under oil immersion. spiral bacteria are readily seen if present. at least 10 oil-immersion fields on two slides should be examined before determining a specimen is negative for helicobacter-like organisms. 28 since helicobacter produce abundant urease, a rapid urease test (e.g., clotest, ballard medical products, draper, utah) may be used for diagnosis. 38 the kit consists of an agar gel impregnated with urea and a ph indicator. a gastric biopsy sample is applied to the gel, and if urease is present, ammonia will form and change the ph (and thus the color) of the gel. the gel may change color rapidly (within 30 minutes), but 24 hours must elapse before the test can be considered negative. 28 the more rapidly the color changes, the higher the bacterial load. both false-positive and false-negative results are possible with rapid urease testing for various reasons, giving the test a sensitivity of 70% to 90%. 28, 37 histopathologic examination of gastric biopsy samples using hemotoxylin and eosin (h&e) or silver stains is highly sensitive and specific in human studies for detection of helicobacter-like organisms. the organisms are not equally distributed; so, examination of biopsy specimens from multiple sites will increase sensitivity. the bacteria may be seen in mucus on the surface epithelium as well as in the gastric pits, glandular lumen, and parietal cells. organisms may also be seen submucosally within gastric lymphoid follicles. 46 histopathologic examination of biopsy samples also allows for assessment of other abnormalities. mild to severe lymphocytic-plasmacytic or lymphocytic gastritis may be present. in humans combination therapy with antibiotics and antisecretory drugs is recommended to reduce the risk of gastric ulcers and cancer from h. pylori infection. treatment is highly successful at eradicating both clinical signs and histologic changes in the gastric mucosa. since helicobacter infection is common in cats, yet no clear pathogenic role has been established, it is difficult cases. 29 depending on the underlying cause and severity of disease, abdominal pain, anorexia, lethargy, pale mucous membranes, and drooling may also be seen. cats with neoplastic disease may have prolonged clinical signs and are more likely to present with anorexia and weight loss. 29 cats with perforated ulcers may or may not present with signs of shock. diagnosis may be problematic because the clinical signs and physical examination findings are often not specific, even in cats with perforated ulcerations. 8 the causes of gastric ulceration in cats are not well characterized. in dogs the most common cause is the administration of ulcerogenic drugs, particularly nsaids, either alone or in combination with corticosteroids. several cases of nsaid-induced gastroduodenal ulceration or perforation have been reported in cats. 8, 34, 45 additional cases may be reported in the future, because long-term administration of these drugs is gaining in popularity for treatment of chronic diseases such as osteoarthritis. nsaids cause direct mucosal damage and interfere with prostaglandin synthesis. although inhibition of the cox-1 enzyme is thought to be the cause of adverse effects, such as gastric ulceration, even cox-2-selective drugs have been associated with adverse effects, and safety in sick cats is not well evaluated. recently, guidelines for the long-term use of nsaids in cats were published by the international society of feline medicine and the american association of feline practitioners. 51 the recommendations include administering nsaids either with or shortly after food, withholding therapy if inappetence or anorexia develops, determining dose based on lean body weight, and titrating to the lowest effective dose. neoplastic causes of gastric ulceration include systemic mastocytosis, mast cell tumor, lymphosarcoma, adenocarcinoma, and gastrinoma (zollinger-ellison syndrome). cats with chronic renal disease may suffer mucosal damage from uremic toxins and increased gastric acid production secondary to hypergastrinemia (because of decreased renal metabolism of gastrin). 18 hepatic disease is a cause of gastric ulceration in dogs but is uncommonly reported in cats. 23 recent anesthesia and surgery have been implicated as a cause of gastric ulceration and perforation, perhaps through hypovolemia, hypoperfusion, or stress. 8, 29 other non-neoplastic causes reported for gastric or gastroduodenal ulceration in cats include parasites (e.g., ollulanus tricuspis, toxocara cati, aonchotheca putorii, gnathostoma spp.), bacterial infections, toxins, inflammatory bowel disease, and foreign bodies. one case report describes a cat with severe gastric ulceration caused by intoxication with dieffenbachia leaves. 36 in some case reports, the cause for the gastric ulcerations could not be determined. a minimum database should be collected for cats suspected of gastric ulceration, to identify underlying diseases. anemia, usually regenerative, may be present. eosinophilia, or hypoproteinemia. survey and contrast radiographs are often normal. the most common finding on histopathologic examination of biopsy samples is lymphocytic plasmacytic (lp) gastritis ( figure 23-14) . some patients will also have concurrent evidence of lp inflammation in the small intestine, pancreas, and/or liver. such patients will be treated for their concurrent problem; treatment of inflammatory bowel disease, pancreatitis, and cholangiohepatitis is covered elsewhere in this chapter. some cats with chronic lp gastritis respond to treatment for dietary intolerance or hypersensitivity with a limited antigen diet (see chapter 17) . patients with moderate to severe lp gastritis may be best treated with a limited antigen diet and immunosuppressive therapy (prednisolone 1 to 2 mg/kg/day, po tapering to every other day at the lowest dose that controls clinical signs). patients that fail this initial treatment approach may require additional immunosuppressive therapy, such as chlorambucil (see table 23-9) . occasionally, cats with chronic gastritis are diagnosed with eosinophilic inflammation on histopathologic examination of biopsy specimens. treatment is similar to that for lp gastritis, although such patients should be evaluated for evidence of hypereosinophilic syndrome and eosinophilic enteritis. eosinophilic fibrosing gastritis was suspected to be caused by toxoplasmosis in one case report. 33 gastric or gastroduodenal ulcerations are uncommon in the cat compared with the dog and may be caused by a variety of disorders, both gastric and nongastric. 29 classical clinical signs include vomiting, hematemesis, and melena. however, in one review of eight cats, hematemesis and melena were present in less than one third of suturing of the ulcer site as well as collection of biopsy samples for histopathologic examination. the prognosis for recovery was excellent in two studies, particularly for cats with non-neoplastic causes of gastric or gastroduodenal ulceration. 8, 29 in one study of seven cats with perforated gastric or duodenal ulcers, the survival rate was low (14%). 23 disorders of gastric motility are better characterized in dogs than in cats. the most common clinical sign is vomiting of undigested food 8 hours or more after a meal. if outflow obstruction is present, vomiting may be projectile. there may also be a history of recurrent trichobezoars. various disorders are associated with impaired gastric motility, such as chronic gastritis, drug therapy (e.g., anticholinergic and narcotic drugs), dysautonomia, gastric neoplasia, metabolic disorders (e.g., hypokalemia), and temporary postsurgical gastroparesis. in some cases of chronic motility dysfunction, no cause can be identified. outflow obstruction may be caused by neoplasia, foreign bodies, and extragastric masses. pyloric stenosis is infrequently documented in young cats, often siamese cats. 4, 40, 55 since the range of underlying disorders is diverse, the diagnostic approach should allow for detection of both gastric and nongastric disorders. a minimum database (cbc, serum chemistries, urinalysis, feline leukemia virus [felv] and feline immunodeficiency virus [fiv] serology) is used to establish overall health status. radiographs are used to confirm presence of food in the stomach for longer than 8 hours. ultrasonography may detect gastric lesions, such as masses. endoscopy is used to identify outflow obstruction as well as other lesions, such as ulcers, and evidence of gastritis. assessment of gastric emptying using nuclear scintigraphy is the most accurate method but is limited to referral centers. gastric emptying times for liquids, canned food, and dry diets have been established using nuclear scintigraphy. 11, 16, 17 however, emptying times are variable, depending on the amount and type of diet fed as well as the amount of water ingested. even the shape of kibble affects emptying time. 2 radiographic contrast series are widely used, but gastric emptying times are variable for barium in either liquid form or mixed with canned food. contrast radiography using liquid barium (8 to 10 ml/kg) is performed in a fasted patient. radiographs are taken immediately after administration of the barium and again at 15 and 30 minutes, in some cases, also at 1 and 3 hours. liquid barium is expected to enter the duodenum no more than 30 minutes after administration, and the stomach should be completely empty of barium within 3 hours. the clinician should be aware that some cats with gastric motility disorders will have other findings will be dependent on the presence of underlying diseases; for example, azotemia and isosthenuria may indicate renal disease. electrolyte and acidbase abnormalities may be because of chronic vomiting and anorexia. survey and contrast radiographs and ultrasonography are primarily useful to rule out other causes for the clinical signs, such as foreign bodies. cats with perforated ulcers may have evidence of pneumoperitoneum (sometimes severe) on plain radiographs or ultrasonographs, and this is an indication for surgical exploration. 6, 8, 24, 31, 34 evidence of peritonitis on imaging studies should be followed with peritoneal fluid analysis. a definitive diagnosis may be made using endoscopy, which allows direct visualization of lesions and collection of biopsy samples. however, some cats with gastric ulceration present in poor condition, which may preclude the use of endoscopy because of anesthetic risk and risk of ulcer perforation. 29 the location of ulcers is typically pyloroantral or fundic in cats with nonneoplastic disease. 8, 29 areas of erosion may appear pale or hemorrhagic; the mucosa is often friable and bleeds easily. fresh or clotted blood may be seen in the stomach lumen. in some cases, mucosal ulceration must be distinguished from ulcerated tumors. nsaid-induced ulcers are typically found in the antrum and do not have marked mucosal thickening; ulcerated tumors frequently have thickened edges and surrounding mucosa. 49 biopsy samples should be taken at the periphery of the ulcer to avoid perforation. treatment should be directed at any underlying disorder. treatment for nsaid toxicity is described in chapter 31. general supportive measures include fluid therapy and electrolyte replacement; blood transfusion may also be required (see chapter 25) . gastric acid production can be decreased with the use of h 2 -receptor blockers or proton pump inhibitors, and sucralfate is used as a mucosal protectant (see table 23 -5). sucralfate may inhibit absorption of other oral medications and should be given 2 hours apart from other drugs. if vomiting is severe or persistent, antiemetic therapy is warranted (see table 23 -3). analgesia should be provided for painful patients; a good choice is the opioid buprenorphine (see table 6 -1). broad-spectrum antibiotic therapy is indicated for patients with significant mucosal barrier dysfunction, perforation, leukopenia and/or neutrophilia, fever, and melena. surgical intervention is warranted for patients with life-threatening hemorrhage, failure to respond to medical management, or evidence of perforation. 29 the entire abdominal cavity and gastrointestinal tract should be thoroughly explored to locate extragastrointestinal lesions, non-perforated ulcers, and multiple ulcers. in one case series, nonperforated ulcers were detected at laparotomy by association with adhesions or a gastric mass. 29 surgical management includes débridement and months. physical examination findings are nonspecific, although occasionally a gastric mass or gastric thickening may be palpated if the stomach is markedly enlarged. results of routine diagnostic testing are generally nonspecific; anemia may be associated with ulceration. survey or contrast radiography may reveal a mass ( figure 23 -15, a); other findings include delayed gastric emptying, impaired motility, and mucosal ulceration. ultrasonography is also useful for diagnosis and can be used to guide needle aspirates of masses ( figure 23-15 , b). endoscopy allows for visualization of lesions as well as the ability to obtain partial thickness biopsy samples. problems with interpretation of endoscopic biopsy samples include detection of necrosis, inflammation, and ulceration rather than the primary lesion. in dogs some neoplastic lesions are submucosal, making it very difficult to obtain diagnostic samples by endoscopy. therefore several biopsies should be taken and masses should be biopsied multiple times in the same place to sample deeper tissues. the center of ulcerated lesions should not be biopsied. surgical biopsies are more reliable for diagnosis. a normal gastric emptying time with liquid barium. barium can also be mixed with canned food and fed as a meal; retention of barium-containing food in the stomach for more than 8 to 12 hours is abnormal. gastric emptying time may also be established with the use of barium impregnated polyspheres (bips; med i.d. systems, grand rapids, mich.) and radiography. gastric emptying times for bips have been established in healthy fasted and fed cats as well as in sedated cats, 10,52 but the values do not correlate well with scintigraphic studies. 17 a mixture of small (1.5 mm) and large (5 mm) spheres are administered with food, and two to four radiographs are taken over the next 24 hours. the small spheres are intended to mimic liquid transit time and the large spheres solid transit time. however, studies assessing the clinical relevance of this method are lacking. one review concluded that bips are probably sufficiently sensitive to detect grossly delayed gastric emptying. 60 treatment of gastric emptying disorders is directed at identifiable causes. treatment for gastric ulcers, chronic gastritis, and foreign bodies is described elsewhere in this chapter. pyloric stenosis is managed surgically. if no outflow obstruction exists, treatment with prokinetic agents, such as metoclopramide or cisapride, may be beneficial (see table 23 -3). gastric tumors account for less than 1% of malignancies in dogs and cats. 30 benign gastric tumors are even less common than gastric malignancies. gastric smooth muscle hamartoma has been reported in one 11-year-old cat. 50 although adenocarcinoma is the most common gastric cancer of the dog, lymphoma is the most common gastric cancer in the cat. feline gastrointestinal lymphoma occurs as two major types: small cell (lymphocytic) and the more aggressive large cell (lymphoblastic) form. small cell lymphomas are more frequently enteric. 57 in one study of 12 cats with gastric lymphoma, diffuse large b-lymphocyte tumors of immunoblastic nuclear type predominated. 42 gastric lymphoma is not associated with felv, and the role of helicobacter in the development of gastric lymphoma in cats requires investigation. 7 adenocarcinoma, 12,13,54 plasmacytoma, 62 and gastric carcinoid 44 have also been described. the siamese cat may be predisposed to adenocarcinoma. 12, 54 as would be expected, most cats with gastric neoplasia are older cats. as for most gastric diseases, vomiting is the most common clinical sign of neoplasia. the vomitus may contain blood and melena may be present. other clinical signs include anorexia, weight loss, bloating, and depression. perforation of the tumor may occur, leading to pneumoperitoneum or septic peritonitis. clinical signs present gradually and are often present for weeks to surgical resection is the most common treatment for gastric neoplasia other than lymphoma ( figure 23 -16). the prognosis for most patients is poor, typically because of debilitation, concurrent diseases, and recurrent or metastatic disease. 30 the success of chemotherapy for lymphoma depends on cell type, with small cell tumors carrying a better prognosis than large cell tumors. in diarrhea can be defined as increased volume and/or increased frequency of defecation of stools with increased water content. approaches to diarrhea, as for any clinical sign, need to take into account the individual animal. for example, neoplasia is much less likely to occur in a kitten than in a geriatric cat. in many cases, the precise diagnosis of gastrointestinal disease cannot be reached without biopsy samples. the decision to obtain biopsy samples should follow a logical pathway that is appropriate to the cat's condition. these are summarized in figure 23 -17. for example, many cases of acute diarrhea in a well cat can resolve with limited or no intervention, and so do not require a precise diagnosis. the diagnostic steps are 1. signalment and clinical history 2. physical examination 3. fecal assessment 4. blood and urine testing 5. imaging (radiography, ultrasonography) 6. biopsy samples these steps do not include treatment/diet trials or other empiric therapies that are appropriate in many cases. steps 3 and 4 are often undertaken at the same time, and there is no definite order for these steps. they are divided here for reasons of clarity. in a younger cat, where infectious causes are more likely, thorough fecal testing is more important; in an older cat, extragastrointestinal diseases, such as hyperthyroidism, are more likely; so, blood and urine testing is more important, but fecal assessment should not be neglected. the decision to proceed to step 4 (and each subsequent step) should take into account several considerations. the main considerations in assessing and managing a cat with diarrhea are • is there an acute onset or a chronic time course? • are there any dietary changes or indiscretion? 36 consider treatment trials: antibiotics (e.g., amoxicillin/ clavulanate ؉ metronidazole). food trials with novel proteins (e.g., rabbit, kangaroo, venison). • is the cat well or unwell? • is there primary or secondary gastrointestinal disease? • is there small or large bowel diarrhea? the components of the clinical history for cats with diarrhea are detailed in table 23 -12. after establishing the cat's age, breed, vaccination, and deworming history, it is important to establish the duration and nature of the diarrhea. chronic diarrhea is usually defined as greater than 3 weeks in duration and mostly warrants at least some degree of a diagnostic workup, whereas acute diarrhea is often self-limiting in a well cat. a description of the feces helps determine whether the diarrhea is small or large bowel in origin (table 23 -13); this will affect how any investigations might proceed. important questions to ask concern frequency of defecation (and how this compares with the normal state), tenesmus (straining usually indicates large bowel diarrhea, since an irritated colon leads to urgency), volume of feces (smaller volumes are typical of large bowel diarrhea; larger volumes are more typical of small onset and duration of diarrhea acute versus chronic? acute diarrheas are abrupt in onset and of short duration, and generally they are self-limiting. chronic diarrheas persist usually longer than 3 weeks and fail to respond to symptomatic therapy. appearance of diarrhea quantity and quality of the stool (color, consistency, character, presence of blood or mucus)? loose to watery feces that contain fat droplets, undigested food, melena, and variable colors suggests small intestinal disease. the volume is always increased with small intestinal disease. loose to semisolid feces containing excess mucus and fresh blood (hematochezia) indicates large intestinal disease. the volume may be normal to slightly decreased with large intestinal disease. description of defecation process tenesmus (straining) and dyschezia (painful defecation)? these are hallmarks of large intestinal disease (e.g., inflammatory or obstructive lesions of the colon, rectum, or anus). frequency is normal to slightly increased with small bowel disease, but greatly increased with large bowel disease. associated physical signs vomiting, anorexia, weight loss, and dyschezia may help localize the disorder to a specific part of the gastrointestinal tract. clinical signs relating to problems in other organs or body systems should be noted and may suggest a more generalized disease. vomiting may occur as a consequence of small intestinal inflammation in some cats with diarrhea. weight loss may result from decreased caloric intake (anorexia), decreased nutrient assimilation (maldigestion/malabsorption), or excessive caloric loss (protein-losing enteropathy or nephropathy). weight loss is observed uncommonly with large bowel disease. in many cases, the answers to these questions are obvious. for example, a cat may seem well but has had access to lilies (the author has seen diarrhea as a primary presenting sign for this!) or has a palpable abdominal mass. substantial weight loss is an indicator that further investigations are warranted sooner rather than later. if the decision is made for empiric management and outpatient care, it is vital to follow up either by scheduling a recheck visit or calling the client, because simple acute problems can turn into complicated chronic problems. if the diarrhea has been present for less than a week and the cat has no weight loss, dehydration, fever, or palpable abdominal abnormalities, it is appropriate to manage the cat as an outpatient. even in the absence of fecal testing, it is appropriate to deworm the cat (see the section gastrointestinal parasites). the cat should be fasted for 24 hours (12 hours, if less than 4 months old) and then fed a bland diet (such as plain, cooked, skinless chicken, or low-residue prescription diets designed for cats with gastrointestinal problems). it is appropriate to maintain the cat on the low-residue diet for at least 7 to 10 days and then slowly reintroduce the regular diet. fecal assessment is mostly used to assess infectious agents, such as parasite-associated diarrhea, but the importance of assessing feces, even when parasitic or bacterial infections are not suspected, should not be underestimated. gross examination of feces can determine if melena or fresh blood or mucus are present to help distinguish large from small bowel disease when the owner's observations may be misleading. occult fecal blood can be an indicator of gastrointestinal inflammation in cases of subtle disease, and undigested starches and fats can indicate maldigestion or malabsorption. 8 for assessment of feces for parasites, the fecal sample should ideally be fresh (<1 hour old). refrigeration (for no longer than one week) can preserve ova, oocysts, and cysts but not protozoal trophozoites. feces should be assessed by a. to assess for trophozoites bowel), how formed the stool is (from soft stool to cow-pat consistency to liquid tea; usually more watery stool relates to small intestinal disease), color (darker indicates digested blood), and presence of any mucus or blood (presence relates to large bowel). most household toxins, such as plants, cause signs additional to diarrhea such vomiting or neurological signs, 11 but it is important to ascertain if the cat has had access to anything unusual. likewise, it is important to find out if the cat has had any possible exposure to dietary indiscretions; this can include if the cat has been seen with or is known to hunt prey including insects. cockroaches carry pathogenic bacteria 12, 18 and other prey such as birds and rats can carry salmonella; salmonellosis in cats has been dubbed songbird fever. 6 simple causes of self-limiting diarrhea include dietary change (either a new flavor or a new style of food, such as dry food for the first time); so, the owner must also be quizzed if anything new has been offered, either new cat food or treats (such as greasy fish or chicken). although the physical examination will usually determine how unwell a cat is, the owner's impressions are also important, because cats can hide signs from strangers, particularly in a practice setting. lethargy and inappetence are important signs, as ill cats typically do not eat well. the cat's general demeanor can be an indicator of how unwell a cat is and therefore dictate the extent of diagnostic testing required. this can be noted by assessing how interested the cat is in its surroundings or any behavior changes from previous visits, such as if a normally difficult-to-handle cat is placid. body weight should be assessed and, if possible, compared with that of previous visits (even those noted on a clinical record from another veterinarian). the body condition score (bcs) should also be assessed and can be very important when there is no prior weight information. dehydration is usually a sign that a cat needs more involved management. abdominal palpation should be performed to assess pain (where?), any masses (foreign bodies, lymph nodes, or even focally thickened intestines, such as with neoplasia), or turgid intestines. fever often indicates infection but can also reflect neoplasia or other inflammatory changes. a thorough examination of all body systems should always be performed, no matter what a cat presents for. in the case of diarrhea, extragastrointestinal signs can be of vital importance, such as a palpable thyroid and tachycardia suggesting hyperthyroidism. after the clinical history has been taken and the physical examination performed, the veterinarian must make the important decisions of whether any interventions are required and whether the patient should be factors affecting interpretation include whether the growth is a heavy and pure growth of a known pathogen, such as salmonella, campylobacter, yersinia, or clostridium difficile. further information about the relevance of culture and pcr results is contained below in the section infectious enteritis. investigations begin by assessing if the diarrhea is the result of primary gastrointestinal disease or secondary to another process, by performing routine serum/plasma biochemistries, hematology, urinalysis, and total t 4 (for older cats). in most cases of secondary gastrointestinal disease, diarrhea is not usually the primary presenting complaint, but since the approach to investigations and management diverge so much, this is an important step to take. biochemistry and urine tests may also show the consequences of diarrhea, such as dehydration and electrolyte abnormalities. a. can aid in the visualization of internal structures of some protozoa 3. fecal flotation (preferably with centrifugation) a. to find cysts, oocysts, and ova fecal culture should be undertaken with the understanding that bacteria will be cultured; so, interpretation is based on the relevance of the positive culture result. used to evaluate the smear for the presence of trophozoites, such as giardia spp. and tritrichomonas foetus. 1. place peppercorn size amount of feces on a warm slide and mix with a drop of 0.9% saline (smear must not be too thick, because trophozoites will be easily missed). 2. apply coverslip. 3. evaluate systematically for motile organisms using the 10× magnification. 4. confirmation at 40× magnification. adding iodine to a wet mount through the edge of the coverslip can aid in the visualization of internal structures of some protozoa. the direct wet preparation must be examined without any stain for motility first, because staining the preparation kills the organism. methylene blue is useful for identifying trophozoites, particularly those of entamoeba histolytica. this method has little to no diagnostic value for the diagnosis of bacterial-associated diarrhea. used to find cysts, oocysts, and ova in feces. standing (gravitational) flotation methods are easier and quicker but have much poorer sensitivity than centrifugation methods. 2 solutions used in centrifugation flotation methods include zinc sulfate and sheather sugar. 1. weigh out 2 to 5 g of feces. 2. mix feces with approximately 10 ml of flotation solution. 3. pour mixture through a tea strainer into a beaker or fecal cup. 4. pour strained solution into a 15-ml centrifuge tube. 5. fill tube with flotation solution so that a slight positive meniscus forms, being sure not to overfill the tube. 6. place a coverslip on the tube, and put the tube in the centrifuge. 7. make sure the centrifuge is balanced. 8. centrifuge at 1200 rpm (280× g) for 5 minutes. 9. remove the tube and let stand 10 minutes. 10. remove the coverslip, and place it on a glass slide. systematically examine the entire area under the coverslip at 100× magnification (i.e., 10× objective). you may wish to use the 40× objective lens to confirm your diagnosis and make measurements; however, with practice, most parasites can be identified using the 10× objective (100× magnification). (fpli) are useful markers of intestinal and pancreatic disease, [14] [15] [16] [17] but it is important to note that they typically do not give a precise diagnosis. cobalamin and folate are water-soluble vitamins and are readily found in commercial cat foods so that dietary insufficiency is rare, and decreased levels are almost always because of gi disease. these vitamins are taken up by specific receptors in different areas of the small intestine. chronic inflammatory gastrointestinal disease may damage the receptors and lead to decreased serum concentrations of one or both vitamins, provided the disease process is severe and long standing enough to deplete body stores. serum cobalamin and folate concentrations may also be decreased in cats with exocrine pancreatic insufficiency (epi). trypsin-like immunoreactivity is a pancreasspecific marker, and assessment of serum tli is used for diagnosis of epi and pancreatitis in the cat, although the sensitivity of the assay for pancreatitis is low. pli is a marker for pancreatic inflammation and is more sensitive than tli for the diagnosis of pancreatitis. since inflammation of the small intestine may be seen concurrently with pancreatitis, serum tli and pli are useful adjunctive tests in the diagnosis of diarrhea. tli, pli, and cobalamin are stable in serum at room temperature for several days, but folate is unstable so that samples for cobalamin/folate analysis should be frozen (table 23-14) . samples submitted for folate concentration should not be hemolyzed, because red blood cells contain high levels of folate. in addition, folate is light-sensitive, and samples should be wrapped to exclude light. severe lipemia may interfere with common assays for tli and pli. the main utility of these tests are to indicate that further investigation of gastrointestinal disease is warranted. when a cat presents for weight loss with no overt signs of gi disease, decreased cobalamin or folate can indicate that further investigations with imaging and, ultimately, biopsy sampling are warranted. many clients are more willing to proceed with hematology can be normal in some cats, with changes expected, and so should not be used to rule out any condition. it can be useful, for example, if there is a left shift neutrophilia, indicating acute infection, or eosinophilia, reflecting parasitism. monocytosis can suggest chronic disease that was not suggested by the clinical history. in the case of acute onset diarrhea, the cat may be unwell as a consequence of the diarrhea (e.g., from dehydration) and not because of the cause of the diarrhea. if rehydration is required (with intravenous or subcutaneous fluids, depending on severity of illness), then it is important that biochemistry tests are performed before fluid administration so that any diagnostic clues are not lost by alteration of the profile from the fluid therapy. fever and neutrophilia may indicate the need for antibiotic therapy. if infection is suspected, fecal sampling (see step 4) should occur before starting antibiotics. if a cat is unwell from dehydration, then further testing may not be warranted. the clinician should be alert that linear foreign bodies can result in diarrhea (see the section intestinal obstruction). diarrhea of chronic duration (greater than 3 weeks) does require a more thorough investigation at the outset. however, if clinically well, the cat can be managed as an outpatient in the first instance, at least while waiting for results of diagnostic testing. a diet trial with a novel protein is appropriate for a well cat with stable weight. as with any patient managed as an outpatient, follow-up is vital and, in this scenario, includes scheduling revisits. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity invasive diagnostics when a specific marker of the disease in the organ involved has been recognized. caution should be exercised, because either cobalamin or folate may not be reduced with gi disease. in one study of small cell lymphoma, only 78% of cats were hypocobalaminemic, 7 meaning that if this was the only instigating factor to investigate, nearly one fourth of cats would not have been investigated further. also, cobalamin may be reduced in nonalimentary illness. 1 2. to detect hypocobalaminemia that may indicate the need for supplementation for clinical improvement. 13 to recognize pancreatic pathology when fpli is increased. 17 it is important to note that an elevated value gives no indication of the nature of the pancreatic pathology. 4. to make a diagnosis of epi when the ftli is low. 15 it should be noted that epi can result from other pathology that may require further investigations. veterinarians if the patient is new to the practice) are usually helpful. body condition scoring (using a 5-point or 9-point scale) for every cat seen is helpful in recognizing those that are underweight. weight loss often occurs with loss of muscle mass in cats, and muscle mass can be assessed over the ribs and pelvis as well as scapulae and nuchal crest. thickened intestines are also a subjective finding; it is the author's opinion that thickened intestines are actually intestines with increased turgidity, since differences between normal intestines and those with inflammatory infiltrates can be as little as 0.5 mm. perhaps more important during the history taking and physical examination are those signs that can point to extragastrointestinal disease. when confronted with a cat showing weight loss or vomiting or diarrhea (or a combination of signs), the clinician should start with trying to distinguish the signs as being either primary gastrointestinal or secondary signs. examples of clues pointing to extragastrointestinal diseases include tachycardia and palpable thyroid nodule, indicating hyperthyroidism, or polydipsia/polyuria, which has a variety of causes but is not typical of primary intestinal disease. inflammatory bowel disease has traditionally been considered an immune-mediated disease. the local immune system of the intestinal mucosa no doubt plays an important role, but recent work has also shown the importance of the normal bacterial population in perpetuating and, perhaps, even initiating pathology. it is known for certain that ibds are an expression of an overanxious immune response, with a recent study 104 indicating increases in inflammatory (il-6), type-1 immunity (il-12 p40), and immunomodulatory (transforming growth factor [tgf]-beta, il-10) cytokines. other researchers have found an association with bacterial counts (enterobacteriaceae, e. coli, and clostridium spp.) and abnormalities in mucosal architecture, indicating that mucosal bacteria are involved in the etiopathogenesis. 65 we can summarize these theories by saying that ibds are likely to be a consequence of hypersensitivity reactions to antigens from the intestinal lumen (e.g., bacterial, parasitic, or dietary antigens). this hypersensitivity may occur because of failed immunoregulation (suppressive function) of the gut-associated lymphoid tissue (galt). it is known that granulomatous colitis in boxer dogs is associated with infection, 143 and pathogens may well be found in at least some cases of ibd in cats that cause the immune response and subsequent inflammatory infiltrate of the lamina propria typically seen. although not described specifically in cats, chronic intestinal inflammatory change can impair motility. inflammatory bowel disease (ibd) refers to intestinal inflammatory infiltrates of the small or large intestine (or both) of unknown etiology. the term ibd should strictly be applied to mean idiopathic ibd, thus excluding inflammatory enteritis because of food sensitivities, although common usage has led to ibd referring to intestinal inflammatory infiltrates of both known and unknown causes. ibd is not a diagnostic end point but a description of a series of intestinal diseases that have similar histopathology. recent efforts by the world small animal veterinary association (wsava) gastrointestinal standardization group have led to both diagnostic and classification guidelines 35, 168 ,169 that encompass chronicity, nonresponse to symptomatic treatment, no specific cause found, as well as histologic confirmation of non-neoplastic intestinal inflammatory changes. there are no obvious breed or gender predispositions, and although cats of any age can be affected, inflammatory intestinal diseases are more likely to occur in middle-aged to older cats (5 to 10 years of age or older) than in younger cats. presenting clinical signs include vomiting, diarrhea, and weight loss with increased or decreased appetite. these signs can occur in isolation or together. weight loss without vomiting or diarrhea deserves special mention because not only have several studies 38, 58 shown this to be the most common presenting sign for ibd, but many veterinarians do not consider primary intestinal disease without the presence of vomiting or diarrhea. weight loss despite normal to increased caloric intake can represent poor absorption of food because of small intestinal disease, although it can also represent maldigestion associated with exocrine pancreatic insufficiency or increased metabolism associated with hyperthyroidism, or even lack of energy utilization associated with diabetes mellitus. conversely, appetite may be reduced, most likely because of nausea. if the large bowel is affected, signs are typically discomfort when defecating, resulting in frequent small volumes of diarrhea, often with mucus and blood; if the large bowel alone is affected, there may be no weight loss. physical examination findings are often nonspecific, but the most consistent findings for small intestinal disease are weight loss (or being underweight in a cat not seen previously) and palpably "thickened" intestines. noting a cat as underweight can be subjective, and prior recorded weights (even from previous paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma than those with ibd, but this change was also seen in 12% of cats with normal small intestine. 177 inflammatory bowel diseases require histologic findings obtained from biopsy samples for diagnosis, but diagnosis should not be made solely on these findings. the wsava international gastrointestinal standardization group has proposed "an all encompassing definition of inflammatory bowel disease" that comprises clinical criteria, imaging criteria, as well as pathophysiologic criteria. 169 the clinical criteria for the diagnosis of ibd include there are no typical laboratory findings in ibd, and many cats may have entirely normal results from routine biochemical and hematologic investigations. moderate liver enzyme elevations may be seen 5,38,58,66 even in the absence of recognizable hepatic pathology, and this may reflect subclinical secondary hepatic disease, secondary cholestasis, or showering of the liver with inflammatory cells from the small intestine through the portal circulation. 66 other changes can reflect consequences of the intestinal disease, such as azotemia or hemoconcentration reflecting dehydration, 58 or hypokalemia reflecting inappetance. 38 the chronic inflammation may be reflected by neutrophilia, monocytosis, 5, 38, 58, 66 or hyperglobulinaemia. 38 hypocobalaminemia can reflect ileal inflammation, and low serum folate can reflect proximal small intestinal inflammation. 149 typical ultrasonographic findings consistent with ibd are focal or diffuse intestinal wall thickening ( figure 23-19) ; normal wall thickness is less than or equal to 2.8 mm for the duodenum and less than or equal to 3.2 mm for the ileum, 50 and large mesenteric lymph nodes with hypoechoic changes may be seen. one study found that ultrasonographic findings correlated with histologic grade of ibd. 5 there is no clear distinction between ultrasonographic changes from ibd and those from small cell lymphoma. one recent therapeutic trial, follow-up visits are vitally important. many cats with small intestinal disease may show initial improvement simply because of the diet having lower residue, since there is decreased substrate for intestinal bacteria to digest and lower osmotic potential. the corollary of this is that failure of one novel protein diet does not mean that all novel protein diets will fail. when food sensitivities are responsible for gastrointestinal clinical signs in cats, the responsible food ingredient is usually a dietary staple. commonly incriminated ingredients are beef, fish, wheat, and corn gluten. 55 a careful dietary history is therefore important. large bowel inflammation typically improves with higherfiber diets, 38, 58 and attempting a trial with such a diet is certainly appropriate. immune suppressive therapy is the mainstay of ibd treatment, and glucocorticoids, such as prednisolone, are most commonly used. sulfasalazine use for large bowel signs has not been critically evaluated but seems safe and effective. in cats with substantial weight loss or severe clinical signs, such as chronic diarrhea, the author prefers to start with corticosteroid therapy, even if dietary causes have not yet been ruled out. the diet should also be changed to one containing a novel protein, and, if and when clinical signs resolve, an attempt is made to wean the cat from corticosteroid therapy, hopefully to the point of being discontinued. a diet challenge can then be used to confirm the diagnosis of food sensitivity. there are no universal guidelines for doses of corticosteroids. the author prefers the use of orally administered prednisolone to reduce the chance of side effects and will choose the starting dose based on the severity of disease. the starting dose is usually 2 mg/kg, once daily, po (10 mg/cat/day for most cats) starting 10 days after biopsies have been obtained to allow time for the mucosa to heal. if there is an improvement noted after a recheck at 2 weeks, the higher dose is maintained for a further 2 to 4 weeks, at which point, many cats are back to their normal weight and are not exhibiting clinical signs. if this is the case, the corticosteroid dose can be weaned down to 1 mg/kg, po (often 5 mg/cat/day) for several months, with continued rechecks scheduled to assess weight, clinical signs, and diet. the goal is to wean down to the lowest effective dose. if hypocobalaminemia is present, cobalamin supplementation may be required. 144 cobalamin is administered parenterally at 250 µg/cat subcutaneously weekly for 6 weeks, then every second week for 6 weeks, then monthly. owners can be shown how to inject their cats (as practitioners routinely do with diabetics). clinicians often consider the assessment of histologic samples to be out of their hands; however, it is important to work with the pathologist by providing good quality samples and a good clinical history, as well as having an open dialogue if the findings are not within expectations. for example, with lymphocytic/plasmacytic infiltrations, the pathologist has the difficult task of distinguishing diseased from normal tissue in a site that is laden with lymphocytes in the healthy state. once deciding the tissue has pathology, the pathologist's next task is distinguishing inflammatory infiltrate from neoplastic infiltrate with normal, mature lymphocytes (as seen in small cell lymphoma). inflammatory change also results in changes to normal tissue architecture, with thickened villi, edema, or erosion of the epithelium being typical changes. clinicians should expect morphologic descriptions as well as assessments of degree and type of inflammation. these difficulties are further compounded with the recognition that histologic grading of mild, moderate, or severe does not necessarily correlate with severity of clinical signs. this means that a cat with severe clinical signs of weight loss and vomiting or diarrhea may have only mild histologic changes (and vice versa). concurrent inflammation of the pancreas and liver with intestinal inflammation was first described in the mid-1990s, 171 and despite constant reference to this phenomenon at conferences and veterinary websites, there has been little description since then, though one study found 70% of ibd cases had liver inflammation and 30% had pancreatic inflammation. 6 the term "triaditis" has frequently been used, but the author prefers to spell this "tri-iditis" to distinguish it from inflammation of the hepatic portal triads. there has been no assessment of prognosis when the pancreas and/or liver are involved, but the author has found no difference in prognosis. many cases diagnosed with intestinal inflammatory infiltrates have these changes because of dietary sensitivity. in one study, 29% of cats with histologic gastrointestinal changes improved with dietary elimination therapy alone. interestingly, improvement was noted within 4 days compared with the longer duration of 8 weeks often recommended for improvement of dermatologic manifestations of food sensitivities. this careful study made note of the cat's prior diets and likely dietary causes of sensitivities. 55 another study found dietary therapy to be unsuccessful in 52 of 60 cats but no specifics of diets tried are noted. 58 as with any national cancer institute working formulation (nci wf) system. 116 for most veterinarians in practice, the most important distinction is the histologic grade, because low-grade (lymphocytic or small cell) lymphoma has a much better prognosis (and requires different treatment) compared with high-grade (often lymphoblastic) or intermediategrade lymphoma. for the purposes of simplicity and practicality, only small cell lymphoma and high-grade lymphoma will be addressed here. the prognosis and treatment for intermediate-grade intestinal lymphoma should be considered as for high-grade lymphoma. small cell lymphoma was first described in human pathology in 1966. 122 earlier, small lymphocytes were considered end-stage cells without the ability to divide. in cats small cell lymphoma is most commonly associated with the gastrointestinal tract or skin. 163 small cell neoplasia can be a confusing concept, since our traditional ideas of malignant neoplasia focus on rapidly dividing cells. the confusion is compounded by various terms used in the literature, such as lymphocytic lymphoma, low-grade lymphoma, well-differentiated lymphoma, or diffuse lymphoma; another term, epitheliotropic malignant lymphoma predominantly applies to small cell lymphoma, and other papers fail to distinguish these lymphomas from lymphoblastic lymphosarcoma (the traditional, aggressive form). "small cell lymphoma" seems to be most widely used term, though the author prefers "lymphocytic lymphosarcoma," since it is more descriptive. intestinal small cell lymphoma can be considered as a severe lymphocytic intestinal infiltrate, the most common form of which is commonly called ibd. not only is lymphocytic ibd hard to distinguish histologically from lymphocytic lymphosarcoma, but the approaches and treatments are similar. several reports have suggested a relationship between the two conditions in that inflammatory infiltrates may become neoplastic over time. 27, 43, 90 prevalence the true prevalence of intestinal small cell lymphoma is unknown, but several recent studies have indicated similar rates to inflammatory bowel diseases, with kleinschmidt et al noting 10 small cell lymphoma cats compared with 14 with intestinal lymphocytic infiltrates, 74 evans et al reporting 10 cases compared with 12 with ibds 40 , and baral et al diagnosing 8 cases compared with 10 with ibds. 6 traditionally, 90% of feline lymphosarcoma is regarded as intermediate or high grade, 163 but this may not be the case within the gastrointestinal tract. fondacaro et al found 75% of gastrointestinal lymphoma to be lymphocytic 43 ; a more recent paper found some cats seem resistant to conventional therapy. if this is the case, the diagnostic findings should be re-assessed to ensure no steps were missed or findings disregarded; the cat should be reexamined to look for emergence of other signs; and the pathologist who reads the histology should be contacted to recheck the findings. some cases of apparently resistant ibd are actually food sensitive, but it can be difficult to find the incriminating diet source, and commercial diets are not always effective. if underlying infectious causes have been entirely ruled out and the practitioner is certain of the diagnosis of idiopathic disease, immune suppressive therapy can be increased by either increasing the dose of prednisolone or using other agents, such as chlorambucil, typically at 2 mg/cat, po, every second day. it has been suggested that cats with eosinophilic inflammation may be more likely to be refractory to standard therapy. side effects of immunosuppressive therapy are rare but include inducing diabetes mellitus, immune suppression, delayed healing, and gastrointestinal ulceration. reported doses of sulfasalazine to manage large bowel ibd are 10 to 20 mg/kg, po, once daily for 7 to 10 days. 174 because this drug is usually only available as 500 mg tablets, one eighth of a tablet, providing a dose of 62.5 mg, is usually appropriate for most cats. in some countries, it is possible to have a compounding pharmacist formulate the drug into more convenient tablet sizes or as an oral suspension. cats are generally regarded as susceptible to salicylates, and possible side effects include vomiting or diarrhea, or anemia. the exact pharmacodynamics of this drug are not known; so, caution for extended use should be exercised and the drug withdrawn if any possible adverse signs are noted, but there are anecdotal reports of extended use of this drug without adverse consequences. a survey of the online veterinary cancer registry (http://www.vetcancerregistry.com) identified 6% of all submitted feline tumors to be intestinal tumors. approximately 74% of reported feline small intestinal tumors were lymphomas. adenocarcinomas accounted for 17%, and other tumor types reported included mast cell tumors and leiomyosarcomas. 135 "lymphoma in veterinary medicine: no longer a oneword diagnosis" was the title of an editorial in a recent issue of the veterinary clinical pathology journal, 95 and this is nowhere truer than in the feline gastrointestinal tract! a recent study classified 50 cases of feline gastrointestinal lymphoma both histologically and immunophenotypically, and it found eight different categories according to the revised european and american lymphoma/world health organization (real/who) classification system and six categories according to the approximately equal numbers of high-grade and lowgrade gastrointestinal lymphoma. 84 older cats are more at risk of small cell lymphoma, with mean or median ages reported from 9 to 13 years. younger cats with the disease have, however, been recognized. 40, 43, 73, 84 no breed or gender predispositions have been definitively recognized. two larger studies have suggested a skew to males with 28 males compared with 22 females in one report, 43 and 24 males compared with 17 females in the other 73 ; most other studies looking at gender and breed did not clearly distinguish between lymphoblastic and lymphocytic neoplasia. clinically, it is impossible to distinguish cats with ibds from cats with small cell lymphoma. this is hardly surprising when even histologic distinction can be difficult! therefore cats will present with weight loss or vomiting or diarrhea at a similar frequency to those with ibd. weight loss has been recognized as a presenting sign in 82% to 100% of cases, diarrhea in 25% to 60% of cases, and vomiting in 25% to 73% of cases, with various combinations of these signs also possible. other variable signs are lethargy and inappetence or, conversely, polyphagia. 40, 43, 73, 84 these findings can be summarized by stating that cats with gastrointestinal small cell lymphoma can present with any combination of signs relating to the gastrointestinal tract. intestinal small cell lymphoma is typically a diffuse disease, and therefore multiple areas of the alimentary tract are usually affected. in studies where different locations of the small intestine were assessed, the jejunum was most commonly affected (100%), with the ileum frequently affected (93% to 100%), and duodenal pathology slightly less prevalent (83% to 90%). 40, 84 although the numbers of cats assessed in these studies are small, the important fact that the duodenum is not always affected needs to be recognized, which has important implications for how biopsy samples are obtained, because lesions beyond the duodenum are likely to be beyond the reach of an endoscope. further difficulties in precise diagnosis may arise, since non-neoplastic lymphocytic infiltrates (e.g., ibd) are often found in other locations along the intestinal tract. 27, 40, 84 the stomach is also affected in 14% to 40% of small cell lymphoma cases. 40, 43, 84 although not fully assessed, involvement of the colon appears rare. 84 local lymph node involvement is common, being noted in up to 59% of cases. 84 this percentage may be even higher, because many studies assessed lymph node cytology from ultrasound-guided fine-needle aspirates, which may miss spread to the lymph node, because the population of neoplastic lymphocytic cells is indistinguishable from the normal population of lymph node cells. histology is required to assess changes in lymph node architecture. liver involvement is not uncommon but not thoroughly assessed. one study noted liver lymphocytic neoplasia in 8 of 38 cats with small intestinal lymphocytic neoplasia, 73 another found 5 of 15 affected cats in which the liver was biopsied, 84 another noted 2 of 4 cats had liver involvement, 27 and a further study detected neoplasia "in the lymph nodes, liver, or both" in all 10 cats with intestinal small cell lymphoma. 40 the pancreas may also be involved. 73, 84 this may be akin to the noted association of lymphocytic inflammation of intestine, pancreas, and liver 171 that has been dubbed tri-iditis. ultrasound findings may not suggest extragastrointestinal involvement. in the case of liver pathology, ultrasonography may show no changes in as many as 75% to 80% of cases. 40, 84 focal nodular changes and he patomegaly have been recognized as ultrasonographic signs of hepatic small cell lymphoma. 7 both lymphocytic ibd and lymphocytic neoplasia are often recognized simultaneously in the same cat, 40, 84 and numerous authors have suggested that lymphocytic ibd may be a precursor to intestinal lymphoid neoplasia. 90, 125 if this is the case, then antigenic factors, such as bacterial population changes or food sensitivities, could be considered primary initiating factors for small cell lymphoma since they are potential underlying etiologies of ibds. 79 however, neoplasia also requires genetic mutations to occur (often affecting regulation of cell death and cell survival), and these may be initiated by the inciting antigenic factors or the ongoing inflammatory changes. 154 as opposed to other feline lymphoid neoplasia, no association has been made with felv infection. 27, 43, 73, 125 intestinal lymphocytic lymphosarcoma begins in the superficial mucosa and progresses to involve the entire mucosa and submucosa; then advancing in a perivascular pattern into the tunica muscularis, eventually infiltrating all four intestinal tunics. 43 lymph node and other organ (such as liver or pancreas) involvement likely represent metastasis through lymphatics and perhaps hematogenously. more distant metastasis is not reported. serum or plasma biochemistry and hematologic findings are typically nonspecific. however, this testing is important as part of the diagnostic workup to rule out extra-gi disease, such as hyperthyroidism or diabetes mellitus. common biochemistry findings are mild to moderate increase of liver enzymes, such as alanine aminotransferase (alt), aspartate aminotransferase (ast), and/or alkaline phosphatase (alp). 27, 40, 43, 84 as with ibds, these liver enzyme changes may or may not represent overt hepatic disease. 67 albumin may be reduced 43 but is normal in most cases 27, 43, 84 ; azotemia may be present and may be of prerenal origin or represent concurrent renal disease. in one study, 25 of 32 cats were hypocobalaminemic; 1 of 27 cats had low folate, but 10 of 27 had elevated folate; and 12 of 16 cats had increased ftli. 73 hematologically, a mature neutrophilia with or without monocytosis is sometimes present, representing the inflammatory response; lymphopenia may be present as a stress response. anemia may be present and may occur as a result of chronic slow gi blood loss, and in some cases, ulceration, or it may be because of chronic disease; hemoconcentration is also possible, reflecting dehydration. 27, 40, 43, 84 palpable or ultrasonographically visible thickened intestines (30% to 41% of cases) 27, 40, 43, 84 or mesenteric lymph nodes (20% to 50% of cases) 27, 40, 43, 84 are no more or less likely to be present in comparison with ibds. there are no defined ultrasound guidelines for cats with intestinal small cell lymphoma, because most prior papers do not distinguish between small cell and lymphoblastic neoplasia. 54 ,113 a more recent paper found 9 of 15 cats undergoing ultrasound examination had diffuse small intestinal wall thickening, with a mean of 4.3 mm (range, 3.4 to 5.0 mm; median, 4.5 mm), and focal mural thickening of 20 mm was noted in one cat. 84 in many cases, against expectations, intestinal wall layering was preserved. these findings also mean that 5 of 15 cats had ultrasonographically normal intestinal wall thickness (≤2.8 mm for the duodenum and ≤3.2 mm for the ileum). 50 if affected, jejunal lymph nodes may appear as hypoechoic and enlarged; in the same study, 12 of 15 cats had lymph node changes with a mean diameter of 15.9 mm (range, 6.5 to 30 mm; median, 10 mm) 84 compared with the normal diameter of less than or equal to 5.0 mm. 132 none of these findings can definitively distinguish small cell lymphoma from ibds; although one recent paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma (figure 23 -20) than those with ibd, this change was also seen in 12% of cats with a normal small intestine. however, thickening of the muscularis layer together with lymphadenopathy was recognized in 26% of those cats with small cell lymphoma compared with 4% of those with ibd and 2% of cats with no small intestinal pathology. 177 biopsy samples and histopathology are required for definitive diagnosis. an example of jejunal and mesenteric lymph node appearance at laparotomy is shown in figure 23 it is difficult to distinguish between lymphocytic inflammation and small cell lymphocytic neoplasia in any location; some histopathologic features that might help in differentiating the ends of the spectrum may include therefore become known as the fondacaro protocol. this consists of a combination of prednisolone and chlorambucil given orally by the client at home (table 23-15 ). the rationale is that a slow alkylating agent, such as chlorambucil, is more appropriate to use for the slowly dividing, well-differentiated lymphocytes that cause disease. this can be contrasted to the aggressive chemotherapeutic agents required for the rapidly proliferating cells in lymphoblastic neoplasia that is typically associated with lymphosarcoma. reported response rates to this protocol are excellent, with 59% to 76% of cats achieving complete clinical remission, reported median survival times ranging from 20 to 30 months for those cats responding to therapy, and reports of individual cats surviving as long as 76 months. 43, 73, 84 the original reported protocol comprised prednisolone (10 mg/cat, po or 2 mg/kg, po) given daily with chlorambucil pulsed by administration of 15 mg/m 2 for 4 days every 3 weeks. a more recent study 73 dosed prednisolone similarly, but chlorambucil was given as continuous therapy of 2 mg/cat, po every second or third day. no mucosal congestion, edema, or fibrosis in lymphocytic neoplasia, 43 compared with ibd 4. epitheliotropism, or homing of neoplastic t lymphocytes to the mucosal epithelium in lymphocytic neoplasia 27 these features can be seen in figure 23 -22. each of these criteria may be useful but are unlikely to be definitive. further studies that may not be routinely available but which may be helpful are immunophenotyping; most reports have found purely t lymphocytes in most cases of intestinal small cell lymphoma 27, 73, 84, 116 (figure 23-23 ). 2. clonality; the detection of a clonal population of cells, as recently described for intestinal lymphocytic lymphosarcoma, 101 would be closest to providing the basis for definitive diagnosis. effective treatment of feline intestinal small cell lymphoma was brought to light by fondacaro et al 43 and has cat to be weaned off corticosteroids, with chlorambucil continued as monotherapy (as is often the case with humans). iatrogenic diabetes mellitus usually needs to be managed with insulin therapy, at least initially (see chapter 24) . high-grade lymphoma or lymphosarcoma is the traditional style of aggressive, rapidly dividing lymphoid neoplasia that carries a much poorer prognosis than small cell lymphoma. most early studies do not distinguish grade of neoplasia; so, the prevalence of low-grade and high-grade alimentary lymphoma are difficult to assess. several recent studies found a similar prevalence of each, 84 ,116 but the seminal paper describing small cell lymphoma found only 17 cases of lymphoblastic lymphoma compared with 50 cases of small cell lymphoma. 43 this ratio of approximately one high-grade gi lymphoma case for every three low-grade cases more closely approximates the rate found in the author's practice. the reported median ages of affected cats range from 10 to 12 years, but cats as young as 1 year old have been diagnosed. most papers note that males are overrepresented, and siamese cats may also be overrepresented although most affected cats are domestic shorthairs. 43, 49, 90, 116, 176 precise signalment is difficult to determine from the literature, because many papers assess all anatomic locations of lymphoma without necessarily breaking down epidemiologic data for each anatomic site. also, there are few comparisons to a reference population. the association of lymphoma with felv infection is well established and documented 139 and is covered in chapter 28; fiv has also been shown to be lymphomagenic. 12, 139 since the control of felv through vaccination began in the 1980s, nonretroviral-associated lymphoid neoplasia has become more common, and the rates of intestinal lymphoma have, in fact, increased since felv infection rates have decreased. 86 the underlying causes for this increase are not known. the association with inflammation from ibds was noted for small cell lymphomas, and perhaps there is a spectrum from lymphocytic ibd to small cell lymphoma to high-grade lymphoma. that some cats are more likely to have inflammatory changes become neoplastic is suggested by a paper noting higher lymphoma rates in cats with vaccine-associated sarcomas (a neoplastic condition where the role of chronic inflammation is well noted). 89 similar protocols are used in humans with both lowgrade (i.e., lymphocytic) lymphosarcoma and chronic lymphocytic leukemia. 117,151 some studies with humans have indicated that continuous therapy with chlorambucil results in prolonged survival, 64 although metaanalyses have not been able to determine optimum dosing and scheduling of administration of chlorambucil or other alkylating agents in these conditions in humans. 20, 72 although we do not have enough data to critically compare pulsed therapy to continuous dosing, the study assessing continuous dosing appeared to have a lower number of cats completely responding, although those cats that did respond had a longer median survival 73 than those in the studies assessing pulsed chlorambucil dosing. 43, 84 the differences may also relate to the definitions used for complete response. the chlorambucil dose of 2 mg/cat, po every second day (or third day) is often chosen because of the ready availability of 2 mg coated tablets, the breaking of which can expose the owner to these cytotoxic medications. chlorambucil can be compounded into smaller doses, thus allowing daily dosing of 1 mg capsules. the author has used this dose to apparent good effect, but there has been no critical assessment. it is unknown whether involvement of lymph nodes or other organs, such as the liver, affects prognosis. the only study of substantial size to include extra-gi locations found anatomic location was not prognostic for response or survival time. 73 in another study, of the five cats with liver involvement, two cats did not survive more than 5 months, yet the other three lived longer than 2 1 2 years, with two surviving longer than 4 1 2 years. 84 a study of hepatic small cell lymphoma suggests the density of neoplastic lymphocytes may influence survival, and density may relate to the stage of the disease when diagnosis occurs. 7 adverse effects of chlorambucil are rare, but gastrointestinal signs, myelosuppression, and myoclonus have all been reported. gastrointestinal signs, such as vomiting, diarrhea, or inappetence, can be difficult to distinguish from continuation of the gastrointestinal disease diagnosed. these signs are usually self limiting. myelosuppression is also possible with thrombocytopenia reported. 57, 84 monoclonus has been reported on one occasion. 17 it is ideal to check hematologic parameters every 2 months for cats receiving chlorambucil. continuous therapy using lower doses of chlorambucil may be less likely to lead to these adverse effects. high doses of corticosteroids can induce diabetes mellitus, and thus blood glucose should be checked regularly. if diabetes occurs, the author has found that budesonide (1 mg budesonide is generally considered to be equivalent of 5 mg prednisolone) can be substituted for prednisolone, since it has reputed lower systemic effects (though no assessments of this drug's effectiveness in cats have been made). an alternative is for the of distinction of intestinal layering as shown in figure 23 -24. the area of lymphomatous infiltration is hypoechoic, because it contains a uniform cell population without much reactionary fibrous tissue. mesenteric lymphadenomegaly is common (figure 23-25) , as are changes in other organs, such as kidney, liver, or pancreas. ascites may also be seen. 54, 113 although ultrasonographic distinctions predominate, there is considerable overlap between ultrasonographic findings with small cell lymphoma and high-grade lymphoma. the clinician must not lose sight of the fact that microscopic distinctions are required to diagnose either condition. cytologic diagnosis of high-grade lymphoma from fine-needle aspirates (fna) is much more likely than with small cell lymphoma. this is because there is usually a focal lesion, and the neoplastic cells are a monomorphic population of large, immature cells (i.e., that are not normally seen in tissue). sometimes, mixed lymphoid whether the underlying cause is retroviral or chronic inflammation or anything else, the pathogenesis of highgrade intestinal lymphoma, as with small cell lymphoma and other neoplasia, depends on chromosomal changes that affect regulation of cell growth and death, resulting in malignant transformation and clonal expansion of immature lymphocytes. 152 metastasis can occur in one third to two thirds of cases, 90,92 with involvement of mesenteric lymph nodes most commonly noted, but spread to liver, spleen, kidneys, and thorax is also possible. 90 a recent survey of gastrointestinal lymphoma found that most cases (37 of 50) involved the small intestine (including 3 that also involved the stomach and 4 that also involved the large intestine), and 4 of 50 cases involved the large intestine only. 116 cats with high-grade alimentary lymphoma often present similarly to those with other gastrointestinal diseases. typical clinical signs are weight loss, anorexia, lethargy, vomiting, diarrhea, or a combination of these signs. repeated studies have found cats with no vomiting or diarrhea; in one study, 13 of 28 cats had only anorexia or weight loss on presentation. 90 cats with large bowel pathology usually present, as with other causes of colitis, with increased urgency, and small, frequent amounts of diarrhea, often with blood or mucus. cats with large bowel neoplasia of any form can present for constipation caused by intestinal obstruction. palpation of an abdominal mass has been recognized in 59% to 85% of cases, 43, 90 but the corollary of this is that 15% to 41% of cases did not have a palpable mass. it is also important to note that up to 50% of cats with intestinal small cell lymphoma, and a number with ibds, have palpable mesenteric lymph nodes; so, a palpable abdominal mass is not a specific indication of high-grade neoplasia. many cats have palpably thickened bowel loops. 124 hematology and plasma or serum biochemistry findings are also nonspecific. increased liver enzymes may or may not indicate liver involvement. anemia may be recognized and can be non-regenerative, reflecting chronic disease or slow blood loss, or regenerative if there is more substantial blood loss associated with mucosal ulceration. hypoalbuminemia can be because of blood loss or intestinal protein loss. hypercalcemia of malignancy is a possibility but not commonly reported. despite nonspecific signs, laboratory testing is important to rule out extra-gi diseases and help manage consequences of enteric disease, as with small cell lymphoma and ibds. ultrasonography commonly shows a focal intestinal thickening (of 5 to 25 mm) with partial or complete loss although noted as the next most common intestinal neoplasia, after the various forms of lymphoma, adenocarcinoma is seen relatively infrequently in practice. most cats are more than 10 years old, 32,76,126 males may be overrepresented, and several studies have recognized a distinct overrepresentation of siamese cats. 32, 76 three distinct forms have been described 140 : cats typically present with nonspecific signs of gastrointestinal disease but can present with obstructive signs. cats with large bowel neoplasia can present for tenesmus or hematochezia and even constipation, if the lesion is obstructive (or partially obstructive). on physical examination, an abdominal mass is palpable in approximately 50% of cases, but other findings are usually nonspecific. anemia can be found if mucosal ulceration has occurred, but there are no distinctive laboratory findings. lesions can occur anywhere along the intestinal tract. one study of 100 cases found 40% of feline intestinal adenocarcinoma lesions were present at the ileum or ileocolic junction. 32 twenty-five to 50 percent of cases have metastasis at the time of the diagnosis, and this is a poor prognostic indicator. 32, 76, 145 radiology may show a mass lesion or intestinal obstruction, and ultrasonography can localize lesions to an intestinal origin. the ultrasonographic appearance of the proliferative, circumferential, outwardly expansile populations (of immature lymphoblasts and mature lymphocytes) are seen if a germinal lymphoid follicle is aspirated, and precise diagnosis may be difficult if there are a large number of lymphoblasts. 160 fna samples are best obtained with ultrasound guidance. the cytologic sample quality is greatly improved by not aspirating when the needle is visualized in the mass but merely "pecked" into the mass so that the needle is merely acting to finely "core" the mass. on removing the syringe and needle, the hub of the needle is removed before drawing air into the syringe, the hub is replaced, and the sample within the needle is expressed onto a slide. usually, the decision to diagnose by cytology from fna is based on the ultrasonographic appearance of a mass. since there is substantial crossover of ultrasonographic appearance of intestinal masses, laparotomy for excision is often performed with the affected bowel submitted for histology. except when intestinal obstruction has resulted, there is no therapeutic benefit of excising a gastrointestinal lymphoma (which requires excision and anastomosis), but there is minimal room for doubt when a histologic diagnosis is achieved. the response to therapy for high-grade intestinal lymphoma is significantly worse than that for small cell lymphoma. 43, 124 further, response to therapy for highgrade intestinal lymphoma appears to be worse than for lymphoma in other anatomic locations. 142 precise remission rates and survival times are difficult to quantify, because many studies assess lymphoma from multiple locations and do not necessarily differentiate response of gastrointestinal lymphoma or report the grade of lymphoma. with remission rates reported from 18% to 80% 43,92,176 and a median survival time of up to 41 weeks (range, 4 to 120 weeks), 176 it can be said with some certainty that some cats respond to therapy for reasonable durations. multiple authors have noted that the best prognostic indicator is response to an initial treatment cycle, 92,100,176 which should prompt clinicians to encourage owners to start therapy and decide whether to continue based on the cat's response. there are several published chemotherapeutic protocols, 43 ,92,100,142,176 but all follow the same principles of using medications to target specific phases of the cell division cycle (such as l-asparaginase and vincristine) with other medications that interrupt multiple phases of the cell cycle (cyclophosphamide and doxorubicin). targeting the cancer cell in different ways enables more cells to be killed, reduces the toxicity of the individual drug used, and reduces the likelihood of resistance to a specific drug. several authors have noted increased success with the addition of l-asparaginase and doxorubicin to protocols. 92, 162, 176 chemotherapy for lymphoma is covered in more detail in chapter 28, oncology. look promising, but only two cats assessed had gastrointestinal mast cell neoplasia. lomustine was used unsuccessfully in one cat with sclerosing mct; another cat with sclerosing mct received eight treatments of vinblastine and had a survival time of greater than 4 years. 56 adenomatous polyps have been reported in the duodenum 87 and ileum 106 and can result in intussusception. 133 cats of asian ancestry, predominantly siamese, are greatly overrepresented, and most reported cases have been males. 87 cats usually present for vomiting or hematemesis that, surprisingly, can be very acute in onset; complete intestinal obstruction may result. 87, 106, 133 resection is curative, with survival times of more than 4 years reported. 87 eosinophilic sclerosing fibroplasia has recently been described in a series of 25 cases and is not strictly neoplasia. 29 the ulcerating mass lesions that can occur anywhere from the stomach to the colon are often grossly and histologically mistaken for neoplasia. there appears to be no breed predisposition or age predisposition (with ages ranging from 14 weeks to 16 years), but 18 of 25 cases (72%) were castrated males cats compared with 7 of 25 (28%) female spayed cats. eighty-four percent of cats presented for vomiting, 68% presented for weight loss, and 7 of 12 (58%) cats had peripheral eosinophilia. all cases had a palpable abdominal mass. the pyloric sphincter was the most common site, and lesions in this location were mostly considered unresectable. fourteen of 25 cats (56%) had bacterial colonies within microabscesses and necrotic foci within the lesion. the bacteria recognized were predominantly gram-negative rods, but antibiotics did not seem to be clinically effective. the bacteria are suspected to initiate the lesions, having been embedded after foreign body penetration. there are no specific treatment recommendations, but excision, where possible, would be prudent; corticosteroids appear to be helpful adjunctive therapy. survival times are difficult to estimate since many cats were euthanized because neoplasia was suspected and follow-up times were short (up to 6 months) for the remaining cats. 29 there are very few reports of intestinal leiomyosarcomas in cats, 8, 159 which have been reclassified as gastrointestinal stromal tumors. 99 these tumors may be more likely to arise from the ileocecocolic junction. resection, if possible, is usually recommended, with survival times of 3 to 4 months reported before recurrence. the author owns a cat with this tumor where resection was not form is better described than the annular, constrictingband form with minimal outward enlargement. in these cases, sonographically, a solitary segmental intestinal mural mass is present and characterized by circumferential bowel wall thickening with transmural loss of normal sonographic wall layers. the thickening can vary in echogenicity but may be hypoechoic and may be symmetric or asymmetric. there is no definitive distinction, however, from lymphosarcoma, mast cell tumor, smooth muscle origin tumors, or even segmental benign inflammatory bowel disease. 126 surgical resection is the treatment of choice. there seem to be two distinct groups in terms of survival time postresection: 1. short-duration survival (euthanasia or death within 2 weeks of surgery) 2. long-duration survival (mean survival time of 15 months, 76 with a number of cats surviving greater than 2 years) 76, 109 because clean margins improve prognosis, for large bowel adenocarcinoma, subtotal colectomy may be required for complete excision. 145 because of the potential for success after resection, it is recommended to excise unidentified masses at the time of surgery. 145 other forms of intestinal neoplasia are recognized infrequently, and include intestinal mast cell tumors, adenomatous polyps, eosinophilic sclerosing fibroplasia, gastrointestinal stromal tumors (leiomyosarcoma), and hemangiosarcoma. mast cell tumors (mct) are often cited as the third most common form of feline gastrointestinal tumor, 85 but the intestines are a far less common site than cutaneous, splenic, or hepatic mast cell neoplasia. 85, 124 masses are usually segmental nodular thickenings that occur in older cats. 140 the masses are indistinguishable ultrasonographically from other tumors, such as lymphoblastic lymphosarcoma. 126 a recent series of 50 cases described a variant of feline intestinal mast cell tumor, dubbed sclerosing mast cell tumor, for which neoplastic cells form a trabecular pattern with dense stromal collagen. additionally, eosinophilic infiltrates were moderate to marked in most cases. these cases can be confused histologically with eosinophilic enteritis, gastrointestinal stromal tumor, or fibrosarcoma. 56 surgical resection is recommended, but lesions are commonly infiltrative or metastasize widely, and there are few reports of successful treatment. lomustine (dosed 50 to 60 mg/m 2 , po every 4 to 6 weeks) has recently been assessed as adjunctive chemotherapy for mast cell neoplasia in various locations, 123 and results recognized in that many intestinal bacteria can be found in healthy animals. 70 further antibiotic administration can result in increase of other bacteria. 69 fungal causes of diarrhea are usually recognized from histology of biopsy samples. it remains to be seen whether the recent ready availability of pcr panels looking at a number of infectious causes of diarrhea will be beneficial for recognizing pathogens that had previously been misdiagnosed or a hindrance for readily recognizing commensal organisms not necessarily causative of the clinical signs being investigated. the most common viral, bacterial, and mycotic causes of diarrhea in cats are described below. parasitic gastrointestinal diseases are covered later in this chapter. viral causes of diarrhea are not usually specifically diagnosed, since, with the exception of the canine fecal elisa possible, and the cat appears healthy 24 months past diagnosis (figure 23-27 ). cats with intestinal hemangiosarcoma often present with anemia, and the disease appears to be highly metastatic. 33 the intestines appear grossly thickened by dark red tissue. 138 the small and large intestines seem to be affected with similar frequency. removal of macroscopic disease is recommended, but often the full extent of the severity is only recognized at surgery. 33 the prognosis is poor. suspicions of infectious causes of diarrhea should be aroused in younger cats, cats from shelters, or cats with immune suppression. when considering infectious causes of diarrhea, clinicians should assess whether the diarrhea is large bowel or small bowel in origin and correlate this with specific pathogens that are likely to cause clinical signs as shown in table 23 -16. to increase the diagnostic yield of fecal examination for parasitic causes of diarrhea, wet smears and appropriate fecal flotations should be performed on fresh fecal samples (<1 hour old). it is appropriate to administer broad-spectrum anthelminthics, even if fecal tests are negative. bacterial and viral causes of diarrhea should be considered when the cat is systemically unwell with fever. fecal culture should be performed in these circumstances, but the limitations of this testing need to be yersinia enterocolitica current theory of fip pathogenesis involves initial infection with fecv and then mutation to fipcv in small numbers of susceptible individuals. 112,165 routine serologic testing for fecv in cats with diarrhea would neither prove correlation with the clinical signs nor affect how the disease is managed and so is not recommended. cats with fecv diarrhea should be managed with symptomatic therapy of fasting, then reintroducing a bland diet and supportive care with fluid therapy if necessary. other viruses, such as astrovirus, reovirus, rotavirus, and torovirus-like agent, have been recognized to cause diarrhea in cats, but their roles as pathogens are unclear. they are not routinely recognized in practice, since electron microscopy of fecal samples is necessary for diagnosis and is not routinely performed. management is supportive care with appropriate fasting, then reintroduction of bland diets and fluid and electrolyte replacement if necessary. 51 successful identification of a known bacterial pathogen from a fecal sample does not necessarily mean that the agent found is the cause of disease in the cat. although a number of bacterial pathogens have been demonstrated to cause diseases when specific pathogen-free (spf) cats are experimentally infected, 42 these same organisms can be found in healthy cats. 93 the differences between healthy and diarrheic cats that have bacteria found in their feces may relate to virulence factors of the organism, or host factors (local or systemic immunity) of the cat. there is no definitive answer for this quandary. the author's opinion is that • if a diarrheic cat is systemically unwell and has a fever, then feces should be cultured. • if an organism is isolated that is known to cause signs consistent with those the cat is showing, the cat should be treated appropriately. campylobacter diarrhea is usually caused by c. jejuni. clinical signs of infection are poorly documented, but most cats are asymptomatic. younger cats are more likely to have clinical signs and hemorrhagic, mucoid diarrhea has been reported. diagnosis can be from culture of feces or swabs, and the organism is quite hardy; so, it usually survives transport to the laboratory. 28 in individual cases, the organism has not been cultured after antibiotic treatment, 45,46 but it is not definitively proven that antibiotic therapy affects the natural course of the disease. antibiotics that can be used are amoxicillin-clavulanate (15 mg/kg, every 12 hours, po) for parvovirus, routine definitive tests are not available. clinical signs of panleukopenia (feline parvovirus infection) are more likely to occur in kittens, with the highest morbidity and mortality occurring between 3 and 5 months of age. subclinical cases in older (susceptible) cats probably go unrecognized. the organism is very stable in most environments, and infections mostly occur from environmental contact. peracute cases can result in death within 12 hours, with little or no warning signs. acute cases often have fever, depression, and anorexia, with signs beginning approximately 3 to 4 days before presentation. vomiting is usually bile tinged and unrelated to eating. diarrhea does not always occur, and when it does, it is usually later in the course of the illness. leukopenia is not pathognomonic, because this can also occur with acute bacterial infection (e.g., salmonellosis can present identically). 53 commercially available elisa tests for canine parvovirus antigen in feces can detect feline parvovirus; however, shedding may have ceased by the time clinical signs occur, and vaccination can result in positive test results for up to 2 weeks. 111 aggressive fluid therapy, usually at twice maintenance rates, is usually required. broad-spectrum antibiotic coverage is used to prevent or treat secondary bacterial infection from viral injury of intestinal mucosa. parenteral antibiotics are preferred to prevent the possibility of further gastrointestinal irritation. the author recommends calculating iv doses and introducing appropriate amounts of antibiotics to the fluids bag to create a constant rate infusion (cri); cefazolin can be used in this way at 100 mg/kg/24 hours, and betalactam cris are commonly used in human medicine. 127 aminoglycosides or fluoroquinolones can be used concurrently at routine doses if fever persists after 24 hours or the cat is moribund on presentation, but care must be used with these agents. aminoglycosides are potentially nephrotoxic, and fluoroquinolones have been reported to result in cartilage damage in growing animals, although this has not been demonstrated clinically in cats. 134 fluoroquinolone retinal toxicity has been seen in all animals. cats that survive the first week usually recover, and prior infection imparts lifelong immunity. 28 vaccinations are highly effective for disease prevention. feline enteric coronavirus (fecv) mostly causes mild, self-limiting diarrhea and must be distinguished from feline infectious peritonitis coronavirus (fipcv), which is essentially always fatal and for which diarrhea is not a typical sign (but is possible). the most widely accepted or fluoroquinolones, such as enrofloxacin (5 mg/kg, once daily, po) for durations of 14 to 21 days. 44 macrolides, such as erythromycin (10 to 15 mg/kg, every 8 hours, po), are regarded as the drug of choice for humans but can cause gastrointestinal side effects. 93 clostridium difficile has been recognized in up to 5% of diarrheic cats. 93 clinical signs are typically acute onset watery diarrhea and anorexia. diagnosis has been made with detection of toxin a or toxin b in fecal samples using elisa. although these tests have not yet been validated for cats, they may prove to be a useful aid to diagnosis 94 and are available for testing of equine feces at some commercial laboratories. nontoxigenic strains exist; so, positive culture alone does not ensure diagnosis. metronidazole (10 mg/kg, every 12 hours, po) for approximately 7 days is the treatment of choice. 93 clostridium perfringens typically results in large bowel diarrhea with tenesmus, mucus, and hematochezia, but small bowel signs can also be seen. 42 pcr testing for enterotoxin a is commercially available and may prove to be a useful adjunct in diagnosis. antibiotics that can be used include metronidazole (10 mg/kg, every 12 hours, po), tylosin (10 to 20 mg/kg, twice daily, po), or amoxicillin-clavulanate (22 mg/kg, every 12 hours, po) for 7 days. 94 escherichia coli is a ubiquitous organism within the feline intestinal tract, and it would be unusual not to successfully culture e. coli from the feces of both healthy and unwell cats. when e. coli is associated with clinical signs of gastrointestinal disease, it is mostly as an opportunistic pathogen, with overgrowth resulting from changed environmental conditions, such as inflammation from other pathology or another pathogen. there are also specific strains of e. coli that are true pathogens because of virulence factors not present in commensal e. coli; these include enteropathogenic e. coli and enterotoxigenic e. coli, which both induce a watery diarrhea, and enterohemorrhagic e. coli, which produces a diarrheal syndrome with copious bloody discharge and no fever. 77 pcr testing is commercially available to identify pathogenic strains of e. coli 16,148 ; although not offered at routine veterinary laboratories, this testing is available to veterinarians, and laboratories offering these services can readily be found online. diagnosis should also document histologic lesions corresponding to the strain of e. coli identified. 77 there is emerging resistance to e. coli worldwide in all species of animals, including humans. this includes the typical therapies for gram-negative bacteria of beta-lactamenhanced penicillins and fluoroquinolones. 167 a major risk factor is prior antibiotic usage, because commensal organisms are exposed to antibiotics. pcr testing does not enable antibiotic sensitivity testing, and fecal culture may not be able to distinguish pathogenic from nonpathogenic strains; so, sensitivities may not be an accurate reflection of the pathogenic organism. pcr testing for genes that impart resistance to e. coli have recently been described but are not yet commercially available. 34 in some circumstances, supportive care with fluid and electrolyte replacement may be all that is required while the cat's immune system combats the infection. empiric therapy could include beta-lactam-enhanced penicillins (such as amoxicillin-clavulanate at 20 mg/kg, every 12 hours, po), fluoroquinolones (such as enrofloxacin at 5 mg/kg, once daily, po), or cefovecin (8 mg/kg, every 2 weeks, sc), but the clinician must be aware of possible drug resistance. salmonella typhimurium infection is possible from ingestion of infected prey, infected food sources, or from a contaminated environment, including the veterinary hospital. the resulting clinical signs depend on the number of infecting organisms, the immune status of the cat, and the presence of concurrent diseases. infection rates in cats (and humans) have been correlated with seasonal bird migrations, 153 and the illness has been dubbed songbird fever, 52 but there is no distinction between this and other salmonella infections. clinical signs usually begin 3 to 5 days after exposure, starting with fever (often >40° c [104° f]), malaise and anorexia, and progressing to diarrhea, vomiting, and abdominal pain. hematology can show leukopenia with a left shift and nonregenerative anemia, and biochemistry results are usually nonspecific. diagnosis is based on isolation of the organism by culture or identification with pcr, but care should be taken to correlate pathogen identification with clinical signs since, as with most gi pathogens, the organism can be isolated from healthy animals. 147 as with e. coli, antibiotic resistance is widespread, 120 with one united kingdom survey finding the multiple drugresistant strain dt104 to be the most frequent bacteriophage type identified. 115 treatment should be reserved only for those cats showing systemic signs, because routine antibiotic use in treating salmonellosis induces drug-resistant strains and prolongs the convalescent excretion period. 52 antibiotic choice should be based solely on sensitivity findings, since resistances are so widespread and unpredictable. 98 this means that if the organism has been identified by pcr, then culture of feces must also be undertaken. the duration of treatment must be long enough to eliminate fecal excretion of the organism, prevent the chance of relapse, and reduce the chance of resistance developing; up to 28 days has been advocated. 4, 164 these cautions are particularly important because of the zoonotic potential of salmonellosis. linear foreign bodies have traditionally been considered more common than discrete foreign bodies in cats, 10,14,41 but a study from a primary care facility indicated only 33% of foreign body cases were because of linear foreign bodies. 60 the larger case load of linear foreign bodies at referral institutions noted in earlier studies may indicate the abilities of primary care practitioners to recognize and effectively deal with discrete foreign body obstructions. most studies have found that cats with intestinal foreign bodies are generally younger (mean, 1.0 to 2.7 years), with a notable exception being obstruction from trichobezoars where three of five cats in one study were 10 years or older; the greatest risk factor appears to be length of hair coat. 9 no specific breed predispositions have been described but siamese and siamese-related cats have been noted to have oral fixations 13 and so may be expected to be overrepresented with intestinal foreign bodies. clinical signs will vary depending on the type of foreign body (linear or discrete), the position of obstruction, and the time since obstruction. most cats present for anorexia or vomiting. partial obstruction can result in diarrhea (which can be bloody). foreign body obstruction is typically considered an acute condition, with duration of obstruction because of a linear foreign body, measured from the onset of clinical signs to diagnosis, reported to range from 1 to 10 days. 10,41,60 however, one paper demonstrated chronic, intermittent, gastrointestinal disease from a linear foreign body of a 1-month duration 175 demonstrating that partial obstruction can result in a chronic course. physical examination may or may not reveal abdominal pain, palpable abdominal mass (or plication), dehydration, or fever. all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of a linear foreign body. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see figure 3-8) . life-threatening consequences can result from the interactions of local and systemic factors that arise from intestinal obstruction. locally, damage to the mucosa from traction and pressure of the foreign object can cause hemorrhage, ischemia, and necrosis. systemically, hypovolemia, toxemia, and acid-base and electrolyte imbalances can ensue. complete intestinal obstruction by discrete masses results in gas and fluid distention of the lumen proximal other bacterial causes of diarrhea have been reported in cats, such as yersinia enterocolitica, 48 yersinia pseudotuberculosis, 63 clostridium piliforme (tyzzer's disease), 71 and anaerobiospirillum sp. 36 specific diagnosis of these (and other bacterial infections) may be found in the course of investigation. management follows the principles of supportive care and appropriate antibiosis based on sensitivity testing. small intestinal bacterial overgrowth (sibo) has not been specifically described in cats. the criteria defined for dogs is a fasting bacterial count in duodenal juice of greater than 10 5 organisms/ml 11 and is often recognized with other chronic gastrointestinal diseases. 130 healthy cats appear to have at least this number of upper intestinal bacterial with a range of 10 5 to 10 8 /ml recognized. 68 bacterial overgrowth could potentially occur with ileus or intestinal inflammation of any underlying cause. foul-smelling small bowel diarrhea with no specific pathogen recognized may be an indicator of this condition, as could an increase in bacterial metabolites, such as folate. if suspected, it is appropriate to manage with broad-spectrum antibiotics, such as metronidazole (10 to 15 mg/kg, every 12 hours, po) or amoxicillin (10 mg/ kg, every 12 hours, po) for an extended duration such as 21 to 28 days. alterations in intestinal flora have been recognized after such treatment 69 ; however, any advice for this "condition" is entirely empirical. all efforts should be directed at identifying a precise underlying cause. mycotic and other infectious agents are only rarely recognized as intestinal pathogens in cats. diagnosis is made by histologic and microbial analysis of samples obtained at biopsy. possible agents include histoplasma capsulatum, 24 aspergillus spp., candida albicans, 140 and pythium insidiosum. 119 intestinal obstructions arise most commonly as a result of neoplasia in older cats and foreign body ingestion predominantly in younger cats. 10, 41, 60 less common causes include intussusception 83 and granulomatous inflammation (e.g., from fip) 59 ; tapeworm infection, with greater than 30 worms acting as a linear foreign body, has also been reported. 173 other listed causes are volvulus, intestinal torsion, incarceration of bowel in a hernia, adhesions or stricture, intramural abscess or hematoma, and congenital malformations. 140 may not occur if the obstruction is partial or intermittent, or if vomiting results in less fluid present. since most foreign body obstructions in cats are proximal, identifiable dilatation may not be recognized for this reason. 78 linear foreign bodies present further challenges for radiographic recognition; the following typical radiographic signs 129,137 may or may not be present: to the obstruction. most gas accumulation is a result of swallowed air, which is predominantly nitrogen that cannot be absorbed by the intestinal mucosa. gas also arises from bacterial fermentation. fluid accumulates as a result of increased secretions (saliva, bile, and secretions of gastric, pancreatic, and small intestinal origin) and retention of fluid already ingested, and it can be augmented by local hemorrhage. 39 since most intestinal obstructions in cats do not reach the midjejunum, 60 reabsorption of fluids that normally occurs at the jejunum and ileum is impaired. 39 linear foreign bodies, such as string, dental floss, or elastic toys, require proximal anchoring, usually under the tongue or in the pylorus (for example, by part of a toy attached to elastic). peristalsis moves the free end of the "string" through the intestinal tract, resulting in pleats of intestines around the foreign body. as the foreign body is forced against the intestinal mucosa, the mucosa becomes edematous, and even partial penetration affects mucosal integrity, allowing systemic entry of bacteria. intraluminal bacterial populations increase for both discrete and linear foreign bodies as a result of stasis. mucosal permeability can be affected by prolonged luminal distention, allowing entry of bacteria and toxins systemically or into the peritoneal cavity. direct entry of bacteria to the peritoneal cavity, causing septic peritonitis, can result from perforation of the intestinal wall from linear foreign bodies or sharp discrete foreign bodies, such as toothpicks or plastic toys. definitive diagnosis requires identification of the foreign body retrieved at surgery or in some cases, by endoscopy. this may be aided greatly prior to surgery by diagnostic imaging. however, imaging findings, particularly in the case of partial obstructions, may be subtle enough that obstruction of no identifiable cause is recognized or no overt signs are apparent. laboratory findings are not helpful in the precise diagnosis but are important to assess fluid and electrolyte balances that must be corrected. cats rarely help practitioners by ingesting radiopaque objects, but on the rare occasions that they do, these can be observed easily on plain radiographs. nonopaque foreign bodies depend on dilatation of the intestine from gas and fluid accumulation proximal to the obstruction for radiographic recognition (figure 23-28) . one study has suggested that if the jejunal diameter is greater than 2.5 times the length of the cranial end plate of the second lumbar vertebra, then intestinal obstruction is the most likely abnormality. care must be taken that the jejunal and not duodenal diameter is measured and that the radiographs must be positioned strictly lateral, because an oblique view can alter the measurement of the lumbar vertebra. 1 however, dilatation of an obstructed intestine successful treatment of foreign body obstruction requires evacuation or removal of the foreign body as well as correction of any bacteremia or endotoxemia, acid-base or fluid imbalances. discrete foreign body obstruction requires surgery or endoscopy to remove the object. in some specific circumstances, linear foreign body obstruction may be managed conservatively by cutting the anchor point below the tongue and allowing the cat to pass the foreign body by peristalsis. however, the decision to manage a cat conservatively must be done with the cat hospitalized, with fluid therapy and antibiotic coverage and a clear recognition on behalf of the practitioner and the owner that surgery may subsequently be required. cutting a sublingual linear foreign body may be achieved in a conscious cat by applying pressure with the thumb of one hand in the intermandibular space to elevate the tongue and gently grasping it using gauze with the other hand while a second person cuts the line with a suture cutter. there is a chance of a small nick on the sublingual surface. if the cat will not tolerate the procedure, sedation is appropriate. when cutting the line, the nature of the linear foreign body should be assessed (i.e., is it more or less likely to cut mucosa). in one study, 10 19 cats with linear foreign bodies were managed conservatively with 10 cats subsequently requiring surgery. the authors of that paper created guidelines that will be adapted here. conservative management should be attempted if the cat • is presented acutely (within 2 days) after known ingestion of a linear foreign body • has a sublingually fixed linear foreign body that can be cut • has no overt signs of peritonitis • altered gas pattern with luminal gas collecting in small bubbles instead of normal curved tubular columns. this can be subtle when there is only minimal involvement of the intestine but overt when involving the entire small intestine. commashaped gas patterns are more likely to occur with linear foreign bodies. 1 contrast radiography can aid diagnosis for both discrete and linear foreign bodies but should be used with caution because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium is irritating to the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. 137 ultrasonography is a very useful diagnostic tool, particularly for discrete foreign bodies, where, in most cases, there is overt distention of the small intestines with intraluminal fluid apparent (figure 23-31) . this modality has not been extensively assessed as an adjunct to diagnosis of foreign body intestinal obstruction in cats specifically, although there are several papers assessing dogs and small numbers of cats that agree with its utility. 114,156,161 linear foreign bodies are more difficult to assess ultrasonographically, but plicated bowel can be recognized, sometimes with the foreign body seen as a hyperechoic line centrally. 156 si a technique has been described for removal of linear foreign bodies by making a single enterotomy incision proximally and passing a red rubber catheter over the linear foreign body aborally, milking the foreign body within the catheter through the colon for retrieval from the cat's anus by an assistant. 2 this technique is not always effective, because it can be hampered if the foreign body is knotted or does not run smoothly through the red rubber catheter. 102 if the affected bowel segment demonstrates evidence of necrosis or perforation on the mesenteric border of the intestine, resection and anastomosis should be performed. necrosis is indicated by dark discoloration, thin intestinal wall, poor arterial pulsation, poor capillary bleeding, or lack of peristalsis. end-to-end anastomosis can be accomplished using a simple interrupted appositional pattern or a modified simple continuous appositional pattern with the same type of suture material used for enterotomy closure. 14,88 intraabdominal masses causing intestinal obstruction are often presumed to be neoplastic but can also be of infectious origin. resection, where possible, is always recommended, because resection of neoplasia (if no metastasis) can offer a good prognosis, 76, 87, 109, 145 and infectious causes may be managed with adjunctive therapy after definitive diagnosis. intestinal obstruction in older cats is more likely to be secondary to neoplasia. any neoplasia can cause obstruction, but adenocarcinoma 32,76 and adenomatous polyps 88, 106 are reported to cause obstruction more often surgical intervention is mandatory if • clinical signs (e.g., vomiting or anorexia) persist or deterioration occurs with conservative management • the cat has overt signs of peritonitis • the linear foreign body is fixed at the pylorus some authors disagree with attempting conservative management, since a perforated intestine from a linear foreign body reportedly carries a 50% mortality rate, 41, 88 and early surgical intervention is never an incorrect decision. this should be balanced with the observation that cats can carry a linear intestinal foreign body, such as an elastic cord for a 1-month duration without intestinal perforation. 175 however, fishing line, for example, would not be so forgiving! surgery to remove an intestinal foreign body (figures 23-32 and 23-33) should be considered an exploratory laparotomy. that is, the aim of the surgery is not only to remove the foreign body but to assess the entire intestinal tract and abdomen for other foreign bodies or pathology. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction, thus delaying healing and creating the potential for surgical dehiscence. linear foreign bodies require multiple enterotomy incisions, since pulling the object out through a single incision could create iatrogenic intestinal perforation. the anchor point (either sublingual or pylorus by gastrotomy) must be released in the first instance. enterotomy incisions are closed with 5/0 synthetic, monofilament, absorbable suture material, such as polydioxyanone (pds) or equivalent, in either a simple interrupted or simple continuous pattern. 14,88 removal of a discrete foreign body (a piece of leather) at laparotomy. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction. this is the same cat as in the radiology image in figure 23 -28. affected bowel is required, with anastomosis of the healthy tissue. there appears to be no benefit to enteroplication, which can result in significant ileus. there is no benefit to performing resection-anastomosis if the intussusception does reduce manually. 15, 25 the prognosis depends on the underlying disease process and the chronicity of the intussusception, and therefore how debilitated the cat is at presentation, however, prognosis is mostly good, with survival reported in up to 80% of cases, though recurrence can occur in some cats with idiopathic disease, often at different locations of the intestinal tract. 15 constipation is defined as infrequent or difficult defecation associated with retention of feces within the colon and rectum. prolonged constipation results in harder than other types of neoplasia. please refer to the sections on intestinal neoplasia earlier in this chapter for more details. granulomatous inflammation causing a single focal intestinal lesion can lead to obstruction in the same way that neoplastic change can. feline infectious peritonitis (fip) can present as focal lesions, 59 often in the colon or ileocecocolic junction. in the case of fip, the focal lesion is usually an indicator of multisystemic disease; so, resection does not help prognosis. the fungus-like organism, pythium insidiosum has also been reported to cause granulomatous lesions, resulting in intestinal obstruction 119 from large extraluminal masses that are approximately fist sized. resection with adjunctive itraconazole (10 mg/kg) for 2 months after surgery was a successful treatment. intussusception refers to invagination or prolapse of one portion of the intestine into the part of the tract that either precedes or follows it. there is a bimodal age distribution with intussusceptions in older cats, most likely associated with neoplasia (or ibd in some cases) 25 ; underlying causes for younger cats are ill defined and may be idiopathic in many cases, 15,25 but associations with parasitism and, in one case, a linear foreign body, have been made. siamese and burmese cats seem to be overrepresented. the most common locations are the ileocolic region and the jejunum. 15, 25, 83, 110 affected cats present with nonspecific signs of gastrointestinal disease, such as anorexia and lethargy. vomiting is not necessarily a presenting sign; diarrhea may occur. abdominal palpation reveals a mass in most cases. plain and contrast radiography only show evidence of obstruction and usually do not help define that the bowel has intussuscepted. 15, 25, 83, 110 ultrasonography is very useful for diagnosis, because a distinctive pattern of alternating hypoechoic and hyperechoic concentric rings (figure 23-34) is present in transverse sections. 25, 110 sometimes, the target lesion seen can be hard to distinguish from the pathology of other intraabdominal masses, such as lymph nodes, and in these cases, the size of the lesions can help, because the width will always be greater than 11 mm with an intussusception (because the sum of at least four intestinal wall widths cannot be less) and is often greater than16 mm. 110 surgical correction is always required, and manual reduction is typically not possible because there is usually significant venous infarction, edema, and congestion (figure 23 -35) as well as adhesions from fibrin and effusions from the affected bowel. 15, 25 if the intussusception does not reduce manually, resection of the and drier feces that become impacted, and this is known as obstipation. 140 chronic, recurrent constipation and obstipation can result in megacolon, which refers to persistent increased bowel diameter that is not responsive to therapy. megacolon is not a specific disease entity; it may be considered the most advanced stage in the spectrum of chronic constipation. 18 in most cases, constipation can be managed quite simply if the underlying cause is determined and dealt with. a comprehensive list of causes of constipation is noted in table 23 of course, multiple factors can interact. for example, an older cat may have renal disease and so will be dehydrated to some degree and have arthritic hips and so be reticent to squat. the presenting signs of constipation are usually evident to owners and include straining in the litter box and producing hard dry feces, if at all. sometimes, however, owners can misinterpret signs. cats can strain because of lower urinary tract problems, and, if no urine is produced, some owners assume the problem is because of constipation. some constipated cats can intermittently have diarrhea because of direct colonic irritation from hard dry feces and so may present for diarrhea and not constipation. cats can also present for less specific signs, such as anorexia, lethargy, weight loss, and even vomiting. 140,170 vomiting can occur because of colonic receptors stimulating vagal afferent endings, which, in turn, can stimulate the chemoreceptor trigger zone. 61 sometimes owners are concerned that their cat is defecating less, but the cat has just changed its diet to a much lower-residue diet and so is producing less feces. a full dietary history is an important aspect of the initial assessment. physical examination should confirm presence of feces in the colon and assess the degree of impaction. the presence of feces can usually be confirmed by abdominal palpation. in constipated cats, the colon is often palpated as a long firm tube extending cranially; sometimes, feces can be palpated to and around the colic flexure. alternatively, the feces may be palpated as large, discrete fecal concretions (that can sometimes be hard to distinguish from intraabdominal masses such as lymph nodes). if there is any doubt of the presence or degree of fecal impaction, survey abdominal radiographs should be taken. a lateral view taken in a conscious cat should be adequate to confirm the diagnosis. the physical examination should also assess for contributing causes, including musculoskeletal conditions. any recent trauma should be taken into account. the hips and lumbosacral region should be assessed for pain. the degree of flexion and extension of the hips should be gently assessed. the lumbosacral spine can be assessed by running two fingers on either side of the spinous processes. the cat will flinch in painful areas. any arthritic change is magnified in an underweight cat, since there may be less muscle mass and the joints may bear a heavier load. any suspicions of underlying musculoskeletal abnormalities can be confirmed with radiographs. neurologic assessment should also be performed. subtle changes just affecting colonic innervation will not be apparent on physical examination alone. however, an assessment of proprioception, placing reflexes, and gait should at least be performed to assess for lumbosacral spinal cord disease. anorectal abnormalities or lesions should be evaluated. impacted or infected anal sacs can lead to reticence to defecate; and therefore anal sacs should be assessed and expressed. because this is painful for most cats, the cat should be held by an experienced assistant. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb positioned externally. a rectal exam can be performed with a well-lubricated (gloved!) middle finger, feeling over the pelvic rim for masses as well as assessing if the colon closes over (squeezes) the finger. if the colon feels open around the finger, this can be an indicator of impaired colonic innervation but does not imply that this is a permanent change. if there are impacted feces continuing to the anus, rectal examination is not possible until this has been cleared. if a cat finds anal gland expression or rectal examination too painful to tolerate (based on the clinician's judgment), these procedures should be done under sedation. hydration and electrolyte status are also important factors in the constipated cat. chronic renal disease is defined by azotemia (in conjunction with inadequately concentrated urine), which means the cat must be dehydrated to some degree. plasma or serum biochemistry and urinalysis can be used to diagnose renal disease, assess degree of renal disease, or recognize prerenal dehydration. electrolyte changes including hypokalemia and hypocalcemia may also contribute to reduced colonic smooth muscle function. 140 in young to middleaged cats of apparent good health and hydration, blood difficulty defecating and pass hard, dry stools but do not have fecal impaction at the time of examination. after the obstructing feces have been removed, steps must be taken to ensure colonic motility and smooth passing of feces. medical management of constipation traditionally involves laxatives and prokinetic agents. these may not be required in straightforward cases. as long as there is no obstructive lesion, cisapride at 2.5 mg/ cat, every 12 hours, po 82 is very safe and can be instituted with a view to reducing the dose to once daily after 10 to 14 days and discontinuing if signs remain abated. doses of up to 7.5 mg/cat, every 8 hours, po have been reported. cisapride is only available from compounding agencies in most countries. an osmotic or lubricant laxative (table 23 -18) may be used concurrently at reduced doses as necessary. reducing the fecal bulk produced is an important part of long-term management. traditional dietary recommendations are to increase the amount of fiber. 18, 26, 140, 170 increased dietary fiber results in production of shortchain fatty acids, which have been demonstrated to stimulate feline colonic smooth muscle contractions. 128 however, dietary fiber is also classified as a bulk laxative and so, by definition, will increase fecal bulk. in humans dietary fiber has been considered a mainstay of therapy for constipation, but a recent review concluded that many patients with more severe constipation have worsening symptoms when increasing dietary fiber intake. 103 because megacolon is believed to be the end result of chronic dilatation, 140,170 it is the author's firm belief that initial dietary efforts should be directed to reducing fecal bulk and thus introducing a low-residue diet. reduced dry matter intake reduces stool volume, 31 and the author has found that recurrence rates of constipation reduce greatly when cats are transitioned to entirely wet food diets. wet food diets also help ensure adequate water and urine assessments are usually not required at an initial presentation for constipation. in all cases, the same principles of management apply: 1. ensure removal of obstructing feces 2. ensure colonic motility and smooth passage of feces 3. reduce fecal bulk 4. ensure adequate hydration 5. manage underlying problems the first step is to ensure obstructing feces are removed. in simple cases, the cat will evacuate feces after use of a glycerin or sorbitol pediatric rectal suppository. another option is administration of a microenema, such as microlax (mcneil consumer healthcare, fort washington, pa.), which contains 5 ml of sodium lauryl sulfoacetate. these products act to lubricate the colon wall and therefore facilitate the passage of feces. the author prefers to use one or two of these within the consult room to observe the cat defecating (the cat must be provided with a litter tray!). the outside tube should be lubricated with the suppository contents before carefully inserting and then expressing the rest of the contents. there are also stimulant laxatives (containing bisacodyl) and emollient laxatives (containing sodium docusate) that have reportedly been used. 140 if a rectal suppository vial cannot easily be inserted because hardened fecal content obstructs its entry, a more substantive enema will be required (sometimes requiring sedation or anesthesia), and this is covered in the next section on management. some cats present for the abdominal wall with the other hand, but great care must be taken with this maneuver, because the devitalized colon can be perforated more easily. 140,170 enemas as described are painful for the cat, and opioid analgesia is recommended at the time of anesthesia. opioids can reduce peristalsis in humans, 96 but having evacuated the bowel, the pain relief is more important than this transient effect. an alternative to enemas is administration of an oral polyethylene glycol (peg 3350) solution (e.g., colyte, golytely). a nasoesophageal tube is placed and the solution is given as a slow trickle (6 to 10 ml/kg/hour) over 4 to 18 hours. defecation usually results in 6 to 12 hours. in a retrospective study of 9 cats, median time to defecation was 8 hours and the median total dose of peg 3350 was 80 ml/kg. 26a no adverse effects were noted. a cat that has been obstipated needs supportive therapy when discharged. there are no controlled comparisons of the various therapies noted in table 23-21; the author prefers cisapride 2.5 mg, every 12 hours to every 8 hours, po 82 (first thing in the morning, when the owner returns from work, when the owner goes to bed), and lactulose syrup 2 ml/cat, every 12 hours, po. a cat that has been so severely obstipated that an enema under anesthesia is required can be expected to continue these medications lifelong. to reduce fecal bulk and decrease the opportunity for recurrence, low-residue canned foods (or sachets) are preferred for cats that have become obstipated. some cats may benefit from high-fiber diets. as with simple initial episodes, canned food helps maintain adequate hydration, and at home subcutaneous fluids may be used additionally in cats with chronic kidney disease. with repeat episodes or severe obstipation, investigations for an underlying cause should be thorough and include evaluation for colonic mass obstructions. a review of published cases indicated that 96% of cases of megacolon are accounted for by idiopathic megacolon (62%), pelvic canal stenosis (23%), nerve injury (6%), or manx sacral spinal cord deformity (5%). 170 although most cases are idiopathic, an attempt should be made to identify and treat any specific underlying causes. megacolon is not specifically defined in cats. it has been described as "generalized colonic dysfunction manifesting as severe colonic dilation and fecal impaction," or a "severely and irreversibly dilated and hypomotile" colon 140 and "a subjective evaluation of the diameter of the colon, usually based on radiographic assessment." 18 there are specific radiographic guidelines for humans with megacolon, in that a colonic diameter of more than 6.5 cm at the level of the pelvic brim is considered diagnostic. 118 intake and therefore help maintain hydration. however, increased dietary fiber is beneficial for some cats, and trial and error may be required to determine whether a high-fiber or low-residue diet will be of benefit to each individual cat. in one report, 15 cats with recurrent constipation refractory to traditional medical and dietary management were successfully treated with a psylliumenriched dry extruded diet. 48a after 1 month on the diet, 14 cats had no clinical signs of constipation. the remaining cat was clinically normal after 2 months on the diet. improvement was noted in 10 of 15 cats after only 7 days of dietary therapy. measures should be taken to ensure adequate hydration. maintaining adequate hydration is particularly relevant for cats with chronic kidney disease that have impaired ability to conserve water. changing to wet food diets helps increase water intake. some cats with chronic kidney disease may need additional fluid support, such as subcutaneous fluids administered by the owner at home on a regular basis. underlying problems may be minor and simple to manage, such as an anal gland abscess, or more involved, such as reduced pelvic outflow, as a result of prior trauma. arthritis is a common underlying factor in many older cats and may be managed with prudent use of nonsteroidal agents (see chapter 26) . in cases of obstipation, the cat is more likely to be debilitated to some degree; so, laboratory investigations to assess plasma or serum biochemistry parameters as well as hematology and urinalysis are ideal. any hydration deficit or electrolyte abnormalities should be corrected before the anesthesia that is often required to remove the obstructing feces. rectal suppositories and microenemas are usually ineffective in obstipated cats. enemas are often required to remove impacted feces in such circumstances. the enema solution must be warmed and introduced slowly to avoid vomiting. the typical volume required is 5 to 10 ml/kg (so, up to approximately 50 ml/cat). the enema solution can be an isotonic electrolyte solution or tap water, and mild soap can be added (but any soap used must not contain hexachlorophene, which is neurotoxic if absorbed); mineral oil can be used (5 to 10 ml/cat) as a lubricant or docusate as an emollient (5 to 10 ml/cat), but the two agents must not be used together since docusate promotes mucosal absorption. sodium phosphate-containing enemas must not be used, because they can induce severe hypernatremia, hyperphosphatemia, and hypocalcemia in cats. often, the enema solution alone is insufficient to reduce the fecal mass, and manual manipulation of the feces by abdominal palpation is required. sometimes the feces must be broken down by a gloved finger perrectum while the colon is massaged manually through radiographically, in the lateral view, the normal colon should be approximately the same diameter as the length of the body of the second lumbar vertebra. 81 in cats, however, "there are no published guidelines for determining megacolon, so, diagnosis of abnormal colonic dilatation is subjective." 75 however, one author has suggested that "as a rule of thumb, the diameter of the colon should be less than the length of the body of the seventh lumbar vertebra (l7)." 105 this author continues, "enlargement of the diameter of the colon beyond 1 1 2 times the length of the body of l7 is indicative of chronic large bowel dysfunction and an explanation must be sought." 105 a recent paper found that 15 of 20 cats with no gastrointestinal disease had a colon diameter greater than the length of l7; however, no assessment of constipated cats was made. 1 in practice, many cats with megacolon have a colonic diameter far exceeding this guideline ( figure 23 -36). one study of 11 cats with megacolon found the mean diameter of the colon was 2.7 times greater than the length of the seventh lumbar vertebra (median, 2.4; range, 1.8 to 3.3), 107 but in general, objective descriptions of this condition are lacking in the veterinary literature. the definition of megacolon in cats should include functional as well as radiographic guidelines. in the absence of broadly recognized radiographic recommendations, the author proposes that the o'brien rule-ofthumb guidelines 105 (as noted above) be introduced until a more comprehensive study can establish other radiographic diagnostic criteria (or confirm these). the author therefore proposes to define megacolon as dilatation of the colon, to more than 1.5 times the length of the seventh lumbar vertebra, which is refractory to medical and dietary management. practitioners can expect the radiographic assessment of colonic dilatation to exceed this guideline in cats with megacolon and, conversely, there are likely to be cats having colonic distention greater than this amount that will respond to medical and dietary management and can therefore not be defined as having megacolon. by the definition used above, megacolon is refractory to medical and dietary therapy; so, to be defined as having megacolon, a cat may have had several episodes of obstipation managed by enema as well as dietary trials (with both low-residue and high-fiber diets) and medical therapy with cisapride and an osmotic or emollient laxative; yet the cat will still obstruct with feces. in these circumstances, the only possible therapy is subtotal colectomy. subtotal colectomy refers to surgical excision of 95% to 98% of the colon, whether it is grossly diseased or not with preservation of the ileocolic junction (icj). this approach has resulted in a more favorable clinical response than when the icj is also excised. 22, 170 when preserving the icj, it has been noted that, in some rare cases, it can be difficult to join the proximal segment of colon to the distal piece of descending colon because of the tethering effect of the ileocecocolic blood vessels. in these cases, sacrificing these vessels and removing the icj (i.e., total colectomy) is recommended to facilitate approximation of the ileum to the distal colonic segment. 21 a recently described technique using a biofragmentable anastomosis ring, compared with sutured anastomoses, showed no discernible effect on prognosis. 131 prognosis following subtotal colectomy is generally good. a review of multiple papers, totaling over 100 cats that had undergone subtotal colectomy, found the most commonly reported perioperative complication was diarrhea or loose stools immediately after surgery. in the majority of individuals, stool consistency improves without further treatment so that within 1 to 6 weeks of the surgery soft, formed stools are developed. diarrhea can persist in a small number of cases. in the longer term, in some cats, constipation can eventually return, but this can usually be managed by dietary and medical therapies. 172 pathology of the rectum or anus is relatively rare in cats and therefore poorly described in the veterinary literature. consequently, published information is often not referenced, suggesting it expresses the authors' opinions. readers are directed to surgical texts for details and approaches about surgical corrections. the anal sacs are paired cutaneous evaginations situated between the internal and external sphincter muscles. these sacs store secretions from alveolar and sebaceous glands that reside within the sacs. each anal gland has an associated duct that opens to the skin surface just lateral to the anus. 136,155 normal anal gland secretions have only very recently been described 47 and vary markedly; the color can be white, brown, orange, yellow, tan or gray, and consistency can range from watery to thick and creamy, with two thirds of cats having solid portions within the secretion. on microscopic examination, epithelial cells are commonly seen, with most cats having some neutrophils present. bacteria are commonly recognized as are, on some occasions, yeasts. bacteria seen in this study were mainly gram-positive cocci (63%) or gram-negative cocci (30%). gram-negative or grampositive rods were also seen but were rarely the dominant bacterial population. 47 with such a wide range of normal secretions, it is difficult to diagnose any pathology from the nature of the secretion alone. however, blood is infrequently recognized, and neutrophils are typically present in only small numbers in normal secretions. anal sac diseases described in cats include impaction, inflammation (sacculitis), infection, abscessation, and neoplasia (essentially the same as in dogs). 140,155 it has been contended in dogs that sacculitis and abscessation are an extension of impaction. it is not known in dogs or cats what the predisposing causes are, but suggested underlying reasons are loose stools (that are less effective at expressing the sac during defecation), local swelling or edema occluding the duct, and obesity. 155 the author's observations have also indicated that constipation can result in anal sac impaction because of less frequent expulsion of the sac contents; the resultant pain of the anal sac impaction can lead to further constipation, thus establishing a cycle. the retention of secretions may predispose to sacculitis, but impacted anal sacs do not always result in inflammation. abscessation is a likely sequel to sacculitis. 155 cats usually present for licking, scratching, or biting at the perineal area and can present for scooting (or dragging their anus) as dogs do. other presenting signs can be inability to sit or settle, a lump seen by the owner, or a generally unwell state. expression is the only management required for impacted (and not infected) anal sacs. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb externally. this is painful for most cats; so, the cat should be held by an experienced assistant, and it is sometimes not possible without some degree of sedation. with frequent episodes, underlying causes should be investigated. sometimes, trial-and-error diet change to manipulate the nature of the feces to either more (highfiber diets) or less (low-residue diets) bulk help reduce the frequency of episodes. obesity should be managed by reduced caloric intake, but dietary management for this should also take into account the nature of the feces. overt infection may be recognized by pus secretion from the anal sacs, which will have a high numbers of neutrophils. this can be managed by broad-spectrum antibiotics, such as amoxicillin/clavulanate or cephalosporins. a single treatment with a nonsteroidal antiinflammatory drug, such as meloxicam, can be given in animals with appropriate hydration and without other illness. anal sac abscesses often present already open and draining. many heal well by secondary intention with antibiotic treatment until they are closed over; so rechecks are required before the completion of an antibiotic course. large abscesses may require surgical drainage with the insertion of a penrose drain and management as for a cat fight abscess. it must be remembered that wounds in this area are easily re-infected by fecal contamination. recurrent impaction, sacculitis, or infection may require anal sacculectomy (as in dogs). this procedure should be delayed until infection is cleared. the procedure is similar to that performed in dogs. reports of anal sac/gland neoplasia were confined to sporadic case reports, 97,108 until a large case series was recently published. 141 in this study, 64 cases of anal gland carcinoma were recognized at a private diagnostic laboratory during a 12-year period, with submissions from 62 practices. this indicates that, for most practices, this condition will be seen, at most, once every 12 years. affected cats ranged in age from 6 to 17 years (median and mean, 12 years); female (mostly spayed) cats were overrepresented (61% of cases), and siamese cats may have been over-represented (7.8% of cases). the number of siamese cats with anal sac neoplasia was 3 times greater than the number of siamese cats in the laboratory reference population. affected cats presented for dyschezia, recurrent constipation, change in the nature or volume of feces, b). a low-residue wet diet is recommended to reduce fecal bulk during the healing period. rectal prolapse occurs as a result of a disease process that causes chronic straining, such as intestinal conditions that result in diarrhea and tenesmus 2. conditions that result in constipation or other intestinal obstruction 3. lower urinary tract diseases 4. dystocia and/or perineal swelling or ulceration, sometimes with purulent or hemorrhagic discharge. most tumors were originally interpreted as and initially managed as anal sac abscess. presumptive metastasis in liver, lung, or abdominal lymph nodes was recognized by physical examination or radiography in six cats; one cat was hypercalcemic. excision appeared to be curative (with a 3-to 4-year follow-up period) in 3 of 29 cats undergoing surgery for resection or debulking (others had only incisional biopsy performed). for the remaining 36 cats with known postsurgical outcome, median survival was only 3 months, with a 19% 1-year survival rate (with none of these cats surviving to 2 years). 141 atresia ani is a developmental defect of the anal opening or terminal rectum (see figure 41 -4). kittens usually present within days or weeks of birth with abdominal distention, discomfort, tenesmus, restlessness, vomiting, and/or loss of appetite. there are several anatomic variations 22 : • type i: a membrane over the anal opening remains, with the rectum ending as a blind pouch just cranial to the closed anus. • type ii: the anus is closed as in type i, but the rectal pouch is located somewhat cranial to the membrane overlying the anus. • type iii: the rectum ends as a blind pouch cranially within the pelvic canal (rectal atresia), whereas the terminal rectum and anus are normal. • type iv: occurs in females and atresia ani exists with a persistent communication between the rectum and the vagina (rectovaginal fistula). this fistula can occur with a normal anal opening as well. most reported cases have been type iv, 23, 146, 150, 158, 166 and this has also been recognized with concurrent sacrococcygeal agenesis. 3 surgical correction has been described for type ii 157 and type iv 19,91 atresia ani in cats. the reader should consult these references for surgical advice; possible complications include megacolon after prolonged obstruction, postsurgical anal stricture, and fecal incontinence because of sphincter dysfunction. foreign bodies in cats rarely obstruct the gastrointestinal tract distal to the jejunum 60 ; however, large fecal balls resulting from constipation can, additional to constipation or obstipation, cause distention of the anus. this distention can result in inflammation of the anal sphincter with loss of tone ( figure 23-37, a) , which, in the author's experience, is temporary with correction of the underlying cause of constipation. it can take some weeks for the dilated anus to return to normal (figure 23 -37, with antibiotics, such as cephalosporins, and regular cleansing. prolapses are usually classified in three ways. 62 first degree: prolapse of only mucous membrane 2. second degree: prolapse of full rectal wall thickness 3. third degree: prolapse is sufficient to bring mesorectum outside the anus the prolapsed rectum is obvious but must be differentiated from ileocolic intussusceptions, which have been described with neoplasia. 37 this distinction can be made by inserting a thermometer through the anus alongside the prolapsed mass. insertion will not be possible for an intussusception but will be for an anorectal prolapse. the prolapsed tissue must be assessed for viability, and management must include determining and managing the underlying cause as well as management of the prolapse. in simple cases where the mucosa is viable, the prolapse can be reduced with lubrication and gentle pressure. a temporary purse-string suture may be required to prevent recurrence. perineal dermatitis is often confused with gastrointestinal or urogenital disease, because there are often copious sebaceous secretions that can mimic fecal or urinary secretions. perineal dermatitis can result from flea or other allergies but also fecal or urine scalding associated with diarrhea or urinary incontinence, respectively. skin fold dermatitis can also occur in obese cats ( figure 23-38 ). episioplasty has been described to correct this, 121 but the author has found that stringent dieting can result in improvement while managing the skin fold dermatitis these populations. 48 the reported prevalence for each parasite varies greatly with the population studied, the geographic location of the population, and the sensitivity of the diagnostic test used to study that population. 48 the presence or absence of diarrhea is not a reliable predictor of whether a particular cat is infected with or shedding a parasite. 42 in fact, most cats with diarrhea do not harbor enteric protozoa. 48 on the other hand, most cats with diarrhea because of enteric pathogens will shed those organisms, often intermittently. it is important to remember that infection with most gastrointestinal parasites may not cause clinical signs. therefore detection of a pathogenic parasite in a cat with diarrhea does not necessarily prove causation. 48 a search should always be undertaken to identify other causes of diarrhea prior to convicting a cat of having diarrhea because of a particular parasite. in addition, co-infections or the presence of other noninfectious causes of diarrhea can result in more severe diarrhea that is often refractory to treatment for the parasite. treatment will be more rewarding if all potential causes of diarrhea are identified in the patient. enteric parasites with zoonotic potential occur commonly enough that cats, particularly those with diarrhea and who are owned by immunocompromised persons, should be evaluated for those pathogens. 26, 27 the following is a discussion of the most common enteric parasites found in cats. for more on parasite prevention and control, see chapter 8, and for more on zoonotic enteric parasites, see chapter 34. ollulanus tricuspis is an almost microscopic nematode worm infecting the stomach of domestic and wild cats. 5 the worm measures less than 1 mm long. 3 the larvae of o. tricuspis develop and hatch within the uterus of the female worm. they develop to maturity in the stomach of the cat where it is capable of re-infecting the host. 3 the worm is transmitted to other cats that ingest the vomitus of an infected cat. 41 clinical signs shown by infected cats include vomiting, anorexia, and weight loss. 2, 5 histologic findings in infected cats include lymphocytic-plasmacytic gastritis, lymphoid hyperplasia, and mucosal fibrosis. gross lesions may be absent, or the cat may develop nodular gastritis. 41 one report suggested the parasite may have been a contributing factor in the carcinogenesis of a gastric adenocarcinoma in an infected cat. 10 160. a common theme when discussing the prevalence of most gastrointestinal parasites in cats is that they occur more commonly in younger cats and in cats housed in crowded conditions, such as catteries and shelters. it is likely an increased chance for transmission exists in lungs. after further development in the lungs, the parasite migrates up the trachea and is swallowed. adult s. felis and s. planiceps burrow into the wall of the small intestine, while adult s. tumefaciens lives in the colonic mucosa. ova may be shed in the feces or hatch in the intestinal tract. autoinfection occurs if larvae become infective and penetrate the intestinal wall before being shed. ova and larvae that are shed develop into freeliving adult worms. 11 the prepatent period is between 7 and 10 days. 1, 11 clinical signs and diagnosis signs of a strongyloides spp. infection are usually absent. 1, 40 lung migration may cause cough or respiratory distress. the presence of the parasite in the intestinal tract may result in diarrhea and weight loss. 11 strongyloides tumefaciens is associated with the formation of small, worm-filled nodules in the colon. 40 identification of strongyloides spp. larvae using the baermann fecal concentration technique is required to diagnose most infections. unless the infection is heavy, examination of a fresh fecal smear is insensitive for identification of these larvae. 1 the nodules formed by s. tumefaciens infection can be visualized during colonoscopy. histopathology of the biopsied nodules should reveal many adult worms. 40 infection with strongyloides spp. can be treated with fenbendazole, 11 pyrantel pamoate, 40 thiabendazole, 1,11 or ivermectin. 3 to evaluate efficacy, repeat a fecal examination 2 to 3 days after the treatment ends. because of the presence of free-living adult worms in the environment and the ability of larvae to cause infection by penetrating intact skin, prevention is difficult. keeping cats indoors in warm, humid climates may be an owner's only means of preventing infection with strongyloides spp. parasites. infections with trichuris vulpis rarely occur in cats and are considered to be clinically unimportant. 3, 14 the two species of roundworms commonly infecting cats are toxocara cati (figure 23-39) and toxascaris leonina (figure 23-40) . the latter also has the ability to infect dogs. 14 cats are infected with t. cati in several ways. most commonly, infection is by ingestion of contaminated food, water, or infected paratenic hosts such as rodents. transuterine transmission has not been reported. 14 the diagnosis of infection with o. tricuspis is difficult, because ova are not shed in the feces; rather, the vomitus must be examined for worms or larvae. the worms may also appear in gastric mucosal biopsy samples. 41 a report of 131 cats undergoing endoscopic examinations found the parasite in gastric biopsy samples from 4 cats. 5 fenbendazole may be effective in treating infections with o. tricuspis. 41 preparations with febantel may also be expected to successfully treat these infections. transmission can be prevented by appropriately treating infected cats. other cats should not be allowed to ingest infected vomit. this parasite is of no zoonotic concern. physaloptera another parasite rarely inhabiting the stomach in cats is in the genus physaloptera. larger than ollulanus tricuspis, this blood-sucking worm infects cats that have ingested intermediate hosts, such as cockroaches, crickets, or flour beetles. 11 preying on transport hosts, such as mice that have eaten an intermediate host, is another way cats become infected with this parasite. clinical signs of infection with physaloptera spp. include vomiting, anorexia, and melena. a diagnosis of physaloptera infection can be made after identifying the ova in the patient's feces or adult worms in the vomitus. occasionally, the worms may be seen during gastroscopy. the adult worms must be differentiated from ascarids. 11 infection can be treated with ivermectin, pyrantel pamoate, or fenbendazole. 3 because there is no migratory phase of the life cycle, the treatment does not need to be repeated. 11 three species of strongyloides infect cats. strongyloides felis infects cats in india and tropical australia, 1,43 s. tumefaciens is a rare parasite of cats in the southeastern united states, 3 and s. planiceps is found in cats in malaya and japan. 1 strongyloides stercoralis, found in dogs and humans, produces experimental infections in cats, but natural infection with this species has not been observed. 1 feline infection with strongyloides spp. is considered by most to be rare. however, one report from australia identified s. felis in 169 of 504 necropsied cats. 43 infection with strongyloides spp. occurs after ingestion of infective larvae. infection can also take place after the larvae penetrate the skin of the cat. 11 ingested larvae penetrate the intestinal wall and migrate through the diaphragm into the lungs. after cutaneous penetration, the larvae enter the venous circulation and enter the clinical illness because of roundworm infection is uncommon. illness, when it does happen, most often occurs in kittens 26 signs may be mild and can include vomiting, 14 diarrhea, weight loss, poor growth, and a "pot belly." 26 a heavy infection with t. cati can result in catarrhal enteritis. severe infections can lead to intestinal obstruction and, possibly, perforation. 26 much less dramatic changes arise after infection with t. leonina, although enteritis may occur. 14 roundworms are frequently diagnosed with a fecal floatation. the centrifugal floatation technique is more sensitive than the simple fecal floatation technique many hospitals use. 14 occasionally, adult worms will be passed with the feces. the goals of treating roundworms include disease prevention in an individual cat or kitten, prevention of environmental contamination by cats defecating outside, and the prevention of zoonotic infections. many effective and safe anthelmintics are available (table 23-19) . benzimidazoles, such as fenbendazole, act on the parasite's microtubular structure, leading to disintegration of the worm's intestines, muscular layer, and hypodermis. 14 pyrantel in the pamoate formulation is poorly absorbed and causes paralytic parasite death. macrocyclic lactones, such as milbemycin, also lead to paralytic parasite death. these compounds act on the parasite's gamma-aminobutyric acid (gaba)-and glutamate-controlled ion channels. these channels are lacking in tapeworms, accounting for the lack of efficacy against these parasites. 14 lastly, emodepside (a cyclic octadepsipeptide) has been combined with praziquantel in the product profender (bayer animal health). this topical parasiticide has been shown to be both safe and effective. 14 these drugs appear to be so safe that overdosing is almost impossible. 14 kittens can be dewormed starting at two weeks of age and again at 4, 6, 8, 12, and 16 weeks. 26 older kittens and adults can be dewormed every month to 4 months. 14 because of the safety of these drugs, the possibility of false-negative tests and, more importantly, the zoonotic potential of these infections, perhaps all kittens should be dewormed, not just those testing positive. roundworm ova are very hardy and can remain infective for years. 14 they survive sewage treatment and composting, and there is no practical means of decreasing the ova population once the environment is contaminated. thus it is best to attempt to prevent contamination in the first place. when practical, keeping cats indoors allows appropriate control of potentially transmammary infection occurs, but only if the queen is acutely infected late in pregnancy. chronically infected queens do not pass t. cati ova in their milk. 14 after ingestion, t. cati larvae migrate through the small intestinal wall, into the liver, and then to the lungs where they are coughed up and swallowed. these larvae then infect the small intestine. some of the migrating larvae become encysted in the cat's muscle tissue. larvae from ova ingested through the milk tend not to undergo migration and mature directly in the small intestine. 14 the prepatent period is approximately 8 weeks. infection with t. leonina occurs after ingestion of infective ova or an infected paratenic host. unlike t. cati, very few t. leonina larvae migrate through the cat's tissues. most develop in the wall of the small intestine. the prepatent period is 7 to 10 weeks. toxascaris leonina ova can become infective within 8 days of being passed in the feces when the ambient temperature is 27° c but normally require 3 to 4 weeks. 14 lungs, and kidneys. ocular larval migrans results in granulomatous retinitis that is often misdiagnosed as retinoblastoma in older children. 3 this can lead to unnecessary enucleation. toxocara cati appears, however, to be less important than t. canis as an infection in humans. 3 the species of hookworms that infect cats are ancylostoma tubaeforme and ancylostoma braziliense (see figure 23 -39). they are reported to be an uncommon infection in cats. 26, 34 ancylostoma braziliense can also infect dogs. hookworm infections occur after ingesting food or water contaminated with hookworm larvae or eating contaminated fecal material. if the pet cat is allowed outdoors, attempts at preventing hunting may reduce the possibility of infection. keep children's play areas, such as sand boxes, inaccessible to cats when children are not at play. feeding only well-cooked food can prevent infection by contaminated food. finally, empirical, preventative deworming for cats that go outdoors should be performed 3 to 4 times yearly. any less frequently does not lead to an appreciable decrease in the prevalence of the parasite. 7 roundworms easily infect humans who ingest the ova, particularly children. visceral larval migrans occurs after infection with toxocara canis in humans. infection can lead to the formation of nodules in the brain, liver, tapeworm infections are well tolerated by the cat. usually there are no signs of infection other than finding segments on the feces or attached to perianal hair. because both d. latum and spirometra tapeworms absorb vitamin b 12 across the cuticle, megaloblastic anemia is possible, but unlikely. 11 tapeworm infections are diagnosed by identifying the typical appearance of the segments 8 or the egg packets within the segments. 26 the segments of t. taeniaeformis are flat, while those of d. caninum have been described as appearing like a grain of rice. the segments should be handled carefully, because they are friable and rupture may result in exposure of the handler. 8 the operculated ova of d. latum and spirometra spp. must be differentiated from trematode ova. even though tapeworm infections are well tolerated, cats should be treated for reasons of owner discomfort and public health concerns (see table 23 -19) . these infections are easily treated, because drug treatment is highly effective. re-infection must be controlled using preventative measures, especially flea control to prevent re-infection with d. caninum. praziquantel and infected paratenic hosts. the larvae can survive for months in the tissues of paratenic hosts. 14 infection also occurs after larval migration through the skin. in either case, the worm matures in the small intestine. 14 unlike dogs, transmammary infection has not been reported in cats. 3, 14 the prepatent period is between 19 and 28 days, depending on the route of infection. the time to patency after transcutaneous infection is longer than for direct colonization. infective l3 larva develop 2 to 7 days after the ova are passed. 3 developing larvae attach to the mucosa of the small intestine where they ingest copious amounts of blood. because the worms can remove a significant volume of blood from kittens, weakness from iron-deficiency anemia 34 or blood-loss anemia may be noted. 14 melena and diarrhea may also be recognized. signs are uncommon in adult cats. identification and treatment of hookworm infections are similar to that for roundworm infections (see table 23 -19) . hookworm larvae are not as hardy as roundworm eggs. soil contamination may be a temporary problem in areas that experience a hard frost. 3 hookworm larvae will not develop in temperatures less then 15° c or greater than 37° c. frequent, appropriate disposal of feces, cleaning surfaces with a 1% bleach solution, and deterring hunting may prevent infections. migration through the skin of persons coming into contact with the larvae of a. braziliense is the most common cause of cutaneous larval migrans, particularly in the southeastern united states. 3 this is an erythematous, pruritic skin eruption often found on the soles of the feet of infected children. the tapeworms most commonly found in cats are dipylidium caninum and taenia taeniaeformis. diphyllobothrium latum, spirometra spp., and echinococcus multilocularis occasionally infect cats. the latter is important, because it can lead to alveolar echinococcosis in humans. 8 spirometra tapeworms are found in north america (s. mansonoides) and far-east asia (s. mansoni and erinacei), while d. latum prefers temperate climates. 11 unnoticed, but the cat may cough or experience hemoptysis. 3 diagnosis involves demonstration of fluke ova in the feces. although therapy may be unnecessary, 11 praziquantel or epsiprantel are effective in eliminating the intestinal population of the fluke. platynosomum spp. are flukes living in the gall bladder, bile ducts, 46 and pancreatic ducts. 3 these flukes are most prevalent in the southeast united states and caribbean islands 3 and require two intermediate hosts. the first host is a snail, while the second intermediate host is a lizard, toad, gecko, or skink. 46 cats become infected with this fluke after ingesting an infected second intermediate host. the prepatent period for the fluke is 8 weeks. 46 most infections are subclinical. if clinical signs do occur, they may include weight loss, vomiting, diarrhea, icterus, hepatomegaly, or abdominal distention. diagnosis involves identification of ova shed in the feces using a fecal sedimentation method 46 or by finding adult flukes in the gall bladder or bile ducts during abdominal surgery. treatment involves administering praziquantel (20 mg/kg, q24h, po for 3 to 5 days) and/or surgical removal of the flukes. 3 two species of coccidians are the most common to infect cats, isospora felis and isospora rivolta (figure 23-41) . the genus isospora may be renamed cystoisospora. these are epsiprantel are safe and effective. fenbendazole is effective against t. taeniaeformis, but not d. caninum. without controlling exposure to intermediate hosts, tapeworm infections are difficult to eliminate. flea control is imperative in eradicating infections with d. caninum. controlling predation helps prevent ingestion of t. taeniaeformis-infected rodents. infection with d. caninum occurs in young children who are most likely to eat fleas. infection results in only minimal signs of illness. 8 the larval stage of t. taeniaeformis is of little zoonotic importance. 3 although cats are uncommonly infected with echinococcus multilocularis, potentially life-threatening alveolar damage occurs in north american humans infected with this tapeworm. 8 plerocercoids of spirometra spp. can penetrate the mucous membranes or open skin wounds of humans and migrate around the subcutaneous connective tissue, forming nodules, a condition called sparganosis. 3 megaloblastic anemia, as a result of vitamin b 12 deficiency, may occur in humans infected with d. latum or spirometra spp. tapeworms. 11 alaria marcianae flukes reside in the intestinal tract of cats and the mammary glands of lactating queens. miracidia hatch underwater from ova shed in the feces and penetrate the skin of a snail. after further development, cercariae penetrate the skin of leopard frog tadpoles and are able to survive the metamorphosis to the adult frog. 3 if the tadpole is eaten by a snake, bird, or mammal, the parasite enters the host's tissues but does not undergo further development. after a male or nonlactating female cat ingests the infected intermediate host, the parasite penetrates the wall of the small intestine, passes through the diaphragm, and enters the lungs for further development. finally, the parasite is coughed up and swallowed to complete maturation and reproduce in the small intestine. if, however, an infected host is ingested by a lactating queen, the parasite migrates through the tissues to the mammary glands, rather than the lungs. 3 once shed in the milk, the parasites develop into mature adults in the kittens. some of the mesocercariae remain in the mammary glands to infect future litters. clinical signs associated with worms in the small intestine are uncommon. 11 migration through the lungs often goes species-specific obligate intracellular parasites. 4, 13 they are able to survive in the environment for months. 13 a detailed description of the coccidial life cycle can be found elsewhere. 4, 13 simply put, direct transmission is by ingesting oocyst-contaminated food or water or by grooming contaminated body parts. indirect transmission occurs after ingesting a mechanical vector or the infected tissues of paratenic hosts. 39 after ingestion by a cat, the oocyst excysts in the small intestine and enters the enterocyte where further development occurs. 26 the parasite may also migrate through the intestinal wall to form cysts in mesenteric lymph nodes. these cysts may serve as a source for reinfection. 4, 13 the prepatent period is 4 to 11 days 26 and the shed oocyst becomes infective after several days of exposure to warmth and moisture. 13 infection with isospora spp. is usually subclinical. 26 signs, if they occur, range from mild, transient watery diarrhea to severe mucohemorrhagic diarrhea with vomiting and resultant dehydration and weight loss. 4, 13 signs are most commonly recognized in severely infected neonatal kittens, 26 particularly those with concurrent illness, and arise because of small intestinal congestion, mucosal erosion, or villus atrophy. 39 signs may also be noted in immunosuppressed adult cats. 26 isospora species are readily found in fecal floatation or wet-mount examinations. shedding can be intermittent, but most cats with diarrhea caused by coccidial infection shed large numbers of oocsyts. 39 fortunately, in most cats, the diarrhea from isospora spp. infection is self-limiting. 26 in fact, if a kitten is persistently shedding oocysts despite appropriate treatment 13 or the parasite is identified in an adult cat with chronic diarrhea, attempts should be made to identify co-infections or other diseases that may cause diarrhea. 39 anticoccidial drugs are either coccidiostatic or coccidiocidal (table 2320) . coccidiostatic drugs are the most commonly used drugs for individual pet cats. trimethoprim-augmented sulfadiazine (tribrissen; intervet/schering-plough animal health, summit, nj) or another sulfa-containing antibiotic, sulfadimethoxine (albon; pfizer animal health, madison, nj), can be used. supportive care for severely affected kittens, such as parenteral rehydration, should be used as needed. coccidiocidal drugs are often reserved for use in densely populated situations such as catteries or shelters. 26 however, many veterinarians are now using them as a first-line defense against isospora spp. infection. 13 ponazuril (marquis oral paste; bayer animal health, shawnee mission, kan.), formulated for horses, is effective and can be safely administered to cats. for more on the use of ponazuril in cats, see chapter 46. a related drug, diclazuril, is also available and may be administered once at 25 mg/kg po. 13 while not available in north america, toltrazuril (baycox, bayer animal health) may be administered once at 30 mg/kg po or 15 mg/kg po once daily for 3 days. 32a a second course of therapy 10 days later may be required to completely eliminate the oocysts. sanitation is very important, because the oocyst requires several days to become infective. frequent removal of feces, preferably daily, is recommended to prevent re-infection and transmission to other cats. 39 controlling a cat's ability to hunt reduces the chance of ingesting an isospora-infected rodent. control of mechanical vectors, such as cockroaches and flies, is also useful. 13 since a cat can become infected after grooming an infected cat's perineum, consideration should be given to treating all cats in contact with the patient. 39 in addition, catteries and shelters should ensure all food is well cooked, litter boxes are cleaned daily, and surfaces are well cleaned with steam 13 or 10% ammonia. 39 where recurrent isospora spp. infections are a problem, prophylactic treatment of all 2-to 3-week-old kittens with ponazuril should be considered. 13 despite all wellintentioned efforts at hygiene and treatment, isospora spp. infection can still be transmitted to other cats. 13 because these are species-specific parasites, transmission of i. felis and i. rivolta from cats to humans does not occur. the flagellated protozoal parasite, giardia duodenalis, has seven microscopically indistinguishable genotypes or assemblages. 26 assemblages a and b infect humans, while assemblage f is harbored by cats. cats will occasionally harbor assemblages a and b. 39 infection with g. duodenalis occurs after ingesting cystcontaminated feces, by grooming an infected cat or from contaminated fomites. 37 re-infection may occur by selfgrooming. only a small number of cysts need be ingested to establish an infection. in humans as few as 10 cysts are required to cause infection. 37 after ingestion of infective cysts, trophozoites begin to excyst in the stomach. 37 this process is completed in the proximal duodenum. 26 the trophozoites adhere to enterocytes along the length of the small intestine using the ventral suction disk. intermittent shedding of immediately infective cysts begins 5 to 16 days after infection. 28 proteins released during encystment of the trophozoites are detected by the fecal antigen tests. 37 cysts may adhere to the perianal region, facilitating re-infection by self-grooming. 28 occasionally, trophozoites are found in examinations of fresh, watery feces. these do not survive for long and are not infective. 4 the mechanisms of disease induced by g. duodenalis are still unclear. after the trophozoite attaches to the brush border of the enterocyte, the tight junction between cells is disrupted, increasing intestinal permeability. 37 the brush border becomes attenuated, further exacerbating malabsorption of water, electrolytes, and other nutrients. 39 the alteration in intercellular adhesion results in t-lymphocyte activation and mucosal cell injury. 37 infection also promotes mucosal cell apoptosis (preprogrammed cell death). 28 in addition, small intestinal bacterial overgrowth may accompany g. duodenalis infections, resulting in more severe clinical signs. 39 fortunately, most cats infected with g. duodenalis show no clinical signs. 28, 39 the most common sign is acute, transient, small bowel diarrhea 28 without systemic illness, such as fever or vomiting. 39 less commonly, a cat might have profuse, watery malodorous diarrhea 37 with mucus. 39 also possible, but uncommon, is weight loss 26, 28 or abdominal pain. 37 the severity of clinical signs exhibited in an individual cat depends on the age and general health of the cat. 37 cats co-infected with cryptosporidium felis or tritrichomonas foetus may have more severe diarrhea that is more difficult to control, 39 as will the presence of bacterial overgrowth. the diagnosis of g. duodenalis requires demonstration of trophozoites or cysts in a fecal examination, or detection of encystment proteins or giardial dna in a fecal sample. a reliable diagnosis may be difficult to obtain for several reasons. cysts are small, easily missed, and must be differentiated from plant debris or yeast. 37 trophozoites are short lived outside the body and can only be found in very fresh, watery feces or, better yet, in diarrheic feces collected directly from the cat's rectum. 37 shedding of cysts is usually intermittent, and the intensity of shedding varies greatly. 28, 37 because of these difficulties, the absence of the organism in a fecal sample does not eliminate it as the cause of diarrhea. it is often necessary to test multiple fecal samples, using at least two different techniques in order to find the organism. 37, 39 the easiest test to perform is a fecal smear or wet mount examination to identify trophozoites or cysts (figures 23-42 and 23-43 ). the sample examined should be very fresh, warm, diarrheic feces. 39 one drop of feces is placed on a slide along with a drop of 0.9% saline or lugol iodine. 28 trophozoites are identified by their characteristic structure (table 2321) . the motile trophozoites have a motion described as appearing like the back and forth rolling motion of a falling leaf. since lugol iodine stain kills the trophozoite, there will be no motion to detect. 28 this test is not very sensitive; however, with trained examiners, the test has a high specificity. increased sensitivity can be gained by performing a centrifugal flotation using zinc sulfate. the sample should be warm, fresh feces or feces refrigerated for no more than 2 days. 28 the processed sample is examined for the same structures as the wet mount. the sensitivity of examining one sample is 70% 28 and increases as more samples are examined. the sensitivity of looking at three samples is 95% 26, 28 ; therefore the test is not considered negative until three specimens have been found free of the organism. 39 a fecal antigen test that identifies the encystment protein is available. the snap giardia antigen test (idexx laboratories) uses fresh or frozen feces, or feces refrigerated for less than 7 days. 34 since the antigen is continuously shed, this test avoids the problem of intermittent shedding of the whole organism. 28 the sensitivity of the test is 85%, with a specificity of 100%. 35 by combining the antigen test with a zinc sulfate fecal centrifugal flotation, the sensitivity improves to 97.8%. 11 it is unknown how long the antigen remains in the feces after treatment. thus a zinc sulfate centrifugal flotation examination should be used to evaluate therapeutic efficacy. 28, 39 the use of this test in cats without diarrhea is controversial, because these cats are unlikely to shed cysts. the zoonotic significance of a positive antigen test in a cat not shedding cysts is unknown and may cause confusion. 39 polymerase chain reaction detection of giardia dna is available, but the test has not been standardized across all diagnostic laboratories. one needs to ensure the laboratory performing the test has validated it for assemblage f. the test may also be used to identify cats harboring the zoonotic assemblages a and b. the sensitivity of this test is unknown. 39 two commonly available drugs are used most frequently to treat infections with g. duodenalis (see table 23 -20) . fenbendazole may be effective and can be used in pregnant queens 28 and in cats co-infected with roundworms, hookworms, and taenia spp. tapeworms. 39 however, in one small study, only four of eight cats infected with both g. duodenalis and cryptosporidium felis stopped shedding giardia permanently after receiving fenbendazole. 29 febantel, in the combination product drontal plus (bayer animal health), is converted to fenbendazole. when six experimentally infected cats received 56.5 mg/ kg of febantel q24h po for 5 days, four of them stopped shedding g. duodenalis cysts. 39 metronidazole has been the traditional drug used to treat g. duodenalis in pets. 28 the drug is also useful for treating concurrent small intestinal bacterial overgrowth and clostridial infections. 39 the administration of metronidazole may eliminate shedding in 67% of cats. 26 neurologic side effects may occur at the dose recommended for treatment of giardia (see above, therapeutics for vomiting and diarrhea). the use of a giardia vaccine was ineffective in clearing infection by itself. 44 the combination of fenbendazole and metronidazole has been suggested as the initial treatment of choice for g. duodenalis infections. 39 although controlled studies are lacking, they may work synergistically by acting on two different targets within the parasite. 28 febantel would be expected to have the same synergism with metronidazole. drug therapy may not be necessary in cats without diarrhea that are infected with g. duodenalis, 37 because it is uncommon for a cat to carry the assemblages required to infect humans. the veterinarian may be obligated to treat a healthy cat if the owner wants to treat, the owner is immunocompromised, or the goal is eradication of an infection from a multicat home or prevention of parasite transmission to giardia-naïve cats is attempted. 28 what may appear to be treatment failure is more likely to be re-infection. in addition to drug therapy, steps should be taken to prevent re-infection. all cats with diarrhea positive for g. duodenalis should be treated along with their housemates. 37 sanitation is imperative in the fight against re-infection and transmission of g. duodenalis. dispose of old litter pans and scoops and use disposable litter boxes during treatment. when the infection is eliminated, not just controlled, new litter boxes and scoops may be purchased. bathe all cats during treatment to remove cysts from the hair coat. since giardia spp. cysts are susceptible to desiccation, 28 blowdry all cats using a warm air blower, paying particular attention to the perineal area. disinfect bowls, housing, and other utensils with bleach. 28 in addition to antiprotozoal drugs and sanitation, supportive care may become necessary. probiotics and a highly digestible, bland diet may be offered to cats with small bowel diarrhea, while a high-fiber diet may be useful for those few cats with large bowel diarrhea. 39 where required, hydration and electrolyte imbalances must be corrected and antiemetics used to control vomiting. therapy can be evaluated by retesting feces with a zinc sulfate centrifugal flotation examination 1 to 3 days after the end of treatment and again 3 weeks later. a positive test immediately posttreatment is most likely because of therapeutic failure. if the cat is negative immediately after treatment ends, but is positive 3 weeks later, re-infection is likely. 28 since the fecal antigen test may remain positive long after the infection is eradicated, this test is inappropriate for evaluating therapy. 28, 39 re-treatment of fecal flotation-positive, recovered cats may be handled in a manner similar to the positive healthy cat mentioned above. 28 cats with diarrhea that continue to shed cysts may be re-treated for g. duodenalis infection along with dietary modification and empirical treatment for other common intestinal parasites. however, serious consideration should be given to investigation into other potential causes of diarrhea. 28 the giardia vaccine has been found to be ineffective in preventing infection 37 and production has been discontinued. 39 this means prevention of giardia infection involves avoiding exposure, stress and re-infections. providing a clean environment, feeding only processed foods, and controlling potential transport hosts will help reduce the chances of exposure. isolation of cats with diarrhea may be important, too. 39 municipal sanitation control is difficult as the cyst survives for weeks in cool, moist environments. 28 cysts are also able to survive water treatment and can pass through attempts at water filtration. 37 giardiasis is associated with debilitating diarrhea in some humans, particularly those who are immunocompromised. 35 however, cats do not commonly carry the assemblages needed to infect humans. transmission of g. duodenalis from cats to humans is rare and unproven. 28 still, it seems prudent to consider the owner's health when contemplating management of giardial infections in cats. to avoid human health risks, cats with diarrhea that test positive for g. duodenalis should be treated with the goal of controlling the diarrhea. 39 since no treatment for g. duodenalis is completely effective or 100% safe, treatment of positive cats without diarrhea should only begin after a discussion of the benefits and risks of the treatment with the owner. 39 tritrichomonas foetus is best known for causing bovine reproductive infections. it is an obligate anaerobic parasite 26 that also colonizes the lower intestinal tract of cats. there are enough differences between the two isolates that the feline isolate does not cause disease in heifers and vice versa. 39 the parasite depends on the host's normal intestinal flora and secretions for obtaining nutrition. 7 a report from the united states of purebred cats tested at an international cat show found t. foetus in 36 of the 117 cats tested, a prevalence of 31%. 23 this parasite seems to have a higher prevalence in purebred cats than nonpurebred cats. a study of pet cats visiting veterinary hospitals across the united states reported 12 of 32 purebred cats were positive for t. foetus, while only 5 of 141 nonpurebred cats were positive. in this same study, 12 of the 17 positive tests were from purebred cats. 45 a study from the united kingdom of diarrheic fecal samples sent to a veterinary diagnostic laboratory reported similar results. purebred cats represented 14 of the 16 cats testing positive for t. foetus. the u.k. study also found the siamese and bengal breeds each represented 6 of 14 positive cats; only two other breeds tested positive. 25 transmission like most other protozoal parasites, t. foetus is transmitted by ingestion of the parasite, in this case, the trophozoite. unlike most of the other parasites, t. foetus does not form cysts and only survives up to 3 days outside the body in moist feces. 47 a cat becomes infected through the use of a shared litter box with an infected cat. after walking into the box, the parasite is transferred from the infected feces of one cat to the paws of the other. infection then occurs through ingestion of the trophozoites during grooming. 47 after infection, t. foetus colonizes the distal ileum and colon, 15 followed by shedding of infective trophozoites 2 to 7 days later. 19 there are several mechanisms by which t. foetus causes diarrhea. these include alteration of the cat's normal bacterial flora population, increases in local inflammatory cytokine concentrations, production of enzymes, and direct mucosal injury. the resulting injury leads to plasmacytic-lymphocytic 49 and neutrophilic colitis. 37 although most infections involve only the mucosa of the colon, one study reported two of seven cats with diarrhea and t. foetus infections as having trophozoites in deeper layers of the colonic wall. 49 co-infection with cryptosporidium felis 17 or giardia duodenalis 39 can be associated with increased numbers of t. foetus trophozoites and increased severity of diarrhea. signs of infection are most frequent in kittens and young cats, although infections without clinical signs can occur. 39 adult cats, however, may also show signs of t. foetus infection. the most common sign is a foulsmelling large bowel diarrhea with increased frequency of defecation, 39 mucus, blood, 15 and flatulence. 37 the consistency of the diarrhea may wax and wane, but the presence of diarrhea does not. 47 cats with diarrhea are otherwise in good health and maintain their body condition. 15, 39 severe diarrhea can result in anal swelling and fecal incontinence. 39 diarrhea may respond to the use of antibiotics because of changes in the cat's intestinal microbial flora. however, it always returns at the cessation of therapy. 39, 47 many cats experience a spontaneous resolution of the diarrhea within 2 years of diagnosis. 15, 38 since t. foetus causes reproductive infections in heifers and bulls, there is speculation the parasite also infects the reproductive tract in cats. tritrichomonas foetus was found in the uterus of a queen with pyometra. 9 however, in a study of 60 breeding male and female cats from 33 catteries, no cytologic or molecular evidence of t. foetus was found in the reproductive tract. the authors reported colonic infection with t. foetus in 15 of the 60 cats representing 22 of the 33 catteries. 24 detection of the trophozoites in a sample of feces is the most expedient means of diagnosing an infection with t. foetus (figure 23-44 ). an index of suspicion is required, because the clinical presentation of t. foetus infection is often mistaken for infection with giardia duodenalis. if a cat is not responding to treatment for that parasite, consider t. foetus as a cause of the diarrhea. the sample required for the diagnosis of t. foetus is a fresh, nonrefrigerated sample of watery feces. refrigeration kills the trophozoites, and they are not found in normal feces. 34 the sample may be freshly passed diarrhea, feces collected using a wire loop passed into the colon, or collected by a colonic flush using a red rubber catheter and 10 ml of saline. 47 a wet mount or smear examination of the feces should be performed on all cats with diarrhea. examination of multiple samples may be required to find the t. foetus trophozoites with this technique because it is insensitive. 39 the trophozoites must be differentiated from giardia duodenalis based on structural differences and motility patterns (see table 23 -21). the trophozoites of t. foetus can be cultured using the inpouch tf system (biomed diagnostics). this test is more sensitive than the fecal wet mount examination and detects 1000 trophozoites per sample. 15 the number of parasites shed by a cat with diarrhea is high enough to be routinely detected with this method. 18 the test should be performed in-house, because the parasite is unlikely to survive the trip to the laboratory. 47 the test pouch is inoculated with 50 µg of freshly collected feces, about the size of a peppercorn. 18 any more than this increases the chances of bacterial overgrowth. 15 the pouch is incubated at 25° c and examined under the microscope for motile trophozoites every other day for 12 days. the pouch should be tapped gently to dislodge the parasites, which tend to collect along the seams. 15 the test is considered negative if parasites are not found after 12 days. one benefit of this system is that it does not support growth of giardia duodenalis or pentatrichomonas hominis. 18 if a fecal wet mount examination and culture are both negative and infection with t. foetus is still under consideration, a pcr test can be performed. this test detects dna from live or dead trophozoites, but is more expensive than other diagnostic methods. 47 this test is more sensitive than the other two methods and can detect 10 parasites per sample. 16 the sample size is 200 mg of feces not contaminated by litter preserved in 3 to 5 ml of rubbing alcohol shipped at room temperature. 15 trophozoites of t. foetus are sometimes found in colonic biopsy samples adhered to the surface or in the lumen of crypts. 15 the most effective drug for the treatment of t. foetus in cats is ronidazole. 17 the drug has a bitter taste and should be compounded into capsules. veterinary staff and owners should use gloves when handling ronidazole. 15 if a confirmed relapse occurs, another course of treatment may eliminate the parasite. 39 diarrhea may take several weeks to resolve after elimination of the parasite, because significant colitis is often present. 47 effectiveness of treatment can be evaluated by performing fecal pcr tests 2 and 20 weeks after the end of treatment. 15 apparent treatment failures may occur because of re-infection, co-infection with giardia duodenalis or cryptosporidium felis, or the presence of another concurrent diarrhea-causing disorder. a more worrisome cause for treatment failure is a recent report of parasite resistance to ronidazole in two cats. 22 fortunately, diarrhea ultimately resolved in both cats despite the continued presence of the parasite. if the cat retests negative and the diarrhea is not improving after 2 weeks, consider the possibility that another disease may exist. nonspecific treatment for diarrhea is unhelpful 37 and may prolong the duration of diarrhea. 15 diarrhea may respond to antibiotics as they alter the intestinal flora population; however, once treatment is stopped, the diarrhea will return. 47 an important and potentially serious adverse effect of ronidazole administration in cats is a reversible neurotoxicity. onset of signs often begins within 1 week of the onset of therapy and may last between 1 and 4 weeks after cessation of therapy. 38 these signs can include depression, ataxia, seizures, 47 behavioral changes, weakness, hyperesthesia, and trembling. 38 neurotoxicosis usually requires only supportive care along with discontinuation of the drug. the neurologically affected cat should be retested for the parasite, because it may have been eliminated. 38 because of the potential for neurotoxicity, the use of ronidazole should be restricted to cats with confirmed infections with t. foetus. 47 crowded conditions should be avoided, because transmission of t. foetus trophozoites is more efficient in these settings. 39 cats testing positive should be isolated from other cats during treatment. 37 providing a clean environment will help prevent transmission of trophozoites. although there is a report of an infection in one immunocompromised person, transmission of t. foetus trophozoites from cats to healthy humans has not been reported. 39 still, prudence dictates handling feces infected with t. foetus trophozoites carefully. recent genetic evaluations have shown that most feline infections with cryptosporidium spp. are with c. felis; not, as previously thought, with c. parvum. 36 cryptosporidium parvum seems to be limited to farm animals. 4 cryptosporidium felis is an obligate intracellular parasite infecting the small intestine. 4 infective oocysts are ingested from contaminated feces during self-grooming of contaminated body parts and from contaminated food and water. 32, 39 after infection, the parasite attaches to the brush border of the enterocyte. the prepatent period is 3 to 6 days, 39 and the oocysts are infective as soon as they are shed, making this a very contagious disease. 20 like most intestinal parasites, shedding is often intermittent. the pathogenic effects of c. felis infections are not well understood. direct cytotoxicity and inflammation causes villus atrophy and decreased surface area for absorption of water, electrolytes, and other nutrients. 20, 32 apoptosis (preprogrammed cell death) of the mucosal cells may be accelerated, adding to the malabsorption. 20 most infections with c. felis are subclinical. 39 signs, if present, range from a mild, self-limiting small bowel diarrhea 33 to chronic intermittent small bowel diarrhea. 32 severe diarrhea with weight loss and anorexia may also occur. 32, 33 clinically apparent infections are most common in kittens, adult cats with concurrent gastrointestinal diseases, and cats co-infected with giardia duodenalis or tritrichomonas foetus. 39 cats with co-infections may experience more severe clinical signs. 32 a fecal flotation, which should be performed on all cats with diarrhea, may reveal c. felis if there are large numbers of oocysts (figure 23-45) . the fecal floatation test, however, is often negative 39 because of intermittent shedding. the parasite is small and floats in a higher plane than helminth ova; the high-power lens and appropriate adjustment of the microscope stage is required to find the parasite. 32 the small size of the oocyst makes identification difficult, particularly if the examiner is not specifically looking for them. 34 a modified ziehl-neelsen stain of a thin fecal smear may help in the identification of the oocysts. 39 this technique works well in humans with large numbers of oocysts. 33 once signs resolve or the oocyst numbers decline, a single examination of a stained smear becomes insensitive. when only one sample is available, testing for c. felis antigen is a good choice. 34 the prospect microplate assay (alexon biomedical, sunnyvale, calif.) is more sensitive and specific for the diagnosis of c. felis than is the examination of a stained smear. 6 immunofluorescent antibody testing is available from some laboratories. fecal c. felis dna can be detected using pcr testing. this test is available at many veterinary diagnostic laboratories; however, at present, there is no test standardization among laboratories. 39 the clinical and zoonotic significance of a positive pcr test combined with an oocyst negative test is unknown. 39 therefore a positive pcr test in a cat without diarrhea presents a confusing situation for the attending veterinarian with regard to recommendations for the owner. unfortunately, there are no completely effective and safe treatment protocols available for c. felis. 32, 39 a concerted attempt to find other causes of diarrhea should take place prior to convicting a cat of having diarrhea solely from c. felis infection. most reports on therapy for c. felis are uncontrolled and anecdotal. a number of drugs have been discussed. azithromycin for at least 10 days appears safe but produces variable results. 39 paromomycin, an oral aminoglycoside, may be effective. however, one study reported acute renal failure in 4 of 32 cats receiving the drug. deafness also occurred in three of those four cats. 21 nitazoxanide is a drug approved for treating humans with diarrhea caused by cryptosporidium spp. infections. the administration of nitazoxanide to cats at 25 mg/kg q12h po for at least 5 days 39 up to 28 days 32 may be effective. however, nitazoxanide is a gastrointestinal irritant and commonly results in vomiting and foul-smelling diarrhea. co-infections with giardia duodenalis and/or tritrichomonas foetus are more difficult to control. if diarrhea from c. felis infection improves but does not resolve at the end of therapy, the duration of treatment may be prolonged. 39 additional diagnostic testing should also be performed to ensure the only cause of the diarrhea is infection with c. felis. environmental control of c. felis is difficult, because it is extremely hardy. it is resistant to chlorination and most disinfectants. 32 oocysts remain viable at temperatures above freezing up to 65° c. 4 the parasite is difficult to filter and survives treatment at municipal water treatment facilities. 20 steam-cleaned housing and utensils may be beneficial in controlling parasite numbers, and they are susceptible to 5% ammonia solutions; however, the required contact time is 18 hours. 39 cryptosporidium spp. are relatively species specific, and there are no reports of waterborne outbreaks of human cryptosporidiosis associated with c. felis. 32 cryptosporidiosis can cause life-threatening diarrhea in hivpositive persons. 20 fortunately, humans are rarely infected with c. felis. 39 in fact, the zoonotic species most commonly found in humans (often veterinary students), is c. parvum found in young heifers. 4 regardless of a person's health, feces from a cat with diarrhea should be handled carefully. if a cat infected with cryptosporidium spp. is owned by an immunocompromised person, a pcr test may be useful in determining the species of the parasite and its zoonotic risk. like other coccidians, toxoplasma gondii is an obligate intracellular parasite. 12 domestic cats and other felids are the only animals that shed oocysts. any warmblooded animal, including humans, can be infected with this parasite. toxoplasma gondii can be transmitted by ingestion of infective oocysts in fecally contaminated food or water after ingestion of tissue cysts through carnivorism, or by transplacental or trans-mammary transmission of the parasite. the parasite enters into one of two cycles, depending on the host species. the enteroepithelial cycle only occurs in cats and results in shedding of oocysts after sexual reproduction of the parasite. after a cat ingests an infective oocyst or a tissue cyst, the parasite enters the mucosal cells of the small intestine, where it may undergo development and sexual reproduction, after which oocysts are shed. 12 the prepatent period after ingesting an infective oocyst is 19 to 48 days, while shedding after ingesting tissue cysts starts in 3 to 10 days. 4 fecal shedding, which occurs only after initial infection, lasts for 2 to 3 weeks 4,31 and the oocysts become infective 1 to 5 days after they are shed. 12 the extraintestinal cycle occurs in any animal, including cats. after ingestion, the parasite penetrates the cells of the small intestine and rapidly replicates in the enterocytes and associated lymph nodes into tachyzoites. after hematogenous and lymphatic spread, tachyzoites infect cells in all tissues of the body. 4 tissues most commonly infected include the brain, liver, pancreas, and lungs. 30 if a pregnant queen becomes infected, tachyzoites cause placentitis, after which they infect the fetus. 13 in 3 weeks, the host's immune response slows parasite replication, and the resultant bradyzoites form tissue cysts 30 in the brain, striated muscle, and liver, and they remain viable for the life of the animal. 4 immunosuppressive drugs or disease may dull the suppression of parasite division by the host immune system and allow the slowly dividing bradyzoites in tissue cysts to begin rapid division, thereby reactivating the infection with tachyzoites. 30 none of the forms of t. gondii produces a toxin. rapid replication of tachyzoites within a cell leads to rupture of the cell and necrosis of the tissue in which they are located. 12 the most commonly injured tissues are the brain, lungs, liver, and pancreas. prenatal infection leads to more severe illness, because the immature immune system is unable to slow down replication by tachyzoites, allowing continued damage to tissues. prenatal infection is more likely to result in ocular infections, and neonatal death is usually caused by pulmonary or hepatic infection. 30 type ii and iv hypersensitivities may be involved in the pathogenesis of chronic disease from bradyzoites in tissue cysts. 30 kittens infected perinatally can be stillborn or die shortly after birth. they may also suffer from hepatomegaly and ascites, central nervous system signs resulting from encephalitis, respiratory distress, or uveitis. 12, 13 clinical signs of infection in healthy adult cats are uncommon (box 23-2). 31 diarrhea from enteroepithelial development of the parasite is rare. 39 cats that develop clinical disease often have an episodic course with vague signs 30 that depend on the body system affected. onset of illness may be acute or chronic, and the most commonly affected organs include the brain, lungs, liver, heart, pancreas, and the eyes. 13 signs are the result of spread of tachyzoites after initial infection or after reactivation of tissue cysts. cats suffering from uveitis may develop lens luxation and glaucoma. the best way to identify a cat shedding t. gondii oocysts is to demonstrate them with a centrifugal fecal flotation technique using sheather sugar solution. the oocysts are about a quarter of the size of isospora felis oocysts ( figure 23-46 ). 12 oocysts of t. gondii are morphologically indistinguishable from hammondia or besnoitia spp. oocysts. 13 detection of fecal t. gondii dna using a pcr test can be used to definitively differentiate t. gondii oocysts from similar coccidians. 31 it is probably best, however, to assume suspicious oocysts are those of t. gondii until proven otherwise. proving infection with t. gondii is responsible for a cat's systemic illness is also difficult. finding tachyzoites in cytology samples is uncommon. they are most likely to be identified from body cavity effusions. 13 the most common method of identifying an infected cat is by detecting t. gondii-associated immunoglobulins using immunofluorescent antibody or elisa techniques. since cats are infected for life, a seropositive cat has been infected at some point in its life. however, use of serology alone is insufficient to diagnose an active t. gondii infection. serum immunoglobulin m (igm) is produced within 1 to 2 weeks after infection, but increased igm titers may persist for months to years. serum immunoglobulin g (igg) begins to rise later; in some cats, igg may not be detectable for 4 to 6 weeks. 12 by the time igg is detectable, shedding will have ceased. maternally acquired igg persists in kittens for 8 to 12 weeks. 13 a rising igg titer is associated with an active infection, but the degree of increase is not associated with the severity of the clinical signs. if a cat becomes seronegative, it is more likely the titer has fallen below the sensitivity of the test rather than the parasite has been eliminated from the body. 31 because of the vague nature of the clinical signs, many cats are presented later in the course of the disease. by this time, they may have switched from igm to igg production or passed the time of maximal igg production. thus a negative igm titer or a lack of rising igg titer does not rule out t. gondii infection. 30 also, reactivation of tissue cysts is rarely associated with rising igg titers. 31 ultimately, the diagnosis of an active systemic t. gondii infection requires demonstration of an igm titer greater than 1 : 64 or a fourfold increase in igg titers over a 2-to 3-week period along with signs consistent with toxoplasmosis, the exclusion of other disorders that may cause the clinical signs, and response to appropriate anti-t. gondii treatment. 31 although serum igm titers may be increased in otherwise healthy cats, increased igm titers in cerebrospinal fluid or aqueous humor only occurs in cats with active cns or ocular infections. 30 the goals of treating a cat infected with t. gondii are to reduce shedding of oocysts and to control the clinical signs in sick cats. shedding can be reduced by using ponazuril, 13 toltrazuril, or high doses of clindamycin. the drug options for treating a sick cat include clindamycin, trimethoprim-augmented sulfadiazine, or azithromycin for at least 4 weeks (see table 23 -20) . recurrences are more common if the cat is treated for less than 4 weeks. 13, 30 the antifolate drug pyrimethamine may be more effective than trimethoprim, but megaloblastic anemia develops in many cats. supplementation with folinic acid (5 mg/cat, once daily, po) or brewer's yeast (100 mg/kg, once daily, po) may prevent or reverse the anemia. 12 no drug clears all of the tissue cysts; so, cats remain infected for life. if uveitis is also present, use appropriate topical, oral, or parenteral corticosteroids. for a cat with proven t. gondii-associated uveitis alone, a topical ocular glucocorticosteroid is the only required treatment; no antibiotics are necessary unless the uveitis is persistent or recurrent. 31 • wash hands after handling cats, especially if you are pregnant or immunocompromised. • remove fecal material from the home environment daily, since shed oocysts require a minimum of 24 hours to become infective. • do not have immunocompromised persons clean the litter box. if they must clean the litter box, they should wear gloves and wash hands thoroughly when finished. • use litter box liners, and periodically wash the litter box with scalding water and detergent. • wear gloves when gardening, and wash hands thoroughly when finished. • cover children's sandboxes when not in use to avoid fecal contamination by outdoor cats. • only feed cats cooked or commercially processed food. • control potential transport hosts, such as flies and cockroaches, that may bring the organism into the home. • filter or boil water from sources in the environment. • cook meat for human consumption to 80° c for 15 minutes minimum (because of uneven heating, microwave cooking does not kill all t. gondii 12 ). • freeze meat at −12° c for 24 hours. 12 • wear gloves when handling meat, and wash hands thoroughly with soap and water when finished. clinical signs such as malaise, fever, and muscle pain should begin to resolve in 2 to 3 days. 30 if there is no response within 7 days, switch to or add another drug. 31 if there is still no response, search for another condition that may cause the observed clinical signs. however, ocular and cns signs resolve more slowly and thoracic radiographic changes may take weeks to resolve. 30 some cns changes may never completely resolve. cats co-infected with feline immunodeficiency virus (fiv) do not respond to anti-t. gondii treatment as well as fivnegative cats respond. 12 feeding cats commercially processed cat food and avoiding undercooked or raw meat can prevent exposure to t. gondii. controlling hunting reduces access to paratenic hosts with infective tissue cysts. access to mechanical carriers of t. gondii, such as earthworms or cockroaches, should be minimized. human infection with t. gondii is common, more so in warm, humid climates where the prevalence of t. gondii seropositive persons approaches 100%. the number of persons seropositive for t. gondii is estimated to be around 500,000,000 worldwide. 12 infective oocysts are hardy and may remain viable in the environment for up to 18 months. 12 human infection most often occurs after eating raw or undercooked meat infected with tissue cysts or by transplacental infection. 31 seropositive cats are finished shedding and are unlikely to resume shedding even if the infection becomes reactivated. 31 cats found to be shedding oocysts should be quarantined at a veterinary hospital until shedding ends. oocysts of t. gondii have not been found on the hair coat 13 ; so, transmission of toxoplasmosis does not occur after touching a cat. 31 pregnant women infected with t. gondii for the first time, or chronically infected women who are also hiv positive, can transmit the parasite to their unborn child. transplacental infection can result in stillbirths, cns, or ocular disease. 30 more severe fetal disease may occur if the infection happens in the first half of the woman's pregnancy. 12 toxoplasma gondii infection of immunocompetent humans usually results in a self-limiting fever and malaise. 30 steps useful in preventing transmission of t. gondii to humans can be found in box 23-3. pancreatitis refers to inflammation of the pancreas only, with no implication of the underlying cause or pathology. for example, acute necrotizing pancreatitis (anp) with pancreatic auto-digestion, requiring predominantly supportive care by maintaining fluid and electrolyte balances and pain relief, must not be confused with chronic pancreatitis (cp) caused by lymphocytic infiltration, and commonly associated with lymphocytic inflammatory bowel disease (ibd), and often requires corticosteroids to manage. these two conditions (and others) can only be definitively distinguished histologically. in many cases, the clinical signs of cats with acute pancreatitis will resolve with supportive care before a precise diagnosis is reached and will thus remain undiagnosed. there are no formal classifications for feline pancreatitis, but most authors 78, 89, 90 use the terms • acute pancreatitis • acute necrotizing pancreatitis, characterized by severe peri-pancreatic fat necrosis • acute suppurative pancreatitis, characterized by neutrophilic infiltration • chronic pancreatitis, characterized by lymphocytic infiltration the exact prevalence of feline pancreatitis is unknown. necropsy studies from the 1970s to 1990s reported prevalence of feline pancreatitis ranging from 0.45% to 2.4%. 21,67 a more recent study 17 found 67% of 115 cats had evidence of pancreatitis. however, this included pancreatic pathology in 45% of apparently healthy cats, which suggests that mild pathology is unlikely to cause clinical signs. these studies all show lymphocytic pancreatitis to be significantly more prevalent than acute pancreatitis. this may underestimate the true prevalence of acute pancreatitis, since it is understood that no permanent histopathologic changes are present after resolution of acute pancreatitis. 89 it is also possible that studies assessing pathology in necropsy cases do not reflect clinical practice. there are no specific age, breed, or sex predispositions. although one study reported siamese cats to be at increased risk of acute pancreatitis, 33 subsequent studies have recognized the majority of cases are domestic shorthair cats, suggesting no specific breed predispositions. 22, 29, 60, 71 most studies have indicated older cats (8 to 10 years of age) are more likely to be affected, 22, 29, 60, 71 but these studies most likely underrepresent cats with less severe clinical disease for which definitive diagnosis may not be reached and which may be younger. no association has been made with a high-fat diet or obesity. in most cases of both acute and chronic pancreatitis, no specific cause is found, and the disease is primarily considered to be idiopathic. 22, 90 there are, however, some specific underlying causes that are sporadically recognized. these include infections with herpesvirus, 75 calicivirus, 37,49 feline infectious peritonitis (fip), 44 liver fluke 58 and pancreatic fluke, 26, 77 and toxoplasmosis. 20 however, a recent paper found no association between serum feline pancreatic lipase immunoreactivity (fpli) concentrations and toxoplasma gondii serology. 8 pancreatitis has also been recognized subsequent to trauma 81 and organophosphate poisoning. 33 the association of pancreatitis with inflammatory bowel disease and cholangitis is frequently mentioned (triaditis) but poorly described in the literature. 80 one study found 30% of ibd cases to have histologic evidence of pancreatic involvement, 6 and another found fpli concentrations were elevated in 70% of cases with histologically confirmed ibd. 3 it is the author's experience that many cases of pancreatitis recognized with ibd have no specific clinical signs attributable to pancreatitis and should therefore be diagnosed and treated as intestinal disease. diabetes mellitus is a recognized co-morbidity of pancreatitis in cats. a recent study found fpli concentrations were significantly higher in 29 diabetic cats compared with 23 non-diabetics. no association could be made between fpli concentrations and the degree of diabetic control. 23 one study found 5 of 13 cats (38%) histologically diagnosed with hepatic lipidosis were also histologically diagnosed with acute pancreatitis. it is not known if pancreatitis is a cause, consequence, or coincident disease of hepatic lipidosis. for example, anorexia associated with acute pancreatitis could predispose to fatty infiltration of the liver. however, the high rate of concurrent disease has important implications for ensuring cats with pancreatitis receive adequate caloric intake. 1 ongoing or recurrent pancreatitis may lead to pancreatic cysts 10 or exocrine pancreatic insufficiency, 74 which are both covered later in this chapter. although pancreatitis has been experimentally induced in cats, 18, 41, 56 the pathophysiology of spontaneous pancreatitis remains unknown. acute pancreatitis is initiated by an increase in secretion of pancreatic enzymes that leads to inappropriate cellular activation of trypsin and subsequently other digestive zymogens. these activated digestive enzymes lead to local effects including inflammation, hemorrhage, acinar cell necrosis, and peripancreatic fat necrosis. 43, 78, 86 chronic pancreatitis may result from any of several underlying processes: ongoing, low-grade acute pancreatitis episodes may instigate chronicity; chronic pancreatitis, with a predominance of lymphocytic inflammation has been induced experimentally within 5 weeks by narrowing the main pancreatic duct to approximately 25% of its normal diameter 18 ; and the association with ibd 80 may suggest an immune-mediated cause. the clinical signs of pancreatitis in cats are nonspecific. a review of eight prior series totaling 159 cases of acute pancreatitis in cats found anorexia (87% of cases) and lethargy (81%) to be the most common historical findings. 78 vomiting was recognized in 46% of cases, diarrhea in 12%, and weight loss in 47%. physical examination findings were similarly nonspecific with dehydration (54%) being the major finding; fever was recognized in only 25% of cases and abdominal pain in 19%. it is important to note that vomiting and abdominal pain, key features of pancreatitis in dogs, are not consistently recognized in cats. similar, nonspecific findings indistinguishable from ibd are recognized in cats with chronic pancreatitis. 3, 6 diagnosis because the presenting signs and physical examination findings are nonspecific, the diagnosis of pancreatitis can be challenging, requiring not only clinical suspicion but a combination of diagnostic modalities. for the most part, hematology and plasma biochemistry findings are unremarkable, although a combination of findings may increase clinical suspicion. for example, moderate elevations in liver enzymes, bilirubin, and glucose are present in approximately 50% of cases and hypocalcemia in approximately two of three of cases; hypocalcemia infers a poorer prognosis. hypoalbuminemia is seen in approximately one of three of cases and has important implications for fluid therapy. 78 amylase and lipase elevations are not reflective of pancreatitis in cats. 47 feline trypsinlike immunoreactivity (ftli) is the diagnostic test of choice of exocrine pancreatic insufficiency, but elevations in pancreatitis are not seen consistently enough to warrant use of this test for this purpose. 29, 47, 71 the biggest recent advance in feline pancreatic diagnostics has been the characterization of feline pancreatic lipase, 69 leading to the development of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fpli). 70 it must be remembered, however, that an increase in fpli only tells the clinician that pancreatic pathology is present, but not the cause of pathology, which may be, for example, neutrophilic or lymphocytic pancreatitis or neoplasia, and it may or may not involve the intestines or liver. fpli should therefore be used as a screening test, with elevated results not suggesting a diagnostic end point. further, the high interassay variability of this test 70 would suggest that mild cases may be missed as shown in one study 24 and that the test may not be appropriate for serial monitoring. fpli is currently available as "spec fpl" from commercial laboratories and has a sensitivity of 79% and a specificity of 82% when 5.4 µg/l is used as the diagnostic cut off 25 compared with 3.5 µg/l, which is the listed reference range high point. in an acutely unwell cat (less than 2 days) with only mild to moderate signs of disease, further diagnostics may not be warranted, and many cats will improve with supportive therapy of balancing fluid and electrolytes, pain relief, and antinausea/vomiting therapy. cats with chronic duration of signs and acutely unwell cats that do not improve with supportive therapy warrant further diagnostics. the underlying disease process cannot be assumed from an elevated fpli; in one study of 63 cases, acute necrotizing pancreatitis could not be distinguished from chronic nonsuppurative pancreatitis by signalment, duration of signs, or clinical findings. 22 the major utility of diagnostic imaging is to rule out other differential diagnoses, such as an intestinal foreign body, and perhaps confirm that the pancreas is affected. radiography is non-specific for diagnosis of pancreatitis, but findings may include decreased abdominal detail (sometimes associated with ascites), soft tissue density in the right cranial quadrant of the abdomen, hepatomegaly, or gas-filled intestines 22,60,64 (see . additionally, thoracic radiographs may show pleural effusion. one study found 5 of 20 cats with pancreatic necrosis had such a change 60 ; the mechanisms resulting in pleural effusion are not precisely defined. ultrasonography has high specificity (>85%) but low sensitivity (<35%) for recognizing pancreatitis in cats, 22, 29, 60, 71 with findings dependent on operator skills, quality of equipment, and severity of lesions. typical findings are hypoechogenicity of the pancreas, which may be enlarged or irregular; hyperechogenicity of the peripancreatic fat; the possible presence of abdominal effusion; and abnormal findings with other organs, such as liver or intestine, may add to the clinical picture 22, 60, 64, . one study indicated that contrast-enhanced doppler ultrasonography can provide further diagnostic insights. 54 a recent study suggested that endosonography may be useful in cases where transabdominal ultrasonography is difficult, for example, because of obesity, hyperechoic mesentery, or excessive intestinal gas. 61 for more than 20 years, computed tomography (ct) has been a commonly used modality to confirm pancreatitis in humans, 55 but this reliability has not been demonstrated in cats, where sensitivity may be as low as 20%. 24, 29 definitive diagnosis of pancreatitis, including differentiation of the inflammatory process, can only be made by cytologic assessment of pancreatic tissue. in most cases, ultrasound-guided fine-needle aspiration (fna) of the pancreas is technically difficult because of the small dimension of the feline pancreas; there appears to be no assessment of feline pancreatic fna findings in the literature. gross inspection of the pancreas and samples for histologic assessment can be obtained during laparotomy 22, 64 (see or laparoscopy. 16, 79 because pancreatitis often occurs concurrently with pathology of other organs, 22 thorough evaluation of the abdomen by ultrasonography or gross inspection is recommended, as are multiple biopsies of, for example, intestines, liver, and mesenteric lymph nodes, where appropriate. clinicians may be reluctant to biopsy the pancreas because of perceived risks of deleterious effects. studies of pancreatic biopsy in healthy cats dispel the concern that the pancreas is unforgiving to mild manipulation and biopsy 16,42a and the author's clinical experience is consistent with these findings. supportive care comprising correction of fluid/ electrolyte imbalances, pain management, and nutritional support are the mainstay of therapy for cats with figure 23-49 gross appearance of pancreas at laparotomy; this was histologically diagnosed as chronic pancreatitis (i.e., lymphocytic infiltration was recognized). gross appearance of pancreas at laparotomy; this pancreas was found to be histologically normal. it does look smaller than is typically seen; pancreatic atrophy can look similar to this, grossly. pancreatitis. 78, 86, 89 specific underlying causes, when diagnosed, should be managed, as should concurrent diseases. follow-up evaluation is determined on a case-by-case basis; reduction or resolution of clinical signs is the main criterion for success of therapy. serial fpli values may be monitored when initial results are extremely high but are of limited value for mild increases because of assay variability. dehydration, acid-base and electrolyte abnormalities should be corrected during the first 12 to 24 hours. hypocalcemia, if present, should be treated with a calcium gluconate infusion of 50 to 150 mg/kg during 12 to 24 hours, with continued assessment of plasma calcium concentrations. plasma transfusions can be considered in cats with hypoalbuminemia. 78, 86, 90 although abdominal pain is not commonly described in cats with pancreatitis, it is likely to be present in most cases and may contribute to anorexia. historical concern about exacerbation of pancreatitis with opioids is no longer accepted, and this class of drugs is considered appropriate. meperidine (1 to 2 mg/kg sc or im) every 1 to 2 hours, butorphanol (0.2 to 0.4 mg/kg sc) every 6 hours, or sustained-release buprenorphine (120 µg/kg sc) every 72 hours are alternatives. 67, 78, 86 the author uses one dose of methadone (0.1 to 0.2 mg/kg sc, im, or iv) initially and places a fentanyl patch for longer-term pain management. the traditional recommendation for management of pancreatitis across all species has been nil per os for several days. this recommendation is appropriate for cats with severe vomiting, but there is no evidence to support this approach in cats that are not vomiting and that are eating normally. further, nutritional support is vital for those cats with concurrent hepatic lipidosis. if the cat is not eating voluntarily, nutritional support by tube feeding is often warranted. 67,78,86 a recent paper found nasogastric tube feeding of cats with pancreatitis was tolerated well and resulted in few clinically significant complications. 42 other reported nutritional strategies for cats with pancreatitis incorporate partial parenteral nutrition (ppn; 8.5% amino acids, 20% lipids), or total parenteral nutrition (tpn; 6% amino acids, 20% lipids, 50% dextrose), or both instead of enteral feeding. 14,39,53 cats do not seem to benefit from feeding of specially formulated low-fat diets; commercially available, veterinary liquefied diets appear to be well tolerated despite their high-fat contents. 86 other therapy may be appropriate in individual cases. all cats with pancreatitis that are vomiting should be treated with antiemetics. examples of drugs that can be used are 5-ht 3 antagonists, such as dolasetron (0.5 to 1.0 mg/kg iv or po, once to twice daily); ondansetron (0.1 to 0.2 mg/kg iv every 6 to 12 hours); and maropitant, an nk 1 -inhibitor (0.5 to 1.0 mg/kg sc once daily). these drugs are covered in detail earlier in this chapter under therapeutics for vomiting and diarrhea. dopaminergic antagonists, such as metoclopramide, are less effective antiemetic agents in cats than the other choices mentioned. 78, 89 in most cases, pancreatitis begins as a sterile process, and antibiotic therapy is controversial. pancreatic necrosis and inflammation may predispose to bacterial colonization of the pancreas as demonstrated in experimental models. 82, 84 this has not been demonstrated in spontaneous disease, and no comparison of outcomes has been made of cats with pancreatitis treated with or without antibiotics. cefotaxime (20 to 80 mg/kg iv, im) has been used to prevent bacterial colonization in experimental models. 83 other broad-spectrum cephalosporins or ampicillin may act similarly. antibiotic considerations are possibly more important for acute pancreatitis than for treatment of chronic disease. cats with demonstrated lymphocytic pancreatitis, with or without concurrent ibd or lymphocytic cholangitis, should be treated with corticosteroids (e.g., prednisolone, 1 to 2 mg/kg once to twice daily) with tapering to the lowest effective dose. there is no justification for use of corticosteroids in cats with acute necrotizing or acute suppurative pancreatitis, or cats for which the cause of pancreatitis has not been diagnosed histologically. use of corticosteroids in cats with pancreatic disease creates a risk of iatrogenic diabetes mellitus. surgical intervention is warranted to relieve any bile duct obstruction that may result or for the débridement of pancreatic abscesses or necrotic tissue; in many cases, cats will survive multiple years after such corrective surgery. 65 pancreatic cysts, pseudocysts, and bladders have been described sporadically in cats.* pancreatic cysts are lined by a single layer of cuboidal epithelium and do not communicate with the pancreatic duct; pseudocysts are enclosed by a wall of fibrous tissue, lacking the epithelial lining characteristic of true cysts and can form secondary to pancreatic inflammation; cystic dilations of the pancreatic duct are referred to as pancreatic bladder. true pancreatic cysts have been described in three cats 9,10,15 ; a congenital pancreatic cyst with associated inflammation was described as an incidental finding in an adult cat 15 ; multiple pancreatic cysts were described in a cat with concurrent polycystic disease in the kidney and liver 9 ; and a another cat had multiple recurrent pancreatic cysts with concurrent mild pancreatic inflammation and atrophy associated a with rapid clinical course resulting in diabetes mellitus. 10 cysts, pseudocysts, and bladders may be identified ultrasonographically or by ct. they may be benign, but the associated pancreatic inflammation and other sequelae, such as diabetes mellitus, may need to be managed. pancreatic bladders may result in biliary obstruction, and surgical correction may be required. pancreatic nodular hyperplasia is recognized quite frequently as an incidental finding in older cats or at necropsy. 45 neoplasia of the exocrine pancreas is rare in cats. its frequency was assessed in the 1970s when one study estimated 12.6 cases per 100,000 patients per year at risk, 52 and another found pancreatic tumors in 5 of 800 feline necropsies. 45 a more recent study recognized, from 15,764 feline admissions over a 20-year study period, only two cats with pancreatic adenomas (0.013% of admissions) and eight with pancreatic adenocarcinomas (0.05% of admissions). 62 adenomas appear as small, solitary or multifocal nodules and are not typically associated with adjacent pancreatic inflammation. they do not cause clinical signs, unless large, when any clinical signs result from the physical size and are usually an incidental finding. 45, 86 few generalities can be made about the presentation for pancreatic adenocarcinoma. the age range is large (4 to 20 years), there is no sex predisposition, and no clear breed predispositions are present. 62, 86 only cytology or histopathology can distinguish pancreatitis from pancreatic carcinoma in cats antemortem, yet it is important to differentiate the two conditions, because, in contrast to adenomas, pancreatic adenocarcinoma is associated with a grave prognosis. the presence of lesions consistent with metastases on radiography or ultrasonography may suggest malignancy, but one study could not distinguish neoplasia from pancreatic nodular hyperplasia ultrasonographically based on the appearance of the pancreas alone 32 (figures 23-53 and 23-54) . pancreatic adenocarcinomas in cats can result in a paraneoplastic dermatologic condition consisting of nonpruritic, symmetric alopecia affecting the face, ventral body, and medial aspect of the limbs of cats. the skin is usually glistening but not fragile, and there can be crusty lesions on the footpads.* the pathogenesis of this dermatologic disease is unknown. in one case, surgical excision of the pancreatic carcinoma resulted in resolution of dermatologic disease, indicating that the process is reversible (although signs recurred as the tumor re-emerged). 73 diabetes mellitus is a recognized complication of pancreatic adenocarcinoma. the mechanism is unknown and may simply be secondary to compression or invasion of islet cells by the tumor. in some cats, diabetes is recognized ahead of pancreatic neoplasia. 31, 40, 62 obstructive jaundice has also been described with pancreatic adenocarcinoma. 13 most cases of pancreatic adenocarcinoma in cats have metastasized by the time of diagnosis, and most reported cases die or are euthanized within 7 days of diagnosis. 62 surgical excision is a potential option if neoplasia is confined to one limb of the pancreas, but recurrence is possible even if there is no evidence of metastasis and excision seems complete at the time of surgery. 73 exocrine pancreatic insufficiency (epi) is a condition caused by insufficient synthesis and secretion of pancreatic digestive enzymes from the exocrine portion of the pancreas. 66 in humans it has been reported that 90% of pancreatic acinar cells must be lost before clinical signs of epi are seen. 19 epi is considered rare in cats but is perhaps being recognized more frequently because of increased awareness. there are less than fifty cases described in the veterinary literature* with one of these papers describing only 16 cases from five institutions, with prevalence described as 0.01% to 0.1% of cats seen over a 15-year period. 74 in contrast to this, the gastrointestinal laboratory at texas a&m university recognized 1342 samples with serum ftli concentrations at or less than 8.0 µg/l, which is diagnostic for epi, out of 84,523 submissions, 66 which equates to 1.6% of cats with known or suspected gastrointestinal disease. all studies indicate a wide age range of cats can be affected, from kittens less than 6 months of age to cats more than 15 years old, with a median age of approximately 7 years. there is no apparent breed predisposition. 66, 68, 74 one paper recognized 10 of 16 (62.5%) cats to be male, 74 and another recognized 15 of 20 (75%) male cats, 68 suggesting a possible sex predisposition. chronic pancreatitis is believed to be the most common cause of epi in cats, 66 acinar atrophy (paa) is recognized as the most common cause of epi in dogs, and has been definitively described in two feline cases 74 and mentioned as a cause for three other cases. 85 other potential causes of epi include disruption of pancreatic enzyme flow at the duodenal papilla following duodenal resection 72 and pancreatic fluke infection (eurytrema procyonis), 2,26 and amyloid deposition and neoplasia are other possible causes of pancreatic cell damage that have not definitively been described in cats. 66 congenital pancreatic hypoplasia or aplasia has not definitively been reported in cats, but reports of epi in cats as young as 3 months of age 63, 74 suggest this possibility. since chronic pancreatitis is a common cause of epi and chronic pancreatitis has a strong association with ibd, many cats may have concurrent lymphocytic pancreatitis and enteritis. 66, 74, 85 therefore cats failing to respond to therapy for epi may require further diagnostics and management of an underlying condition. further, destruction of functional exocrine pancreatic tissue can also affect pancreatic endocrine tissue, resulting in concurrent diabetes mellitus. 35 several studies have indicated that all cats with epi will have weight loss when diagnosed, unless a kitten, in which case ill-thrift is recognized. 68, 74 diarrhea is not necessarily present, being described in 50% to 75% of cats; the nature of feces can vary from voluminous, malodorous stools that can be discolored (yellow or pale), sometimes with steatorrhea, to normal feces in other cats. increased frequency of defecation and the presence of mucus in the feces of some cats can lead to the diarrhea being characterized as large bowel. only about 20% to 30% of cats are polyphagic, some described as having a ravenous appetite; conversely, some cats present with anorexia. vomiting has also been described. since cats with epi often have concurrent disorders, such as ibd, the clinical signs recognized may reflect the concurrent disease and not necessarily epi alone. physical examination findings are similarly nonspecific, with thin/ emaciated body condition being the most common finding. hematologic findings are non-specific, but a mild nonregenerative, normocytic, normochromic anemia may be recognized as well as lymphopenia or neutrophilia. plasma biochemistry results may show a mild to moderate increase in alanine aminotransferase (alt) and a mild increase in alkaline phosphatase in some cats. mild to moderate hyperglycemia may be seen, as may mild hypoglycemia or normoglycemia. 66, 68, 74 hypocobalaminemia is recognized in nearly all cats with epi. 66, 68, 74, 85, 87 this may be because of insufficient production of intrinsic factor, a cobalamin-binding protein only produced by the pancreas in cats and necessary for ileal absorption of cobalamin 27 ; it may also be because of failure of pancreatic enzymes to liberate cobalamin from binding by r protein in the duodenum or small intestinal bacterial overgrowth (sibo), not yet specifically described in cats. 74 folate concentrations may be reduced (because of concurrent intestinal malab sorption), 68 normal, 68, 74 or increased, 74 which may relate to reduced pancreatic bicarbonate secretion, secondary to severe hypocobalaminemia, 59 or associated with sibo. 7 none of these presenting complaints, physical examination findings, or routine testing results are specific to epi. therefore epi requires a degree of clinical suspicion and/or thorough diagnostics to ensure the diagnosis is not missed. a low level of serum ftli is diagnostic for epi. 66, 68, 74 samples can be sent to the gastrointestinal laboratory at texas a&m university from anywhere worldwide (with instructions about sample handling requirements on their website: http://vetmed.tamu.edu/gilab/). the reference range for serum ftli is 12 to 82 µg/l, with concentrations at or less than 8.0 µg/l diagnostic for epi. since the clinical signs and routine laboratory findings are nonspecific for epi, it is ideal to test serum for ftli in any cat with weight loss or ill-thrift. the texas a & m gastrointestinal panel also includes testing for levels of cobalamin, folate, and fpli, ensuring concurrent hypocobalaminemia will not be missed and potentially providing indications of other gastrointestinal disease. conversely, although a low level of serum ftli confirms a diagnosis of epi, it is not necessarily a diagnostic end point, since epi is so often recognized concurrently with other gastrointestinal disease. failure to respond to therapy should prompt the clinician to consider and investigate further for concurrent processes. most cats with epi can be successfully managed with dietary supplementation of pancreatic enzymes. commercial products (e.g., viokase [axcan pharma, birmingham, ala.], pancrezyme [virbac, fort worth, tex.], and creon [abbott laboratories, abbott park, ill.]) are available, and powder is considered more effective than tablets or capsules (some capsules can be opened and the contents sprinkled onto food, like powder). the required dose can vary quite substantially from cat to cat. it is appropriate to start with one teaspoon of powder with food twice daily, and adjustments can be made depending on the response; most cats accept the powder readily if it is mixed thoroughly through canned food, but other flavors (e.g., fish oil or brine from canned tuna) can be used to disguise the taste if necessary. raw pancreas (e.g., from beef or pork) may also be used, with 30 to 60 g twice daily an appropriate starting dose. 66 since most cats with epi are hypocobalaminemic, supplementation by subcutaneous injection is required (oral supplementation is not effective since cobalamin deficiency leads to cobalamin malabsorption). an appropriate dose for most cats is 250 µg, and it is usually given weekly for 6 weeks, then every second week for a further six doses; it is appropriate to continue dosing every month beyond that. owners can be taught to inject their cats at home (as owners of diabetic animals are taught to do with insulin). 66 because some cats may have sibo, antibiotics such as metronidazole (15 to 25 mg/kg po every 12 hours for 14 days) may be warranted. an elevation of folate may arouse suspicion of sibo, but it is appropriate to try antibiotics in a cat failing to respond to enzyme and cobalamin supplementation. concurrent diseases, such as lymphocytic, chronic pancreatitis, or ibd may need to be managed with corticosteroids, or diabetes mellitus with insulin. no studies have assessed specific dietary requirements in cats with epi. most cats respond to appropriate treatment, with a return to normal weight and normal feces. with ongoing therapy, cats can lead normal lives for a full life span. 83 the feline liver is a large, complex organ involved in a variety of essential metabolic, functional, and detoxification processes that can be affected, individually or collectively, by disease or dysfunction. cats have a unique set of liver diseases that occur more commonly in this species compared with the typical diseases that occur in dogs, and these include hepatic lipidosis, feline cholangitis syndrome, and infectious hepatopathies (e.g., fip, flukes, histoplasmosis, toxoplasmosis). 2, 15, 34, 58, 61 nevertheless, these conditions often present with characteristic clinical, laboratory, and histopathologic changes that are necessary for proper diagnosis and management. the goal of this section is to review the interpretation of clinical and laboratory changes that occur in these feline liver diseases, provide an approach for separating the more common diseases by their clinical footprint, and then discuss therapy of each liver disease based on our current level of understanding of hepatoprotectants, antioxidants, and drugs used for specific therapeutic purposes. the clinical signs of liver disease in cats are often vague and nonspecific; however, recognition of certain clinical and laboratory abnormalities and their association with liver disease can greatly aid the diagnostic process. the most common early clinical signs observed in cats with liver disease are anorexia, lethargy, and weight loss, which are signs present in many (if not most!) feline diseases. 2, 15 because these early indicators of disease do not point specifically toward liver disease, a delay in diagnosis will occur unless the clinician carefully considers all possibilities and performs other tests to further evaluate the situation. for example, feline hepatic lipidosis is the most common form of liver disease in cats in the united states, united kingdom, japan, and western europe, occurring with a prevalence of nearly 16% in one study. 2 however, the most common, and often only, clinical sign associated with onset of this condition is anorexia; the signs of serious hepatic disease (especially jaundice and vomiting) do not occur until later (days or weeks) in the course of the disease. 2, 21 recognition that anorexia in a cat, even for a few days, is a risk factor for development of hepatic lipidosis is essential, and this risk is increased in obese cats. 11, 21 further, the clinical signs of liver failure develop much more slowly; many cats with hepatic lipidosis present alert and responsive until much later in the course of the disease, thus delaying onset of appropriate therapy. a similar clinical situation exists for the second most common form of liver disease in cats, feline cholangitis syndrome. 15, 28, 58 this complex of diseases in the cat can be associated with signs ranging from anorexia and lethargy to vomiting and jaundice, and these signs can vary in severity and prevalence. the key point is that except for development of jaundice, there is no constellation of clinical signs that are classic clinical indicators of liver disease in cats. 15, 28 as with many feline diseases, the subtle clinical signs of anorexia, lethargy, or inactivity are often the only signs of illness and should be further investigated. there are few changes that occur in the complete blood count that are specific indicators of primary liver disease in cats. the most common finding is the presence of poikilocytes, which are red blood cells with an irregular shape, speculated to be caused by changes in membrane lipids as a result of liver dysfunction. 14 other abnormalities may occur, such as anemia of chronic disease or neutrophilia, but these findings are nonspecific and occur with variable frequency. perhaps the most important reason for obtaining a hemogram is in icteric cats, because this test is essential to help rule out hemolysis as the cause of the hyperbilirubinemia. the serum chemistry profile can be very helpful, but there are several critical points in interpretation of these values that are important to review. the hepatic transaminases (alanine aminotransferase [alt] and aspartate aminotransferase [ast]) are leakage enzymes but do not discriminate among hepatobiliary disorders, nor do they provide an indicator of severity or disease origin. thus although increases in alt may be noted in cats with liver disease, they are also present in a variety of other systemic infectious, inflammatory, neoplastic, and and protein-losing nephropathies can also cause loss of albumin and affect cholesterol, it is essential to evaluate the cat for these problems when interpreting these results. finally, bilirubin metabolism is a critical function of the liver, but interpretation of hyperbilirubinemia requires a careful consideration of bilirubin disposition. hyperbilirubinemia develps because of one of three possible causes: (1) excessive hemolysis of red blood cells (rbc) (also known as prehepatic icterus)-high bilirubin in the blood stream occurs because of an overload of the mononuclear/phagocyte system with heme pigments from rbc destruction, (2) hepatic parenchymal disease or insufficiency (also known as hepatic icterus)-resulting in lack of normal bilirubin metabolism in hepatocytes and regurgitation of the pigments into the blood stream when they are not taken up into cells and excreted in bile, and (3) disease of gall bladder, biliary tract, or pancreatic duct (also known as posthepatic icterus)resulting in obstruction of the bile ducts or loss of bile into the abdomen (duct or gall bladder rupture and bile peritonitis). 41 the bottom line is that in any cat with hyperbilirubinemia, an assessment of the packed cell volume and rbc morphology should be completed to determine whether icterus is caused by hemolysis. once hemolysis is ruled out, then assessment of primary endocrine diseases, including hyperthyroidism, feline heartworm disease, fip, and neoplasia.* alternatively, the cholestatic membrane-associated enzymes alkaline phosphatase (alp) and gamma glutamyltransferase (ggt) are especially useful for recognizing disorders involving biliary or pancreatic ductal components. unlike the dog, these enzymes will only increase modestly in cats, even in severe disease, and there is no glucocorticosteroid or drug induction of the enzymes to influence interpretation. 14, 37 thus increases in alp in the adult cat represent a release of enzyme from the hepatobiliary tree and should be considered clinically important. both alp and ggt are produced in other tissues than the liver, with the highest ggt activity present in the kidney and pancreas; however, sources other than the liver do not contribute to the activity of these enzymes in health. recent studies of the effects on these enzymes in cats with pancreatitis, cholangitis, extrahepatic bile duct obstruction (ehbdo), and hepatic lipidosis reveal some important characteristics in interpreting increases in these enzymes. 14 first, both alp and ggt are increased in cats with pancreatitis, cholangitis, or ehbdo, because inflammation in the biliary tree also affects the pancreatic ducts (and vice versa, figure 23-55) , and if the fold increases in these enzymes are similar, the diagnosis is likely one of the three. 15 conversely, in cats with hepatic lipidosis (without concurrent inflammatory disease of the biliary or pancreatic duct system), large increases in alp are observed, but ggt will remain normal or only slightly increased. thus if the increase in alp is 5 to 10 times, while ggt is not increased or is only increased 1 to 2 times, then the likely diagnosis is hepatic lipidosis. [14] [15] [16] other than enzymes on the biochemistry panel, which are of limited value for assessing liver function, there are several key tests that can be used to help assess liver function cats with elevated liver enzymes. these five tests found on most routine biochemistry panels are helpful functional indicators: cholesterol, bilirubin, glucose, albumin, and urea nitrogen (bun). however, none are immune to outside influences on their interpretation, including bilirubin and cholesterol, which are the most liver specific. in cats with severe liver disease or failure, bilirubin levels tend to be quite elevated, while bun, albumin, cholesterol, and glucose concentrations tend to be significantly decreased, reflecting inability to metabolize urea (lack of arginine), inability to produce albumin or cholesterol, and abnormal metabolization of glucose. however, these changes represent severe loss of liver function and thus are not sensitive indicators of liver function because the changes occur quite late in the course of the disease. nevertheless, in cats with elevated liver enzymes and clinical signs of liver disease, these values should be carefully assessed. because gi disease of hepatic failure. in nonicteric cats with severe liver disease or in young cats suspected of having a portosystemic shunt, serum bile acids are the more reliable indicator of hepatic insufficiency. 18 the measurement of serum bile acid concentrations, preprandially and postprandially, is the most reliable, readily available, and sensitive test of hepatic function in nonicteric cats. 5, 14 that being said, although increases in bile acids are accurate indicators of hepatic insufficiency, the levels cannot be used to assess severity of disease or the type of dysfunction. further, bile acid assays are most effective when paired samples (preprandial-and postprandial) are compared, because single, fasting, or random bile acid samples can result in a falsenegative (normal) result. however, cats will often not eat in the hospital or when they are sick, and this prevents collection of a postprandial sample. however, this does not invalidate the results, because if the result of the single bile acid sample is abnormal, it does reliably indicate liver dysfunction. an alternative to using serum for testing bile acids in cats is urine bile acid analysis. healthy cats excrete a small percentage of conjugated bile acids in the urine 14 ; however, in cats with liver disorders that cause increased serum bile acids (and especially cholestatic liver diseases) a significant increase in urine bile acid excretion occurs. when urine bile acids (uba) were collected 4 to 8 hours after a meal and measured (normalizing the value with urine creatinine: uba/ucr) and compared with serum bile acids in a study of 54 cats with hepatic disease, 17 cats with nonhepatic disease, and 8 normal cats, the results were highly correlated. 47 the utility of the urine bile acid test is that it does not require a paired sample (postprandial test), and it is not as affected as the serum test is by hemolysis or lipemia of the blood sample. normal cats will have an uba/ucr of less than 4.4 µmol/mg, while values greater than 4.4 are considered evidence of significant hepatic dysfunction. 47 it is well known that the liver plays a central role in coagulation homeostasis and is the single site of synthesis of many coagulation proteins, anticoagulant proteins, and fibrinolytic factors. vitamin k is one of the most common factors found to be inactive or deficient in cats with liver dysfunction, and it is essential for normal functioning of factors ii, vii, ix, and x; protein c and s; and thrombin. insufficient or inactive vitamin k can occur for a variety of reasons, including dietary restriction (e.g., anorexia or diet deficiency), disruption of the enteric microflora that synthesize vitamin k (e.g., chronic antibiotic therapy), diseases causing fat malabsorption (e.g., ibd, exocrine pancreatic insufficiency), ingestion of vitamin k antagonists, or liver dysfunction. 20 for example, in cats with hepatic lipidosis, approximately 25% will have an increased prothrombin time (pt), 35% will have an increased partial thromboplastin time (ptt), but 60% of cats will have increased pivka parenchymal disease versus disease of the biliary tree is completed by evaluating the clinical presentation, laboratory values, and imaging of the biliary tree and abdomen for possible evidence of biliary or pancreatic disease. a urinalysis is also an important part of the minimum database, and it is no different in a sick cat with suspected liver disease. in cats the presence of hyperbilirubinuria is abnormal at any urine concentration, because they do not conjugate bilirubin in their renal tubules. 14 however, like bilirubinemia, presence of bilirubin in the urine can occur because of any of the three possible causes of hyperbilirubinemia: prehepatic, hepatic, and posthepatic; thus further evaluation is necessary once bilirubin is detected. ammonium biurate crystalluria suggests the presence of hyperammonemia, which in the cat is either because of a congenital portosystemic shunt (less common in cats than in dogs) or because of severe, end-stage liver disease resulting in portal hypertension, which is typically caused by cirrhosis or advanced polycystic liver disease. 6, 41 the most common feline liver diseases are hepatic lipidosis and feline cholangitis syndrome, which are two diseases that often result in development of clinical or biochemical icterus. thus because hyperbilirubinemia is a more sensitive indicator of liver function than bile acids or other liver function tests, the need for further testing is moot. however, there will be circumstances when further assessment of liver function is indicated, and for this, serum bile acids, blood ammonia levels, and urine bile acids may be needed. there are several situations where liver function testing may be indicated, but the most common indications for additional testing would be a cat with persistently elevated liver enzymes of unknown origin, a cat that develops urethral obstruction because of urate stones (suggestive of portosystemic shunting) or a cat with possible polycystic liver disease. 5 one of the oldest tests of liver function, because of its association with development of hepatoencephalopathy, is measurement of blood ammonia levels. 38 however, although this test is the only practical way to diagnose hepatoencephalopathy in dogs, the test has a number of limitations, including differences in ammonia levels between arterial and venous (lower) samples and significant sample handling issues (ammonia is labile and results are affected by improper sample handling or lack of immediate measurement) that make its use difficult in practice. 43 in cats hyperammonemia is even less common than in dogs likely because of their highfunctioning urea cycle pathways 14 ; the assays have not been validated for feline blood in most laboratories, and as such, the test is not recommended as the sole indicator uncommon in cats, the most common causes are neoplasia (primarily of the pancreas, but cholangiocarcinomas can occur) or chronic pancreatitis, which can occur concurrently with cholangitis in cats, resulting in both intrahepatic and extrahepatic cholestasis in some cats. 23, 41 the bile ducts are affected in cats with chronic pancreatitis, because the feline biliary system and pancreatic duct system merge at the level of the pancreas to form a single duct that empties into the duodenum. thus in cats with either pancreatitis or biliary disease, recent evidence has shown that the inflammation affects both organs. 54, 59 further, in chronic pancreatitis, either persistent inflammation or development of fibrosis can result in dilation or obstruction of the common bile duct. 33 in cats with chronic ehbdo, the common bile duct will become widely dilated and tortuous, a finding easily seen on abdominal ultrasonography but a problem not easily managed (figures 23-56 and 23-57) . interestingly, the gallbladder is often not enlarged, and may in fact be small in cats with this condition, because the remaining fluid in the gallbladder is white bile (highly concentrated mucinous bile from which the pigment has been resorbed). 41 in addition, variable filling of the gall bladder is a normal phenomenon; thus gallbladder size is not an indicator of ehbdo. (proteins induced by vitamin k antagonists or absence). 20 nevertheless, although pivka is a very sensitive test for abnormalities of vitamin k function, most cats with liver disease that have a normal pt/ptt, but abnormal pivka do not represent clinical evidence of bleeding. in any case, abnormalities in the clotting cascade related to vitamin k deficiency in cats with liver disease are common, whether or not they show evidence of active bleeding. and because the balance of the coagulation system in a cat with liver disease can be disrupted by a procedure that initiates small amounts of bleeding (e.g., a biopsy), all cats with liver disease should be given vitamin k as a precautionary measure before and after invasive procedures, even if the clotting times (pt and ptt) are normal. this may be especially important in cats with hepatic lipidosis, because their vitamin k clotting status is likely to be even more affected by the concurrent anorexia and disruption of enteric microflora. 14 the dose of vitamin k 1 (phytonadione, aquame-phyton [merck, west point, pa.]) used prophylactically is 2.5 mg sc, im, or po q12h for 3 to 5 days, then weekly until recovered. see box 23-4 for a summary of the causes of icterus. cholestasis is the reduction of bile flow, which can occur at any point along the biliary tree; bile production occurs in hepatocytes, and flow is connected to the distal concentrating components (gallbladder and common bile duct) by the bile ductules. thus cholestasis can occur inside the liver's biliary tree (intrahepatic cholestasis) or outside the liver in the gallbladder and common bile duct (extrahepatic cholestasis). intrahepatic cholestasis most often occurs in diseases involving hepatocellular damage, leakage, or swelling, such as infections (e.g., bacterial cholangiohepatitis, toxoplasmosis, fip, or other diseases causing inflammation), infiltrative diseases (e.g., lymphoma), metabolic diseases (e.g., hepatic lipidosis), or diseases causing disruption of architecture (e.g., cirrhosis or severe polycystic disease). 41 intrahepatic cholestasis occurs in zone 1 of the liver lobules (periportal zone); at the level of hepatocytes, canaliculi or bile ductules; and is damaging to cells because of the emulsifying properties of lipid on membrane lipids. however, because the liver has a large reserve capacity, clinical icterus (e.g., jaundice) only occurs in the most severe cases when the liver is affected diffusely. thus severe or persistent intrahepatic cholestasis can serve to perpetuate the inflammation and cell damage if it is not corrected. extrahepatic cholestasis or extrahepatic bile duct obstruction (ehbdo) is less common than intrahepatic cholestasis and is most commonly associated with obstruction of the common bile duct. since gallstones are icterus is the result of cholestasis, and the underlying cause can be either hemolysis or hepatobiliary disease, for which further clinical examination will be needed to determine if rbc destruction or liver disease is occurring. in most hepatobiliary diseases of cats, cholestasis is occurring, but there may be no clinically apparent icterus because the degree of hyperbilirubinemia must be at least 2 to 3 times greater than the normal values to exceed the capacity of the liver to process the excess bilirubin. in cats with hyperbilirubinemia not caused by hemolysis, whether it is clinical or subclinical, there is no need for further evaluation of liver function (e.g., bile acid assays), because bilirubin is a more sensitive indicator of liver function than bile acids. the degree of hyperbilirubinemia does not suggest differentiation of intrahepatic versus extrahepatic cholestasis; however, the presence of acholic feces (white feces) is diagnostic for extrahepatic bile duct obstruction (ehbdo), because lack of stercobilinogen (the brown/black pigment in feces) is only found in cats with complete obstruction of the bile duct. finally, the presence of intrahepatic cholestasis and clinical icterus in a cat indicates a diffuse hepatobiliary disease, such as cholangitis or hepatic lipidosis, as focal liver disease, even if severe, will not cause clinical hyperbilirubinemia because of the tremendous reserve capacity of the liver for bilirubin uptake. portal hypertension is an abnormally high venous pressure in the portal system and is typically caused by increased resistance to portal blood flow. there are potentially three regional causes of portal hypertension: prehepatic (disease in the portal vein itself), hepatic (intrahepatic diseases causing compression or decreased flow), and posthepatic (diseases of the caudal vena cava, right heart or pulmonary vasculature). the most common cause of portal hypertension in the cat is cirrhosis or portal venous thrombosis, because portal vein hypoplasia (formerly known as microvascular dysplasia) is known to occur only in the dog, and the other causes of portal hypertension (budd-chiari syndrome, heartworm caval syndrome, pulmonary hypertension) are rare and more likely to occur in the dog. 41, 44 in any case, the clinically recognizable effects of portal hypertension are development of ascites (unusual in the cat), acquired portosystemic shunting (reported in cats), and development of hepatic encephalopathy (less common in cats than in dogs, because of their profound ability to handle protein wastes). 5, 36, 41 most cats and dogs that develop hepatic encephalopathy (he) secondarily to portal hypertension do so because of reduced liver function (because of portosystemic vascular shunting [pss] or cirrhosis and the acquired shunting that develops). cats can develop another form of chronic he because of hepatic lipidosis, but this is believed to be because of the combination of liver failure and prolonged fasting, resulting in arginine deficiency and impaired ammonia detoxification. 2 portosystemic vascular anomalies, also called portosystemic shunts or portovenous shunts (pss), although less common than in dogs, also occur in cats. these vascular anomalies can be either congenital or acquired, single or multiple in number, and occur as extrahepatic vascular shunts or within the liver itself (intrahepatic shunts). 5 the shunting of blood around the liver is the cause of hepatic atrophy and reduced hepatic function that results in an accumulation of toxins, particularly ammonia that leads to the development of hepatoencephalopathy. the two most common veins that serve as the connection point for the shunting portal venous blood are the caudal vena cava and the azygous. 5 in cats a single, extrahepatic, portocaval shunt is the most commonly reported form, and occurs in 75% of cats with pss. 5 as in dogs, specific breeds of cats may have pss more commonly, and these include domestic shorthair cats, burmese, siamese, persian, and himalayan breeds. 5 in contrast to dogs, males may be more predisposed to pss than females, but the clinical signs relate to the three body systems most affected: the central nervous system, gi tract, and urinary tract. the most common presenting complaints in cats are weight loss or poor/stunted growth, and dull, bizarre or lethargic behavior, especially after eating. signs of gi disease common in dogs, such as vomiting, diarrhea, or inappetence, are less common in cats, but in one report, 75% of cats with pss drooled. 5 finally, cats with pss often present with signs of lower urinary tract disease (e.g., hematuria, stranguria, or even obstruction) because of the development of urate uroliths (which are radiolucent, thus difficult to detect). 5 because the most common signs of he are apathy, listlessness, and decreased mental alertness, they are often not recognized specifically as indicative of brain dysfunction but as part of the constellation of signs of the liver disease. however, with progression of the has not been reported. the clinical presentation is typically nonspecific (the most common signs are vomiting, lethargy, and anorexia), and there are no laboratory changes that are suggestive of hepatic neoplasia. thus the diagnosis must be made by identification of disease, other signs will develop, including ataxia, salivation, stupor, or coma. the best and only practical diagnostic test for he is plasma measurement of ammonia levels. 43 however, as previously noted, the test has many technical issues that make its clinical utility in the practice setting difficult at best, and there are few laboratories that have validated ammonia measurement in the cat. cancer of the liver can occur as a primary disease (table 2322) or as a result of metastasis of neoplastic disease occurring elsewhere and, most typically, the abdominal cavity. the most common neoplastic infiltration of the liver that is not a primary liver tumor is lymphoma (figures 23-58 and 23-59), followed by visceral mastocytosis. 4 as with many other types of cancer, hepatobiliary neoplasia is most common in middle-aged to older cats, and it is relatively rare, with a reported incidence of 1.5% to 2.3%. 4 benign tumors, such as biliary cystadenoma ( figure 23-60) , carry a good prognosis if they are amenable to surgical resection. the incidence of metastatic neoplasia (including lymphoma and mast cell tumors) the most common clinical signs are related to spontaneous rupture of the enlarged and friable liver. affected cats may present with lethargy, anorexia, pale mucous membranes, and a heart murmur secondary to anemia. clinical signs of liver disease are usually absent. hepatomegaly and hypotension may also be found. results of routine laboratory testing (mild to marked increases in alt and globulins while alp and ggt are typically normal) and ultrasonographic examination (hepatomegaly, generalized increase in hepatic parenchymal echogenicity) 4a of the liver may be supportive, but definitive diagnosis relies on histopathologic examination of a liver biopsy. fna of the liver is not helpful because amyloid is rarely detected with this method. hemostasis should be evaluated carefully before any biopsy procedure is planned. the most important differential diagnoses are fip, hepatic lipidosis, and hepatic lymphoma. scintigraphic imaging using i-123 serum amyloid p component has potential as a noninvasive test. 39a there is no specific treatment for amyloidosis in cats, so therapy is primarily supportive care (antioxidants, vitamin k, blood transfusion). attention should be paid to identification and control of any underlying chronic inflammatory disease. unfortunately, the long-term prognosis is poor as most affected cats die of intra-abdominal bleeding. survey abdominal radiography is the simplest and most readily available imaging modality to assess structures in the abdominal cavity. radiographs are most useful to assess liver size, will reveal large hepatic masses, and provide evidence of radiopaque masses or other abnormalities in the abdomen. however, the preferred imaging modality used to assess hepatic structures in cats with suspected liver disease is abdominal ultrasonography (aus). the reasons why ultrasonography is a more useful tool for assessment of the liver in cats are numerous, but because feline liver diseases are primarily diffuse, infiltrative, or metabolic diseases that also affect the biliary tree, ultrasonography is the only imaging tool that will give reliable diagnostic information. this widely available diagnostic tool can be helpful in determining liver size and parenchymal echogenicity, in identifying mass lesions, evaluating the biliary tree and gallbladder, quantifying flow (doppler techniques), and identifying vascular anomalies. 29 as with all diagnostic modalities, the skill and experience of the operator is vital to accurate procurement and interpretation of the images. further, it is important to remember that although ultrasonographic images are extremely useful in the clinical evaluation of a cat with possible liver disease, the images themselves do not represent a histologic diagnosis. structural abnormalities by hepatobiliary imaging and subsequent examination of the tissue either by fna or biopsy techniques. historically, amyloidosis has been recognized as primarily a renal disease, especially in abyssinian cats. more recently, cases of hepatic amyloidosis without renal involvement have been diagnosed in siamese and related breeds, as well as in nonpedigreed cats. 4a,10a,30a the majority of cases have been described in australia, the united kingdom, and europe. amyloid a is deposited in the liver, probably in response to chronic inflammation in another organ. in the siamese breed, a genetic component may contribute. 48a the amyloid a protein occurring in the siamese breed differs from that known in the abyssinian breed. 48a contraindicated in cats, because they may cause a lethal shock reaction. 40 a similar reaction may be seen with penetration of the larger bile ducts or gallbladder with a large-bore biopsy needle, because these tissues have a significant autonomic innervation in the cat that may result in bradycardia and shock following the procedure. 40, 48, 54 it is particularly important to recognize this as a risk in cats with ehdbo or dilated bile ducts, and this risk factor reiterates the need for ultrasound examination of the liver prior to making biopsy decisions. nonetheless, owners should be informed of these potential risks, in addition to the risk of bleeding from biopsy sites in any cat undergoing liver sampling. 7, 48 biopsy techniques liver biopsies, whether they are obtained by needle, laparoscopy, or surgical means, should be taken from a location that represents the primary liver pathology, handled appropriately to ensure accurate interpretation of the sample, and the histopathologic description should be interpreted according to the guidelines set by the wsava standards for clinical and histologic diagnosis of canine and feline liver disease. 42, 61 guidelines for obtaining and handling surgical biopsies of the liver are reviewed elsewhere 27 and will not be further discussed. because needle aspirates/biopsies, tru-cuttype biopsies, and laparoscopic biopsies are commonly used to obtain liver tissue in cats, the benefits and limitations of each of these techniques will be discussed. as a general rule, the more tissue that can be obtained, the better the pathologist's interpretation of the tissue abnormalities will be. for example, most pathologists believe that at least six portal areas are necessary to make a diagnosis of inflammation liver disease in cats. 42 this will require either a 16-or 18-gauge needle size or larger piece of tissue than is obtained with smaller needles or an aspirate. the amount of tissues required to view at least six portal areas is approximately 15 mg, and 5 mg will be required for culture of the tissue. 42 if other analyses of the tissues are considered (e.g., metal analysis), approximately 20 to 40 mg of liver is needed. 42 a typical laparoscopic cup biopsy forceps will provide 45 mg of liver tissue, a 14-g tru-cut-type biopsy needle provides 15 to 20 mg, and an 18-g needle biopsy provides only 3 to 5 mg of liver tissue. 42 thus, depending on the clinical circumstances and considered differentials, the best approach for obtaining the needed tissue must be considered prior to planning the procedure. fine-needle aspiration to obtain liver tissue for cytologic examination is commonly performed in cats with liver disease for good reason. the procedure is inexpensive, easy to do, is relatively low risk, and often requires only sedation to complete. 57 further, samples obtained by this method can be diagnostic for hepatic lipidosis, hepatic lymphoma or other round cell tumors, and in for the most common liver diseases of cats (hepatic lipidosis, feline cholangitis syndrome, and neoplasia/ lymphoma), aus examination provides a useful means of obtaining clinical clues and tissue to support or refute the differentials. for example, in cats with hepatic lipidosis, the liver is quite enlarged and typically diffusely hyperechoic, while in cholangitis or other inflammatory diseases, the liver is more often diffusely hypochoic. 29 however, these sonographic findings are very nonspecific and can easily lead to errors in diagnosis if the tissue is not subsequently sampled for confirmation. 24, 35 thus one of the most important utilities of the aus is the ability to obtain liver tissue (either by aspiration or guided-needle biopsy) and for aspiration of the gallbladder to obtain bile for culture. 24, 49 these techniques alone have made the aus an extremely important diagnostic tool in the evaluation of liver disease in cats. the diagnosis of most liver diseases requires a histopathologic sample of liver tissue, and this is particularly true in the most common feline liver diseases, which tend to be diffuse diseases affecting the entire liver. cats with one of these diffuse diseases can be sampled randomly using any one of these commonly employed techniques: ultrasound-guided fine-needle aspirates (fna), ultrasound-guided needle biopsy, laparoscopic biopsies, or biopsies obtained surgically. some types of neoplasia (particularly round cell tumors) and vacuolar hepatopathies (hepatic lipidosis) can often be diagnosed by cytology using fna techniques. however, differentiation of liver cell tumors (adenomas and carcinomas) and inflammatory diseases of the liver cannot be diagnosed without a larger sample of tissue and histopathologic examination. 30, 50 further, even in cats with classic hepatic lipidosis changes, concurrent diseases such as cholangitis or lymphoma can be missed if only fna techniques are employed. 60 thus it is essential to consider that in many liver diseases the lesions, although typically diffuse, may also have focal components; for example, inflammation may be throughout the liver, but fibrosis will be present only in focal areas. thus the results of fna or tru-cut needle biopsies should always be considered in the light of the clinical, laboratory, and ultrasonographic evidence. prior to scheduling a cat for a biopsy, the risk-tobenefit ratio of performing a liver biopsy should always be considered. this is primarily because heavy sedation or anesthesia will be essential in most cats undergoing a liver fna, and for all cats undergoing a liver biopsy (needle or otherwise). in addition to anesthesia risks, the use of automatic spring-loaded biopsy guns to obtain ultrasound-guided biopsies of liver tissue is equipment, the interested reader is referred to several recent reviews on the subject. 48, 54 to maximize the histopathologic accuracy, biopsies taken at laparoscopy or surgically should be taken from both normal-appearing and abnormal areas in the liver. further, if there is a need to obtain samples from the deeper tissues, the laparoscope can be used to direct a tru-cut needle biopsy to the best location for sampling. one of the major advantages of the laparoscopic technique is that it allows the operator to observe the biopsy sites for excessive bleeding, which is unusual, but if observed can be staunched by using pressure on the site, gelatin coagulation material placement, or electrocautery. with experienced operators, the complication rate for laparoscopy is very low (less than 2%), and most complications were because of anesthesia, bleeding, or air embolism. 48 finally, although not necessary to have direct visualization to obtain an aspirate of gallbladder bile, laparoscopy allows easy sampling of bile for culture, which is important in all cats with suspected inflammatory liver disease or hepatobiliary disease. once a diagnosis of liver disease is made in the cat, specific therapy for the cause (if available) should be instituted; however, for many feline liver diseases, no specific therapy is available, and thus hepatoprotective therapy is used concurrently to aid in the recovery of the liver from the insult. in this section, therapy of two of the most common diseases of the feline liver will be considered, with a special emphasis on nutritional aspects of treatment, nutraceutical therapy, and the unique needs of cats. the most common liver disease of cats is idiopathic hepatic lipidosis (figures 23-61 and 23-62) , a disease that results in liver failure because of a combination of factors including hepatic lipid accumulation, insulin resistance, fasting, and protein (especially arginine) deficiency. 2, 8, 9, 11 thus, unlike many diseases of the liver, the primary focus of therapy and the essential component for recovery is nutritional support. as in any patient with serious liver disease, initial therapy is always aimed at correction of any fluid or electrolyte abnormalities that may exist, because these may be profound if the cat has been vomiting. in addition, normalization of electrolytes is particularly important in cats that have been anorexic for an especially long time (1 to 2 weeks), because refeeding syndrome may be triggered with the initiation of feeding, resulting in sudden drops in potassium, phosphate, and magnesium. 1 although this phenomenon is less common and usually less profound in cats fed enterally versus areas where appropriate, definitive diagnosis of certain infectious diseases (e.g., histoplasmosis). 57 however, even with these relatively straightforward diseases, fna of liver tissue has significant limitations, the most important of which is the failure to accurately identify the primary disease. for example, although it is easy to make a diagnosis of hepatic lipidosis using this technique, a paper recently showed four cats that were incorrectly diagnosed with hepatic lipidosis instead of lymphoma because the fna samples were obtained from areas that did not have lymphoma infiltration. 60 in another study, reviewing the agreement between fna cytologic samples of liver and the histopathologic diagnosis, only 51% of the cases had overall agreement. 50 thus although cytology of fna samples of liver tissue in cats with diffuse hepatic disease remains a useful first step, it is important for the clinician to carefully interpret the results and discuss the potential limitations of this technique with owners. there are several needle biopsy techniques available for sampling liver tissue, but not all are suitable or safe for use in cats. the menghini technique is one such approach that is not suitable for use in cats, because it is a blind procedure using a large-bore needle that cannot be used with ultrasound guidance. 42 the second option among the needle biopsy techniques that is not recommended for cats is the biopsy gun device. tru-cut biopsy guns are operated by a triggering device that can result in the induction of a lethal vagotonic shock reaction in the cat immediately following the procedure. 40 for most ultrasound-guided liver biopsy procedures, either the manual or, preferably, the semiautomatic tru-cut device is recommended for use in obtaining needle biopsies from cats. as a general rule, the tru-cut device will advance into the liver to a depth of 2 cm; so, it is essential to carefully note the amount of liver tissue available during the ultrasound assessment before advancing the needle for tissue collection. properly obtained tru-cut needle biopsies are a valuable technique for obtaining a representative sample of liver tissue 61 ; however, because of the risk for bleeding or liver fracture with any movement, it is essential that cats be anesthetized for this procedure. laparoscopy is an intermediate step between needle biopsy and surgical laparotomy for obtaining liver tissue for histopathology in cats. 48, 54 this technique is becoming more widely used as more specialists are trained for this procedure that allows visualization of tissues to be biopsied without opening the entire abdomen. although this technique does require general anesthesia, the limited degree of invasiveness, the large biopsy sample size, and rapid patient recovery make laparoscopy a valuable tool for obtaining liver tissue, 48 and it can be used to obtain biopsies from the spleen, pancreas, kidneys, lymph nodes, or to aspirate the gallbladder. for a detailed discussion of laparoscopic techniques and lipidosis. the echogenicity of the parenchyma is uniformly increased, which is more apparent when compared with other ultrasonographic images presented in this chapter. additionally, the gall bladder is distended. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease. (courtesy dr. randolph baral.) liver gb figure 23-62 gross appearance of liver from a cat with hepatic lipidosis. note the pale tan and exaggerated reticular pattern. in most cases, the edges appear more rounded than is evident here. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease (same cat as in figure 23-61) . (courtesy dr. randolph baral.) those started on intravenous nutrition, it can be a significant source of morbidity if electrolyte replacement and monitoring are not carefully attended. once the cat is hemodynamically stable, the next step in treatment planning in cats with hepatic lipidosis is re-introduction of nutrition, which must include placement of a feeding tube (box 23-5). however, because many of these cats are extremely ill and are not good candidates for anesthesia, placement of a nasoesophageal (ne) tube to allow initiation of enteral feeding is often the most appropriate step for the first few days. when administering food through a feeding tube, there are several important points: 1. the food should be room temperature (not too hot or cold). 2. the tube should be flushed with water following feeding, to remove any particles of food or medication that may cause the tube to clog. 3. if the cat is volume sensitive, it is important to carefully calculate how much water is used for flushing the tube, because a significant volume of fluid can be infused, creating a potential fluid overload. if the cat is fluid sensitive, the total amount of fluids (amount in the food, amount added to food if blenderized, and amount of flush) must be determined, and the amount of fluid used in flushes or food preparation may have to be reduced. force feeding is to be strongly discouraged in these sick cats for several reasons: • it is highly stressful and will further increase the stress response and insulin resistance phenomena that are perpetuating the hepatic lipidosis. • it can be dangerous to the cat (aspiration) or operator (scratches/biting). • it is rarely able to meet the necessary nutritional goals set for the patient. • it may induce food aversion, a phenomenon unique to cats, but creating a profound aversion to the chosen food that can be lifelong. 12 although ne tubes are excellent choices for short-term feeding of cats unwilling to eat, there are several disadvantages to their long-term use, including the nasal irritation that occurs, the relative ease with which cats can (and will) remove them, and the need to use liquid enteral diets. 62 thus once the cat is deemed stable enough for general anesthesia, a long-term feeding tube solution is needed, and this typically is either an esophageal (e) tube ( figure 23 -63) or percutaneous endoscopic gastrostomy (peg) tube. 22, 62 both feeding options are generally well tolerated methods for providing long-term feeding, but e tubes have the advantage of being placed without the need for any specialized equipment, and if complications occur, they are generally easily addressed, because the most common complications are infection at the tube site or premature removal of the tube by the cat. placement of a peg tube, although relatively easy to learn to place, requires having the appropriate endoscopic equipment, and if complications occur as a result of infection or tube removal, more significant morbidity can result. because there is no advantage to placement of peg tubes easiest to use, and are an acceptable choice in most situations. finally, because many cat stomachs are volume sensitive with initiation of feeding, it is very important to start conservatively with small-volume feeding on a more frequent schedule. with prolonged fasting, the stomach volume of a cat with hepatic lipidosis may be reduced dramatically, preventing normal expansion and limiting intake to as little as 10% of normal. thus to avoid vomiting when feeding, the starting volume may have to be as small as 10 to 15 ml every 2 to 3 hours. a good rule of thumb is to start with estimation of resting energy requirement (rer) (40 to 50 kcal/kg is a good estimate of rer), and then attempt to meet 25% of rer the first day. if no problems are encountered, increase the amount to 50% of rer the second day, and so on, but during this period, keep the frequency as high as possible (feed four to six meals per day) so that the volume remains relatively small at each meal. once full rer has been achieved with multiple meals per day, the frequency of feeding can be gradually reduced to three to four meals per day. most cats will eventually tolerate three meals per day well, and some can tolerate two meals per day, but this is quite variable and should not be attempted during the first weeks of feeding. in general, most cats with hepatic lipidosis will require tube feeding for a minimum of 3 to 6 weeks before they will show interest in food and begin eating again on their own. the tube should be retained until the cat has been eating on his or her own for at least 1 week or longer and can be maintained for a longer duration if it is being used to administer medications, because cats can eat normally with the e tube in place. the other therapeutic considerations for cats diagnosed with hepatic lipidosis are directed toward dealing with the complications of the disease and reducing the oxidative stress on the liver with hepatoprotective therapy (table 2323) . in cats that are vomiting, in cats versus e tubes, placement of e tubes is advocated as the best approach for most practice situations. interested readers are referred to several recent reviews on tube placement for specific details on each method and to chapter 18. 22, 62 diet selection is the next step in treatment planning for cats with hepatic lipidosis. in contrast to the belief that animals in liver failure need lower quantities of protein to reduce the workload on the liver, cats with hepatic lipidosis actually need protein to recover. in fact, the work of biourge and coworkers showed that protein was the essential nutrient in reducing hepatic lipid accumulation, was essential to eliminate the negative nitrogen balance, and also appeared to minimize muscle catabolism. 9 further, diets high in protein can improve insulin sensitivity and assist weight loss in recovery from obesity. 8, 11 conversely, although carbohydrates are a readily available energy source, they are often associated with gastrointestinal distress (diarrhea, abdominal cramping) and hyperglycemia (secondary to the insulin resistance in place as a result of obesity and hepatic lipidosis). 2 thus diets selected for cats with hepatic lipidosis should ideally be high in protein (>40% metabolizable energy [me]) and have lower amounts of carbohydrates (<20% me), with the remaining calories coming from fat. the diets that best fit this profile are the diets formulated for diabetic cats; however, kitten food, many adult cat foods, and some of the enteral recovery diets have this high protein/low carbohydrate profile. many of the intestinal diets are not higher protein and are higher in carbohydrates, and so would not be the ideal choice. the key to using any of the foods that are not designed for use in a feeding tube is to blenderize them (and if necessary, strain the food) so that the food will easily go through the 14-or 16-g feeding tube without clogging it. enteral diets designed for use in feeding tubes are the because the primary starting point of the inflammatory disease in cats is the bile ducts (cholangitis), with inflammation extending to the hepatic parenchyma (cholangiohepatitis) only with time and severity, the term cholangitis syndrome has become the preferred terminology. the disease syndrome has been further classified by the wsava liver diseases group into one of three primary types: neutrophilic or suppurative, chronic lymphoplasmacytic (figures 23-64 and 23-65) , and lymphocytic (non-suppurative). 61 each of the forms appears to behave quite differently clinically as well as in their progression and outcome. in general, cats with the suppurative form of cch typically have an acute onset of illness, which often includes fever, anorexia, and vomiting, and they may become icteric quite quickly (figure 23-66) . 28, 52 the nonsuppurative form of cch (lymphocytic form) tends to be a more chronic condition, with affected cats showing nonspecific signs of illness that may include partial anorexia and lethargy, but the signs may wax and wane or are non-progressive. 28, 52 because of the feline pancreatic and bile duct anatomy, it is common for cats with cch to have pancreatitis and vice versa, and in some cases, cats will also have concurrent ibd; the constellation of the three conditions occurring together is called triaditis. 59 this combination is increasingly recognized in cats, and recent reports suggest from 50% to 85% of cats with one syndrome have all three diseases. 25, 51, 54, 59 at this time, the etiology of each of these syndromes and the pathogenesis is not well understood; however, the enteric microflora are presumed to play an important role in the suppurative form, and immune mechanisms are presumed to be the cause of the chronic inflammation found in the chronic nonsuppurative antiemetic therapy is beneficial, because it is imperative that the cat continues to receive some food, and vomiting will complicate this. metoclopramide is often used in cats because of its ready availability and low cost, but it is a very weak antiemetic in cats and thus may not be the best choice. in most cats, the novel nk-1 receptor antagonist maropitant has been a safe and effective choice. 32 the most commonly used antiemetics in the author's feline practice are maropitant (1 mg/kg iv, sc or through the e tube q24h), ondansetron (0.22 mg/kg iv q8-12h), or dolasetron (0.5 mg/kg iv, sc q24h). in addition to control of vomiting, all cats with hepatic lipidosis should be given vitamin k 1 (2.5 mg/cat po, sc) daily for a week, then weekly until the cat has recovered, and vitamin b 12 (cobalamin) (250 µg/cat sc) weekly for 6 weeks, then monthly until blood values are normal. 46 other vitamins may become deficient, such as some of the b vitamins and vitamin e; however, feeding is likely to rapidly replenish these deficiencies if they exist. this is also likely true of amino acid deficiencies, but supplementation of l-carnitine (250 mg/day po) may be beneficial by improving fatty acid oxidation. 10 finally, hepatoprotectant and antioxidant therapy with s-adenosylmethionine (same) (20 mg/kg po q24h) has been advocated to increase glutathione and may be beneficial in cats with hepatic lipidosis. 13, 19, 56 it is important to note that if same is given through the tube (and thus the tablets must be crushed), the dose must be increased by approximately 50% to allow for the loss of absorption from loss of the enteric coating. because drug metabolism is often impaired in cats with hepatic lipidosis, appetite stimulants, such as mirtazapine, cyproheptadine, and clonazepam, should not be used in cats because dosing and side effects can be unpredictable. benzodiazepine agonist drugs (e.g., diazepam) should be completely avoided in cats with possible lipidosis-induced hepatoencephalopathy, because they will exacerbate the signs and may cause fulminant liver failure. 2, 14 fortunately, most cats with idiopathic hepatic lipidosis that receive immediate and aggressive therapy and feeding for their disease will recover completely. cats that develop hepatic lipidosis secondary to other serious diseases (e.g., lymphoma) have a much lower chance of complete recovery and often die of their disease or its complications. the most common inflammatory liver disease in the cat is a complex syndrome with multiple subgroups of disease previously termed cholangitis/cholan giohepatitis complex (cch) but currently recognized under the terminology feline cholangitis syndrome. 61 this disease is quite variable in both its presentation and severity, and it may occur as a primary process or secondary to/ concurrent with other diseases (e.g., pancreatitis, ibd). ultrasonographic appearance of liver with lymphocytic/plasmacytic inflammation. note the varying echogenicity throughout the hepatic parenchyma; areas of hypoechogenicity likely reflect inflammatory cell infiltration. the gall bladder is distended; its shape is distorted by pressure from the transducer. (courtesy dr. randolph baral.) liver gb [10 mg/kg po q24h]), and if pancreatitis is concurrent, pain control with opioid pain relievers (e.g., buprenorphine 0.05 to 0.1 mg/kg po, sq q8-12h). 52 if culture is not possible, combination therapy with enrofloxacin (4 mg/kg po q24h) and metronidazole (5 mg/kg po q12h) is reasonable. in cats with chronic lymphoplasmacytic forms of cholangitis, management must be tailored to the individual situation and often requires therapy with either immunosuppressive doses of prednisolone (2 to 4 mg/kg po q24h) or chlorambucil (4 mg/m 2 po q2d), along with the hepatoprotectants and cholerectics, and concurrent treatment of other diseases (pancreatitis or ibd) that may be occurring. 52 the lymphocytic or lymphoplasmacytic forms of cholangitis may wax and wane in intensity over time, and may require long-term continuous or intermittent therapy to control the disease. there is no specific diet that is recommended for cats with inflammatory liver disease, but protein restriction should not be initiated unless the cat has clear evidence of severe hepatoencephalopathy. the diet should be selected based on other conditions (such as ibd), for which the diet may be more critical in the management. monitoring of serum chemistry values (especially glucose), clotting times, cobalamin levels, and pli/tli concentrations are recommended every few months, as well as careful monitoring of the cbc for all cats on chlorambucil. in all cats with chronic inflammatory liver disease, prior to initiation of immunosuppressive therapy, a careful assessment of the cat for other possible causes of inflammation should be completed (box 23-6). as in dogs, if a cat with pss can have surgical closure of the shunting vessel (ligation, placement of an ameroid constrictor, intravenous coiling), the long-term forms. however, whether or not these syndromes are related, a continuum of disease or completely different diseases remains undetermined. once a definitive diagnosis is obtained by histopathology of the liver tissue and culture of bile, treatment can be tailored to needs of the cat. cats with the more aggressive suppurative form of cholangitis often require intravenous fluid therapy, antibiotic therapy (based on results of culture whenever possible), and supportive therapy (antiemetics, vitamin k 1 , hepatoprotectants such as same [ important antioxidant and stabilizes membrane functions • n-acetylcysteine-a precursor to glutathione and antioxidant, also improves tissues oxygen delivery • ursodeoxycholic acid (tertiary bile acid)-used to replace hepatotoxic, hydrophobic bile acids and increase bile flow • silymarin (milk thistle)-a free radical scavenger and anti-inflammatory/antifibrotic agent • vitamin e-an antioxidant and antiinflammatory vitamin* although few clinical trials of these nutraceuticals have been performed in feline liver disease, a few studies have recently appeared showing that same, ursodeoxycholic acid, silymarin, and n-acetylcysteine all are hepatoprotective, have few adverse side effects, and may be beneficial in many types of liver disease in cats. 3, 26, 39, 53, 55 feline liver disease is a common problem that requires careful consideration of the presenting complaint, clinicopathologic findings, imaging results, and, if available, histopathologic interpretation to be able to provide an accurate diagnostic and therapeutic plan. a variety of insults can be responsible for liver dysfunction or failure, but hepatic lipidosis and feline cholangitis syndrome remain the most common reasons for cats to present prognosis for function and quality of life is generally very good. 5 however, even if surgical correction is anticipated, and especially if surgical correction is impossible or not completely successful, medical management of he is indicated. see table 23 -24 for the basic therapeutic approach to medical management of cats with he resulting from pss. because hepatocytes, by their position in the body between the gi tract and rest of the body, as well as their critical role in metabolism and detoxification, are uniquely susceptible to oxidative injury and reactive intermediates of metabolism, they must be able to protect themselves. the natural defenses of the liver include superoxide dismutase and glutathione, free-radical scavengers such as vitamin e and ascorbate, and other prosurvival signaling pathways that are controlled by hormones and growth factors. 56 however, in injury or overwhelming infection or inflammation, the natural defenses of the liver can be overwhelmed, and then it is essential for medicines and nutraceutical therapy to be included in the treatment plan to help reduce inflammation and fibrosis, protect against oxidant injury, and enhance bile flow. the cytoprotective agents most commonly used in liver diseases to assist in these processes (table 23 -25) are: • s-adenosylmethionine (same)-a precursor in the synthesis of glutathione and an important methyl donor to dna and proteins, is an with icterus or liver failure. therapy must be tailored to the individual, but nutritional support is critical in the management of hepatic lipidosis, and appropriate supportive therapy with hepatoprotectants may be crucial to treatment success. resorption from that space. effusion accumulation is therefore correlated to increased capillary hydrostatic pressure, widening of the oncotic pressure gradient, increased endothelial permeability, increased interstitial hydrostatic pressure, or loss of effective lymphatic drainage or a combination of these factors. 10, 23, 32 peritonitis of any cause results in vascular dilation, increased capillary permeability, and the migration of inflammatory cells into the peritoneum in response to immunomodulatory mediators. the inflamed peritoneum becomes a freely diffusible membrane, allowing a massive outpouring of fluid and plasma proteins from the circulation. 5, 30 ascites is not commonly seen in practice; one study recognized ascites in only three cats out of 1000 admissions to an american veterinary teaching hospital, 34 but the prevalence may be greater in primary care practice. in that study, dilated cardiomyopathy (dcm) was the most common disease associated with peritoneal effusion; however, dcm was diagnosed in most of these cats before 1987, when taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. neoplasia was the most common cause after 1987. 34 feline infectious peritonitis (fip) was by far the most common cause of feline ascites diagnosed over a 10-year period at the feline centre at the university of bristol, comprising 50% of all cats with recognized ascites. 32 cats with ascites usually present with nonspecific clinical signs, such as anorexia or lethargy. the owners may present the cat because they recognize abdominal enlargement ( figure 23-67 ), but in many cases, owners perceive this as weight gain. clinicians should be aware that sudden weight gain in a chronically underweight cat may be because of fluid accumulation (which can be intrathoracic fluid if ascites is not present), particularly if muscle mass seems reduced. ascitic cats presenting subsequent to trauma may have intraabdominal hemorrhage or urinary tract rupture. fever in a young ascitic cat will often suggest fip, and cats with fip may or may not be jaundiced. presence of jugular distention or even a jugular pulse can suggest right-sided heart failure. a palpable fluid thrill can help to distinguish ascites from other causes of abdominal enlargement, such as organomegaly, abdominal masses, bladder distention, abdominal wall weakness, obesity or, occasionally, accumulations of gas within the abdominal cavity 27 (table 23-26) . recognizing a fluid thrill involves gently tapping one side of the abdominal wall with the fingers of one hand while feeling for a sensation of fluid movement the peritoneum is the serous membrane lining the abdominal cavity, as well as covering the organs of the abdomen. it comprises a single layer of squamous mesothelial cells resting on a deeper layer of loose connective tissue. the layer of peritoneum that lines the inner surface of the abdomen is called parietal peritoneum; the abdominal organs are lined by visceral peritoneum. the total surface area of the peritoneum is one to one-and-ahalf times that of the total cutaneous area of the body. 5, 30 the peritoneal cavity contains a small amount of fluid (less than 1 ml/kg body weight) that reduces friction between the abdominal organs as they slide over each other. the fluid is a pure transudate and contains solutes in the same concentration as serum (box 23-7). this fluid is absorbed from the abdominal cavity predominantly through lymphatic vessels lying beneath the mesothelial basement membrane on the surface of the diaphragm. lymphatic drainage occurs predominantly to the sternal lymph nodes. 5, 30 ascites is the abnormal effusion and accumulation of fluid in the abdominal cavity. fluid exchange across the capillary bed is determined by starling forces, that is, the balance between hydrostatic pressure, which causes transudation of fluid out of blood vessels, and the colloid osmotic pressure, which acts to retain fluid within blood vessels. the amount of peritoneal fluid is therefore determined by the balance of these, as well as vascular permeability, with excess fluid drained by the lymphatic system. accumulation of fluid within a body cavity results when the rate of filtration of fluid into a space is greater than the rate of fluid routine laboratory findings are usually nonspecific but may provide clues to the underlying cause of ascites. for example, neutrophilia may point towards septic peritonitis but can also occur with fip; most cats with hemoperitoneum are anemic at presentation 8 ; uroperitoneum often results in azotemia and electrolyte abnormalities; hypoglycemia may reflect sepsis with septic peritonitis, and a recent study recognized 83% of cases of septic peritonitis had ionized hypocalcemia 17 ; elevated liver enzymes may be associated with inflammatory, infectious or neoplastic hepatopathies including fip; elevation of serum globulins occurs in many cats with fip but can also be associated with neoplasia or septic peritonitis; and a finding of hypoalbuminemia (which can cause a pure transudate) should prompt for an assessment of urine protein : creatinine ratio to assess if there is renal protein loss. imaging may be required to confirm the presence of fluid as well as to aid in diagnosis of the underlying cause. radiographic findings can vary greatly depending on the amount of abdominal fluid present and the underlying etiology. loss of normal detail or presence of a "ground glass" appearance to the abdominal cavity is suggestive of the presence of fluid (figures 23-68 and 23-69). very young, thin or dehydrated cats may also have a loss of detail that can mimic the presence of fluid. ultrasonography of the abdomen (figures 23-70 and if a large volume effusion causes discomfort because of abdominal distention, a three-way stopcock may be used so large volumes can be drained from one puncture (figure 23-72) . however, removal of large volumes of ascitic fluid can be detrimental, because it may prevent the subsequent reabsorption of valuable protein and/or red blood cells; the resulting reduction in intraabdominal pressure may encourage further accumulation of fluid; and rapid removal of large volumes can lead to fluid shifts causing cardiovascular collapse. 32 fluid can be collected into ethylenediaminetetraacetic acid (edta) tubes (for total nucleated cell count, packed cell volume, total protein, and cytology), serum tubes (for biochemistry, such as albumin, bilirubin, creatinine, potassium, triglyceride, glucose, lactate, and lipase), sterile tubes for culture, and/or other tubes for effusion-specific tests such as pcr. samples should be prioritized according to the volume of fluid available and to the suspected underlying disease process. 10 initial assessment of fluid retrieved is made on the basis of color and protein concentration, and much information can be gleaned from this simple assessment, even before cell numbers and types are assessed. although this brief, initial assessment is useful to refine the differential diagnoses, a thorough assessment based on underlying etiology and pathophysiology is required for definitive diagnosis and therefore appropriate management (table 23-27) . ascitic fluid, classified according to its pathophysiologic cause, can be divided into transudates, modified transudates, exudates (septic or nonseptic), or effusions (chylous or hemorrhagic). 10,23 can allow the detection of even very small volumes of fluid. it also enables evaluation of the size and structure of intraabdominal organs, such as the liver and spleen, which can help determine the underlying cause of ascites. abdominocentesis confirms the presence of abdominal fluid (in cases of low-volume effusion) and assessment of the fluid is required to diagnose the underlying cause of ascites. most cats tolerate abdominocentesis without sedation and the cat can be held in a standing position or in lateral recumbency (whichever is more comfortable for the cat and familiar to the clinician). the abdomen is clipped and aseptically prepared. a 20-to 22-gauge butterfly needle may be used with a 5-to 10-ml syringe. in cases with low-volume effusion, ultrasonography can help to guide fine needle aspiration from small pockets of abdominal fluid. diagnostic peritoneal lavage can be used if ultrasound-guided aspiration is unsuccessful. for this procedure, 10 to 20 ml/kg of warmed, sterile fluid is infused into the abdomen over 2 to 5 minutes after aseptic preparation of the site. the cat is gently rolled from side to side or allowed to stand; gentle massage of the abdomen also helps distribute the fluid. the fluid is allowed to dwell for a minimum of 2 to 5 minutes before aseptic preparation is repeated before paracentesis. no attempt is made to remove all the fluid. it must be remembered that, since the recovered fluid has been diluted by this procedure, cell counts and biochemical analyses will be affected. 33 transudates are a consequence of altered fluid dynamics. protein-poor transudates (commonly referred to as pure transudates) form predominantly as a result of severe hypoalbuminemia, which causes a lowered colloid osmotic pressure. since there is no change in endothelial or mesothelial permeability, as fluid accumulates, there is no concurrent cell leakage; so, there is a decrease in the cell count through a dilutional effect. consequently, transudative effusions are typically clear and colorless. 10, 23, 32 other pathologic causes of proteinpoor transudates include cirrhosis, lymphatic obstruction, and noncirrhotic portal hypertension (presinusoidal and sinusoidal). since hypoalbuminemia is the most common cause of transudates, serum albumin concentrations must be measured to guide further diagnostics. if the serum albumin concentration is normal (or only minimally decreased), then radiographs, abdominal ultrasonography, and/or echocardiography are indicated to assess cardiac function and for urinary bladder rupture. 10 one review of feline ascitic cases found 24% of effusions were protein-poor transudates, of which 82% were the result of hepatic failure or primary renal disease. 34 modified transudates can result from increased hydrostatic pressure within the postsinusoidal vessels of the liver secondary to right-sided congestive heart failure (e.g., tricuspid insufficiency) or potentially from mass lesions (such as neoplastic masses) obstructing blood flow from the hepatic vein or caudal vena cava into the right side of the heart. the increase in hydrostatic pressure within the vessels of the liver causes a protein-rich fluid to leach out of the liver into the abdominal cavity. since cell membrane permeability does not change, cells do not accumulate in the effusion. 10 modified transudates can also result from increased vascular permeability in the early stages of an inflammatory process, in which case cellularity will be increased. modified transudates were described as the most common type of ascitic effusion identified in cats in one study, with most being resulting from neoplasia and congestive cardiac failure; however, this study partially included cases prior to 1987, when right-sided heart failure associated with dilated cardiomyopathy (dcm) was prevalent. 34 the recognition of the role of taurine deficiency in this condition and the subsequent addition of this amino acid to feline diets now means that right-sided heart failure is only rarely encountered as a cause of ascites in cats. exudates are a consequence of altered mesothelial and/ or endothelial permeability. this permeability results from a cytokine-mediated inflammatory response of any underlying cause (e.g., infectious, neoplastic, immune mediated). exudates have high protein and moderate to high cell concentrations and are classified as nonseptic or septic. exudates are often primarily composed of neutrophils. nondegenerate neutrophils (and the absence of organisms) points to a nonseptic exudate (mostly fip but also neoplasia). fip is the most common cause of exudative effusion in cats and was the most common cause of feline ascites diagnosed over a 10-year period at the feline centre at the university of bristol. 32 the presence of neoplastic cells rules in neoplasia, but the absence of such cells does not rule out this diagnosis since many cases of neoplastic ascites are not associated with exfoliated neoplastic cells. other causes of nonseptic exudates include pancreatitis, lymphocytic cholangitis, and viscus rupture, such as the gall bladder or urinary bladder. degenerate neutrophils typify septic exudates (i.e., septic peritonitis), and their presence should instigate investigation for causes of infection (mostly leakage of gastrointestinal contents). 34 chylous effusions appear as milky or pink opaque fluid, and small mature lymphocytes initially predominate in cell counts. after drainage, more macrophages and nondegenerate neutrophils may be found. chyle is typically classified as an exudate, but its physical characteristics can be consistent with a modified transudate (protein content between 25 and 40 g/l); biochemical analysis of triglyceride and cholesterol levels in the fluid are required to confirm the diagnosis. pseudochylous effusions resemble true chyle both in appearance and cytology but do not contain fat. similar conditions result in both chylous and pseudochylous effusions. chylous abdominal effusions are rarely reported in the cat and only accounted for 7% of cases of ascites in one study. 34 the described causes of chylous ascites in cats are predominantly neoplastic. in a series of nine cats, chylous ascites was associated with nonresectable abdominal neoplasia in four cases (i.e., hemangiosarcoma and paraganglioma), with intestinal and mesenteric lymphoma in two cases and lymphangiosarcoma of the abdominal wall in another. 13 one described case in a 10-year-old cat was thought to be because of fip. 28 figure 23 -71 shows an ultrasonographic image of a cat with chylous abdominal effusion associated with pancreatitis. other potential causes include right-sided congestive cardiac failure, steatitis (inflammation of fat), biliary cirrhosis, and lymphangiectasia. hemoperitoneum in companion animals is categorized as traumatic or spontaneous. traumatic hemoperitoneum is further divided into blunt causes of trauma (i.e., motor vehicle accidents and high-rise falls) and penetrating trauma (i.e., gunshot wounds and bite wounds). 8, 21 inadvertent splenic aspiration, venipuncture, or acute severe hemorrhage should be suspected if the cytology is consistent with peripheral blood including platelets but without erythrophagocytosis or if the blood clots readily. when there is no history of trauma, coagulopathy or spontaneous rupture of a vascular neoplasm should be considered. in one study of 16 feline cases of spontaneous hemoperitoneum, 12 cases (75%) were associated with hepatic pathology such as neoplasia, necrosis, and amyloidosis. 21 in another study of 65 cases of spontaneous hemoperitoneum, 46% (30 of 65) of cats had abdominal neoplasia, and 54% (35 of 65) had non-neoplastic conditions. cats with neoplasia were significantly older and had significantly lower packed cell volumes (pcvs) than cats with non-neoplastic disease. hemangiosarcoma was the most often diagnosed neoplasm (18 of 30, 60%), and the spleen was the most common location for neoplasia (11 of 30, 37%). coagulopathies (8 of 35, 23%) and hepatic necrosis (8 of 35, 23%) were the most common causes of non-neoplastic hemoperitoneum. 8 other nonneoplastic causes of hemoperitoneum include ruptured bladder, hepatic rupture secondary to hepatic amyloidosis, gastric/duodenal ulcer, hepatic hematoma, hepatitis, perinephric pseudocyst, feline infectious peritonitisinduced liver rupture, and feline infectious peritonitisinduced nephritis. 8, 21 the prognosis of cats with spontaneous hemoperitoneum is poor. in two studies, only approximately 12% of cases survived to be discharged from hospital. 8, 21 median survival time for cats that were discharged in one of those studies was 54 days (range, 5 to 1825 days). 8 feline infectious peritonitis (fip) comprised 50% of cats with recognized ascites over a 10-year period at the feline centre at the university of bristol, 32 and, as a rule of thumb, when ascites is recognized in a younger cat, fip should be considered the major rule-out. the abdominal effusion found with fip is typically straw to golden yellow (although the color can be very variable, for example, chyle may be present), may contain fibrin clots, and has a high protein concentration. the total protein content is greater than 35 g/l and often greater than 45 g/l, with globulins comprising 50% or more. 31 one study described an effusion with total protein greater than 80 g/l as 90% specific, 55% sensitive, and having a 0.78 positive predictive value to diagnose fip. 31 the rivalta test evaluates the fluid's globulin content, and was found to be very sensitive but only 80% specific; this test is performed by adding one drop of acetic acid (98%) to 5 ml of distilled water. this fluid is mixed thoroughly, and then one drop of effusion is gently placed on the surface of the mixture. if the drop stays at the top of the fluid or slowly floats to the bottom, the test is considered to be positive. this test can give inaccurate results if inappropriate technique is used or if there is a significant temperature difference between the fluid sample and the acetic acid solution. a positive rivalta test can result from lymphosarcoma, septic, or fip effusions; these can be distinguished by cytology and culture. immunofluorescence staining of coronavirus antigen in macrophages had a positive predictive value of 1.00 but a negative predictive value of 0.57. 15 the potential clinical presentations, diagnosis, and management of fip are covered in detail in chapter 33. one study found neoplasia to be the most common cause of ascites in cats, 34 and neoplasia should be considered the major rule-out in older cats with ascites. the effusion from cats with ascites resulting from neoplasia may be a modified transudate, resulting from compression of hepatic veins or the caudal vena cava, or metastases to the peritoneum; hemorrhage from neoplasia can cause hemoperitoneum; chylous effusions may result from reduced lymphatic drainage or rupture of lymphatic vessels; and raised vascular permeability caused by neoplastic infiltration can result in an exudative effusion. carcinomas, mesotheliomas, and discrete (round) cell neoplasms (e.g., lymphoma, mast cell tumors, malignant histiocytosis) exfoliate cells into effusions more readily than sarcomas, and of these, lymphosarcoma is the most common malignancy of cats. cytology of ascitic fluid reveals neoplastic cells in less than a quarter of cases; so the absence of such cells does not rule out a diagnosis of neoplasia. in these circumstances, the diagnosis may be achieved by ultrasound-guided fine-needle aspiration of affected organs, or even biopsy samples obtained at laparotomy. the specific approaches will depend on the specific neoplasia diagnosed. exudates caused by septic inflammation usually result from bacterial contamination of the peritoneal cavity secondary to gastrointestinal tract leakage or penetrating wounds associated with trauma. gastrointestinal tract leakage may occur as a result of ulceration associated with neoplasia or inflammatory disease or as a result of penetration of a sharp object ingested (such as a toothpick), it can also occur subsequent to prior abdominal surgery. 7, 9, 17, 24 primary septic peritonitis in which no apparent cause can be identified has also been described in cats. 26 septic exudates are usually yellow to tan in color, with yellow particulate matter and are foul-smelling. microscopically, the fluid is characterized by the presence of degenerate neutrophils and bacteria. bacteria are often seen intracellularly within neutrophils. the condition is associated with high morbidity and mortality rates, with survival rates reported between 32% and 80%. 7, 9, 17, 24, 26 the history and clinical signs are often vague and nonspecific but can include abdominal pain, vomiting, lethargy/depression, and anorexia. abdominal pain is an inconsistent finding, being recognized in only 62% of cats in one study 7 and 43% in another. 24 some cats may have an inappropriately low heart rate. 7, 26 hematologic and serum biochemistry findings are also inconsistent; neutrophilia with a left shift may be present, as may neutropenia or a normal neutrophil count. similarly, cats may be hypoglycemic, hyperglycemic, or normoglycemic, and they may be hypoalbuminemic. 7, 24, 26 one study recognized ionized hypocalcemia in 89% of cats with septic peritonitis at the time of diagnosis, 17 and another suggested hyperlactatemia, when present, may be associated with a poorer prognosis. 24 radiographic findings are usually typical of ascites of any cause, but presence of pneumoperitoneum in a cat that has not undergone recent surgery may suggest the presence of gas-forming bacteria or rupture of an abdominal viscus and warrants immediate surgical intervention. ultrasonography does not directly aid the diagnosis of septic peritonitis. 7 exploratory laparotomy to determine and correct an underlying problem, such as full-thickness gastrointestinal perforation (often requiring partial resection) is required, as is copious abdominal lavage with sterile, warmed fluids ( figure 23-73 ). there are no statistically significant survival differences between postsurgical primary closure, open peritoneal drainage, or closed suction drainage postsurgical lavage; however, a trend toward a higher survival rate has been seen in cats treated with primary closure. 24, 26 treatment also involves antibiotics, initially parenterally, based on culture and sensitivity findings. consistent with intestinal contents, most bacteria recognized are gram-negative aerobes, such as e. coli or enterobacter spp., but mixed infections are usually found. 7, 24 anaerobes seem more common in cats with primary septic peritonitis, 26 which perhaps suggests these cases may result from healed over-bite wounds into the abdomen. amoxicillin/ clavulanate would be an appropriate empirical choice of figure 23-73 fulminant peritonitis associated with gastrointestinal perforation. in this case, the effusion volume was low but the high degree of serosal inflammation is evident. antibiotics while awaiting sensitivity results. there are no definitive guidelines for duration of antibiotic treatment; the author uses extended treatment courses of 4 to 6 weeks. supportive care with intravenous fluids to maintain fluid and electrolyte balances is also required perioperatively. bile peritonitis is infrequently reported in cats but has been recognized in association with gunshot 20 or motor vehicle 2 trauma, with biliary obstruction from gall stones 2,22 and subsequent to percutaneous ultrasoundguided cholecystocentesis in a cat with infectious cholangitis. 4 concurrent bacterial infection was recognized in each case; this increases severity of inflammation and worsens the prognosis, although full recovery was achieved in most reported cases. 2, 4, 20 bile peritonitis has the potential to result in small-volume effusions; so, if abdominocentesis does not yield a sample of effusion but bile peritonitis is high on the differential list, then diagnostic peritoneal lavage is appropriate. since repair of or removal of the gall bladder and abdominal lavage are required, exploratory laparotomy is an appropriate means to diagnose this condition. management should be considered as for septic peritonitis of other causes. trauma, including blunt abdominal trauma, urethral catheterization, and bladder expression, is the most common cause of uroperitoneum in cats. 1 it is also recognized as a complication of ureteral surgery. 18 the bladder is the most frequent site of urine leakage after blunt abdominal trauma, whereas the urethra is most commonly injured following catheterization. cats with ruptured bladders may still have a palpable bladder and the ability to urinate. common historical complaints are anuria (53.8%) and vomiting (50%). azotemia is a common finding, and hyperkalemia is seen in around 50% of cases. drainage of urine from the peritoneal cavity seems to improve patient stabilization. morbidity and mortality depended largely on the severity of associated injuries. 1 regardless of the site of injury or the cause of uroabdomen, the first goal of treatment is patient stabilization. isotonic replacement fluids are used for initial resuscitation. treatment of hypovolemic shock, if present, is the first order of fluid therapy. after fluid resuscitation, drainage of urine from the abdomen should be established. continuous passive drainage of the urine is necessary for stabilization and allows effective diuresis to occur. indwelling catheterization of the urinary bladder is recommended to keep the bladder decompressed and reduce urine flow into the abdominal cavity in patients with bladder and proximal urethral injury. if the urethra is traumatized and a catheter cannot be placed, prepubic tube cystostomy may be necessary to achieve temporary urinary diversion. the decision to treat the uroabdomen patient surgically or conservatively should be based on the location and severity of the underlying injury, the condition of the patient at presentation, and the patient's response to initial stabilization. 1, 12 congestive heart failure has become an uncommon cause of ascites in cats since the late 1980s/early 1990s, from which time dilated cardiomyopathy has been largely eradicated. 32, 34 ascites does still result from rightsided congestive heart failure in conditions such as tricuspid insufficiency, 6 arrhythmogenic right ventricular cardiomyopathy, 16 myocardial fibrofatty infiltration, 14 or restrictive cardiomyopathy. 29, 34 concurrent pleural effusion or pulmonary edema is often, but not necessarily, present with cardiac induced ascites. a heart murmur is not necessarily noted. noting a jugular pulse or thrill is helpful diagnostically, if present. the ascitic fluid is typically a modified transudate, but a chylous effusion is also possible. cardiac diseases are covered in chapter 20. in some cases, hepatic lipidosis has been reported to cause ascites, particularly in association with pancreatitis. these cats are often hypoalbuminemic, with the possibility of intravenous fluid therapy contributing to the ascites by raising 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and nonsulfated bile acids as a diagnostic test for liver disease in cats mosby, p 44. 48a. van der linde-sipman j, niewold t, tooten p et al: generalized aa-amyloidosis in siamese and oriental cats bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease accuracy of ultrasound guided fine needle aspirate of the liver and cytologic finding in dogs and cats: 97 cases acute necrotizing pancreatitis feline cholangitis syndrome oxidative stress and neutrophil function following oral supplementation of a silibininphophatidylcholine complex in cats laparoscopic diagnosis of pancreatic disease in dogs and cats s-adenosylmethionine in a feline acetominophen model of oxidative injury therapeutic use of cytoprotective agents in canine and feline hepatobiliary disease liver cytology inflammatory liver disease relationship between feline inflammatory liver disease and inflammatory bowel disease, pancreatitis, and nephritis fine needle aspiration cytology suggests hepatic lipidosis in 4 cats with infiltrative hepatic disease wsava standards for clinical and histological diagnosis of canine and feline liver diseases nutrition for anorectic, critically ill or injured cats the effects of s-adenosylmethionine on clinical pathology and redox potential in the red blood cell, liver and bile of normal cats proteins invoked by vitamin k absence in clotting times in clinically ill cats liver glutathione concentrations in dogs and cats with naturally occurring liver disease critical care nutrition extrahepatic biliary tract obstruction, a retrospective study of 45 cases (1983-1993) statistical relevance of ultrasound criteria in the assessment of different liver diseases in dogs and cats clinical differentiation of acute and chronic feline pancreatitis milk thistle (silybum marianum) for the therapy of liver disease surgery of the liver clinical features of inflammatory liver disease in cats: 41 cases (1983-1993) update on hepatobiliary imaging sensitivity of tru-cut and fine needle aspirate biopsies of liver and kidney for the diagnosis of feline infectious peritonitis generalised amyloidosis in two siamese cats: spontaneous liver haemorrhage and chronic renal failure severe cholestatic liver disease secondary to liver fluke (platynosomum concinnum) infection in three cats safety, pharmacokinetics and use of the novel nk-1 antagonist maropitant (cerenia) for the prevention of emesis and motion sickness in cats pancreatic ductal and interstitial pressures in cats with chronic pancreatitis infectious hepatopathies in dogs and cats abdominal ultrasonographic findings associated with feline infectious peritonitis: a retrospective review of 16 cases complications and longterm outcomes of the ligation of congenital portosystemic shunts in 49 cats clinical, clinicopathological, and histological changes observed in 14 cats treated with glucocorticoids hepatic encephalopathy. current concepts of the pathogenesis effects of oral ursodeoxycholic acids in healthy cats on clinicopathological parameters, serum bile acids and light microscopic and ultrastructural features of the liver uroperitoneum in cats: 26 cases (1986-1995) extrahepatic biliary tract surgery in the cat: a case series and review congenital portosystemic shunts in the cat: a report of nine cases feline cholecystitis and acute neutrophilic cholangitis: clinical findings, bacterial isolates and response to treatment in six cases diagnosis and management of peritonitis in small animals traumatic tricuspid insufficiency in a kitten underlying cause, pathophysiologic abnormalities, and response to treatment in cats with septic peritonitis: 51 cases spontaneous hemoperitoneum in cats: 65 cases primary bacterial peritonitis in dogs and cats: 24 cases a review of the pathophysiology, classification, and analysis of canine and feline cavitary effusions feline hepatic lipidosis uroabdomen in dogs and cats chylous ascites in cats: nine cases congestive heart failure and atrial fibrillation in a cat with myocardial fibrofatty infiltration comparison of different tests to diagnose feline infectious peritonitis arrhythmogenic right ventricular cardiomyopathy in two cats retrospective study: ionized calcium concentrations in cats with septic peritonitis: 55 cases management and outcome of cats with ureteral calculi: 153 cases progressive lymphocytic cholangitis in the cat surgical treatment of bile peritonitis in 24 dogs and 2 cats: a retrospective study feline hemoperitoneum 16 cases (1986-1993) duplex gall bladder associated with choledocholithiasis, cholecystitis, gall bladder rupture and septic peritonitis in a cat the cytologic examination of body cavity fluids a retrospective study of surgically treated cases of septic peritonitis in the cat chronic lymphocytic cholangitis in three cats primary bacterial septic peritonitis in cats: 13 cases pneumoperitoneum in dogs and cats: 39 cases chylous abdominal effusion in a cat with feline infectious peritonitis restrictive cardiomyopathy in a cat with hypereosinophilic syndrome kirk's current veterinary therapy xii: small animal practice feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value differential diagnosis of ascites in cats abdominal paracentesis and diagnostic peritoneal lavage peritoneal effusion in cats: 65 cases (1981-1997) *when present for any length of time, a pure transudate will become modified. this is particularly true of transudates that develop slowly, such as those associated with congestive heart failure or portal hypertension. modified transudates are therefore more common than pure transudates. adapted from tasker s, gunn-moore d: differential diagnosis of ascites in cats, in practice 22:472, 2000. key: cord-023017-k6edtg58 authors: nan title: aasld abstracts (pp. 282a–382a) date: 2006-02-10 journal: hepatology doi: 10.1002/hep.1840380505 sha: doc_id: 23017 cord_uid: k6edtg58 nan of well-characterized human hepatocyte cell lines could facilitate cell therapies such as htx and bioartificial livers. toward this goal, we have developed a tightly regulated human hepatocyte line. methods human hepatocytes were immortalized with a retroviral vector encoding human telomerase reverse transcriptase (htert) and green fluorescent protein (gfp) cdnas flanked by a pair of loxps. after retroviral transduction, a single cell clone was obtained using flow cytometric cell sorting followed by limiting dilution method. the resultant gfp-positive clones were supertransduced with tamoxifen-inducible cre recombinase expression cassette, mercremer. recovery of the reverted form of htert-immortalized cells was conducted by tamoxifen treatment and subsequent gfp-negative cell sorting with moflo. the expression of hepatocyte markers and albumin secretion was compared before and after tamoxifen treatment. transplantation effect of the reverted cells (lx lo9) into the liver were evaluated in a pig model of liver failure induce by an intravenous administration of d-galactosamine (d-gal). resu1ts:a non-tumorigenic human hepatocyte cell line ttnt-16-t3 was established. albumin secretion and gene expression of liver markers were increased in the reverted ttnt-16-t3 cells. transplantation of the reverted cells via the portal vein of pigs treated with d-gal was effective to prolong the survival by providing liver support until spontaneous hepatic regeneration occurred. conclusion: we here demonstrate reversible immortalization of human hepatocytes using tamoxifen-induced crellox self-excision. the resulting cell line would be highly desirable for research and therapeutic applications. acknowledgmenk the work was supported in part by life science project of 2lst century, japan. background and aim: stem cells play a key-role in tissue homeostasis. haematopoietic stem cells (hscs) could migrate into the liver and transdifferentiate, becoming a "liver stem cell reserve''. we aimed to analyse: human hsc (h-hsc) ability to engraft into the mouse liver and to contribute to its regeneration after a toxic damage. materials and methods: nodiscid mice were so divided: a) chimeric not-damaged mice: mice were submitted to irradiation followed by h-hsc intraperitoneally (i.p.) injection; finally they were killed 22, 27 and 31 days after; 8) chimeric damaged mice: mice were irradiated, injected with h-hscs and, 21 days after, damaged using ally1 alcohol (aa), i.p.; killing was performed 1 , 5 and 10 days after the damage; c) damaged not-chimeric mice: mice were damaged (with aa injection) and killed 1, 5 and 10 days after; d) not-chimeric not-damaged mice: mice were killed without any treatment. spleens (s), bone marrows (bm) and livers (l) were tested by flow-cytometry to detect h-hscs and immunohistochemistry (l only) to localize h-hscs and their hepatic derivatives. results: flow-cytometry: group a) presence of low percentage of human cells; groups c) and d) human cells undetectable; group b) presence of low percentage of human cells in bm and s while in the l they represented up to 20% of whole cellular population (the difference respect to the group a has high statistical significance, being alpha<0.001%). immunohistochemistry showed the human cell differentiation into parenchymal cells. conclusions: our results suggest that hscs could be an option to improve thie liver regeneration after a toxic injury, supporting the feasibility of bm-derived liver stem cell hypothesis. we have previously demonstrated that cd8-mediated immune damage of allogeneic liver parenchymal cells is difficult to regulate due to the activity of "immunoresistant" cd8+ t cells. in prior studies we demonstrated that short-term interference with cd401 cd40l (but not cd281b7) costimulation transiently suppressed cdb-dependent hepatocyte rejection. recently, we have determined that targetintg lfa-1 alone preferentially suppresses cd8dependent versus cd4-dependent hepatocyte rejection. in addition, short-term immunotherapy targeting both lfa-1 and cd40lcd40l costimulation produced synergistic effects and resulted in longterm suppression of cd8-dependent rejection such that hepatocyte survival up to 90 days was achieved in the majority of recipient mice. the purpose of this study was to determine whether targeting both cd40lcd40l and lfa-1 mediated signals results in an immunoregulatory state which controls naive cd8+ t cells. methods: 2 x lo6 fvbln (halat, hepatocytes, were transplanted into cd4 ko or c57bl16.scid (all h-2b) mice, and recipients were treated with anti-lfa-1 mab (0.3mg d0-6) and anti-cd4ol mab (lmg, do, 2,4,7) . hepatocyte survival was monitored by detection of serum halat reporter product by elisa. hepatocyte recipients with longterm survival (>60 days) were challenged by adoptive transfer of 2 million naive cdb' t cells (isolated from cd4 ko mice) which are sufficient to initiate rejection of functioning hepatocellular allografts in scid recipients. results: combined treatment with anti-lfa-1 mab and anti-cd40l mab significantly prolonged hepatocyte allograft survival in cd4 ko mice such that 95% achieved hepatocyte survival >60 days posttransplant (mst>105 days, n= 17) in comparison to either agent alone (anti-cd40l mab mst=35 days, n=4, p<o.ool; anti-lfa-1 mab mst=77 days, n=9, p<o.ool). recipient mice induced to achieve hepatocyte survival >60 days (by short-term treatment with anti-lfa-1 and anti-cd40l mab) received adoptive transfer of 2 million nayve purified cd8+ t cells. continued survival of longterm hepatocellular allografts despite adoptive transfer of cd8+ t cells was observed in 60% (3 of 5) recipients. in 2 recipients, adoptive transfer of cd8+ t cells was associated with loss of hepatocellular allograft function by 31 and 56 days following cell transfer. in contrast, control scid mice with functioning hepatocellular allografts which were reconstituted with 2 million naive cd8+ t cells rapidly rejected hepatocytes with mst of 17 days (n=5). conclusion: targeting of both cd40l cd4ol and lfa-1 not only prevents immunoresistant cdb-dependent immune damage of liver parenchymal cells, but also x lo3 vs. 1.321.3 x lo3 and 3.920.4 x lo3 vs. 0.321.0 x 103, respectively) while at 72 hrs it was 2.4t0.8 x lo3 vs. 2.122.0 x 103 (ns) . in the spleen, significantly higher heparanase treated cells were located within the tissue, showing proliferative activity at 48 and 72 hrs post transplantation. already by 24 hrs after transplantation the proliferating index of sec increased from 150.5 in controls to 2256 in heparanase treated rats (i'<o.o2). conclusions: heparanase increases the long-term presence of transplanted cells within portal radicles, thereby facilitating their engraftment. it has also a significant effect on ec proliferation. disc 1 o s u r e s : yaacov baruch -no relationships to disclose orit goldshmit -no relationships to disclose neta ilan -no relationships to disclose gideon shoshany -no relationships to disclose vladislav tsiperson -no relationships to disclose ella veitzman -no relationships to disclose israel vlodavsky -no relationships to disclose 264 cell activation. arunzazu sanchez, peter n u n , insa s schroeder, valentina m factor, snorri s thorgeirsson, national cancer institute, bethesda, m d substantial evidence suggests that proliferation of hepatic stem cell progenies (referred to as oval cells) is responsible for liver regeneration when the replicative capacity of hepatocytes is impaired. it is well established that activity of the tnf-a , nf-kb, il-6, and stat3 pathways is necessary for the regenerative response of hepatocytes following partial hepatectomy. however, much less is known about the signals mediating oval cell proliferation and differentiation. the aim of the present study was to evaluate the involvement of nf-kbistats signaling pathways in regulation of oval cell activation. high activity of nf-kb was found in regenerating liver after aaflph protocol coinciding with the proliferation of oval cells. urification of the oval cell population by magnetic beads sorting using ov1 antibody confirmed that the activation of both nf-kb and stat3 is specific for this cellular compartment. three distinct subpopulations of oval cells were identified based on intensity of ov1 staining by facs and named as ov1 dim, medium and bright. real time pcr analysis showed that they represent oval cells at different stages of differentiation along the hepatocytic lineage. ov1 dim subpopulation was the closest to the hepatic stem cell, as judged by high expression of c-kit. on the contrary, ov1 bright cells displayed the highest expression of hepatic differentiation markers, ttr, albumin, connexin 32 and hnf-4. nf-kb was uniformly activated in the entire oval cell compartment, whereas stat3 was phosphorylated only in the most differentiated ov1 bright subpopulation. furthermore, activation of stat3 tightly correlated with a high proliferative activity in these cells. together, these data provide the first evidence that the transcriptional activity supported by nf-kb and stat3 is required for oval cell activation. the differential induction of these two transcription factors suggests that they may control different stages of oval cell activation. disclosures: introduction: haematopoietic stem cells (hscs) have been shown to have greater plasticity than previously thought, with studies demonstrating that both human and rodent hscs can differentiate into hepatocytes. our group has also confirmed that human stem cell derived hepatocytes are the result of true differentiation and not the result of cellular fusion (newsome et al, gastroenterology 2003) . the extent to which endogenous hscs are naturally mobilized and contribute to liver repair is however uncertain. aims 1) to establish whether liver injury mobilizes endogenous hscs into the circulation & into the liver. 2) to establish if circulating stem cells in liver injury have altered stem cell potential. patients and methods 4omls peripheral blood was analysed from the following groups (n=8): normal adult controls, patients with alcoholic hepatitis, abstinent chronic alcoholic liver disease and paracetamol-induced liver injury. using a flow cytometry sorting machine, circulating cd34 positive hscs were quantified as a percentage of the number of mononuclear cells according to the ishage flow cytometry protocol. equal numbers of cd34 positive cells from each gmup were collected and cultured, to assess their in vitro stem cell properties, as judged by number of colony forming units (cf'us) produced. for patients with alcoholic hepatitis, age, sex, biochemical parameters, maddrey score and outcome (mortality) was recorded. liver biopsies from paracetamol induced liver injury and alcoholic hepatitis patients were stained for cd34 and c-kit markers to quantitate stem cell numbers (number/ portal triad) and compared with normal adult controls. results 1.levels of circulating cd34 positive hscs were significantly elevated in patients with alcoholic hepatitis (0.19% 20.06; p<0 .05) when compared with all other groups. the corresponding cd34 positive values for the other groups are as follows: healthy controls (0.05% 20.01); paracetamol liver injury (0.08% 20.04) and abstinent alcoholic cirrhotics (0.04% 20.002).there was no significant difference in mean mononuclear cell count between these groups. 2.patients surviving their alcoholic hepatitis had significantly higher levels of circulating cd34 positive stem cells (0.26% 20.09; p<o.o5) when compared with those who had died (0.057% 20.03). the surviving alcoholic hepatitis patients had significantly lower maddrey scores (22.1 92.1; p=o.oos) than those who had died (91.67 228) whilst no significant difference in sex or age distribution was demonstrated between these two groups. the total mononuclear cell counts were lower in the deceased alcoholic hepatih group (0.85xlox9l1 2 0.5) when compared with those that had survived (2.7xlox911 2 0.26). if cd34 numbers are adjusted for the total mononuclear cell counts then the increase in circulating stem cells between these two groups became even more apparent (0.44 ? 0.34 and 4.98xlox6 cellsll 24.8 ; p<0 .05). 3.hscs from alcoholic hepatitis patients produced greater numbers of cfus compared with adult control hscs (245 vs.65). 4.liver biopsies from paracetamol and alcoholic hepatitis patients contained greater numbers of stem cells compared with healthy controls (p<0.05). control (0.65 cellsltriad ?o.l) , alcoholic hepatitis (1 cellltriad 20.17) and paracetamol liver injury (1.9 cells/ triad 2 0.35). stem cells were only identifiedwithin the portal triads and not within the hepatic parenchyma in all of these groups. conclusions 1. alcoholic hepatitis is associated with increased levels of circulating hscs both in the blood and the liver. 2. these mobilized cells display in vifro stem cell potential at a level equal to or higher than control hscs. 3. increased levels of stem cell mobilization are associated with improved clinical outcome. disclosures: background klf6 is a ubiquitously expressed kruppel like zinc finger transcription factor identified as a tumor suppressor gene in prostate cancer (narla et al, science 2001) . in addition, klf6 is frequently lost and/or mutated in hepatocellular carcinomas (tal-kremer, aasld 2002-#924) . klf6 is also an immediate early gene that is rapidly upregulated in hepatocytes following partial hepatectomy and in hepatic stellate cells after injury. its expression is regulated in a tissuerestricted manner during development (laub et al, mech dev 2001) . these broad roles yet defined sites of expression suggested a potentially important role of klfg in development. the aim of this project was to examine the role of klf6 in hepatic differentiation using an embryonic stem (es) cell differentiation system in which undifferentiated cells can be driven along tissue specific lineages under defined stimuli. materials: two targeting constructs were generated in which the second exons of klf6 were replaced by a neomycinllac z cassette or hygromycin cassette. klf6 (+/-) es cells were generated by homologous recombination through selection with a klf6 targeting vector containing the neomy&/lac z cassette. to obtain klf6 (-/-) es cells, klf6 (+/-) clones were subjected to the second round of homologous recombination. a l l es cell dones were confirmed to have a normal karyotype after selection. methods and resulk in the undifferentiated state, klfg (-/-)null es cells grew more slowly than wild type es cells, with doubling times up to 50% longer. following differentiation into embryoid bodies (eb) over 6 days in culture, the number of ebs was reduced by > 80% compared with wild type ebs. the disparity in growth rate between wt and (-/-) es cells was greater in later stages than in the initial 2 days of differentiation. this was accompanied by prolongation of the epiblast-like stage, associated with markedly delayed expression of endodermal mesodermal and ectodermal markers. next, we examined the capacity of klfg (-/-) ebs to differentiate into hepatocyte like cells by withdrawing leukemia inhibitory factor after two days, then culturing ebs under serum free conditions for 8 days, followed by growth on gelatinized dishes. basic fibroblast growth factor and dexamethasone were added into the medium starting day 6 and day 10. whereas expression in klfg (+/+) ebs of alpha-fetoprotein and albumin mrna occurred after 10 days of differentiation, their expression was almost absent and significantly delayed in klf6 (-/-) ebs. an impaired ability of klf6 (-/-) cells to differentiate along hematopoietic and neuronal lineages was also observed. in conclusion, these data indicate a broad and proximal role of klfg in tissue specific development with specific role in hepatocellular development. cell transplantation has been recognized as a possible future treatment for liver cirrhosis. one of the major questions is which cell will be of greatest usespleen might be a major source of stem cell, if exist, since splenomegaly is a common in cirrhotic patients and even splenectomy is performed for treatment. recent advance in the isolation technique of stem cells based on the efflux of fluorescent dyes hoechst 33342 has turned out to be an efficient method to purify stem cells called side population (sp) cells. we utilized this method to isolate stem cells from spleen. we also transfused these sp cells into rat liver treated with carbon tetrachloride (cc14) to see whether these sp cells proliferate and differentiate into both hepatocytes or cholangiocytes. methods. c57/6j male mice and female nodlscid mice were obtained from nihon crea. egfp transgenic (tg) rat were obtained from nihon slc. splenocytes were prepared by digesting in 0.5% collagenase solution and forcing tissue through sterile mesh. splenocytes were then resuspended at lo6 cellslml in hanks balanced salt solution (hbss) containing 2% fbs, 1mm hepes (hbss+), and ug/ml hoechst 33342 w or w/o verapamil and were incubated at 37 degrees centigrade for 90min. splenocytes were then stained for 30min. on ice with fitc or pe conjugated various antibodies against sca-1, c-kit, thyl.1, cd45 and cd34. splenocytes were washed in hbss+ alone 3times and then in hbss+ with 2ug/ml propidium iodide. splenocytes were analyzed and sorted on a dual-laser facstar plus flow cytometer. ( excitation:36nm, emission:424/bp44 & 660/bp20 filter & 640-nm short-pass dichroic mirror). we also prepared spcells from donor egfp tg rats' spleen as described above. these spleen egfp positive sp cells were directly injected into liver of anesthetized recipient nodlscid mice. the recipient nodlscid mice were injected with 20 ug. cc14 intrapentoneally every once a week before and after transplantation. immunohistochemical staining were performed to identify engrafted egfp positive cells, liver specific proteins such as cytokeratinl8j9, afp and albumin. results. initial hst333422 staining of low density splenocytes showed the sp fraction to be readily detectable ( 0.3 -1%). these cells were verapamil sensitive and could be stained with hst33342 after verapamil treatment. the frequency of each surface markers positive cells were as follows (table 1 and 2). two weeks after cell transplantation, donor derived egfp positive cells were engrafted. the some engrafted cells distributed in a cluster indicating donor cell proliferation. hepatic differentiation of engrafted cells were shown by double staining of egfp and rat albumin or cytokeratinld. we observed some of afp positive cells in egfp positive cells, but very few of cytokeratinl9 positive cells. conclusions. the adult rodent spleen had consistently detectable fraction of sp cells. the surface marker profiles were different from those of bone marrow sp cells. the splenic sp cells could be engrafted in liver and differentiate into hepatocyte like cells. our results suggest that splenic sp cells could be exploited for the repair of damaged liver. backgroundlaim : it has been reported that bone marrow cells (bmcs) can replace liver in a murine model of tyrosinaemia and correct this metabolic disease through the fusion of donor bmcs to recipient hepatocytes. the aim of this study was to test whether or not this replacement is a general phenomenon in other liver injury models. the following three models were tested; (1) hepatitis b transgenic mouse (tgn(alblhbv)), (2) albumin-urokinase transgenic mouse (tgn(alblp1au)) and (3) carbon tetrachloride (cc14) treatment model. as the selective liver injury models, irradiated tgn(alb1hbv) and tgn(alblp1au) were transplanted with lx106-lx107 bmcs from green fluorescent protein (gfp) transgenic mice (tgn(actbegfp)) or from @-galactosidase transgenic mice (tgn(mtnlacz)). as a non-selective liver injury model, irradiated c57bl/6 mice were transplanted with 1x106 bmcs from tgn(actbegfp) or tgn(mtnlacz) followed by the administration of cc14 (0.02mllkg animal weight, twice a week for 4 weeks). a cci4 protocol without preparative irradiation was also tested, in which c57bl16 mice were first administered ccl, 8 times, then followed by transplantation of tgn(actbegfp) or tgn(mtnlacz) bmcs. after the analysis of the donor cell engraftment in the peripheral blood, these recipient mice were sacrificed and checked for donor-derived hepatocytes at 2-47 weeks post-transplantation. sixty liver sections for each animal (containing approximately 1 . 5~1 0~ hepatocytes), were analyzed for gfp positive cells and the whole livers were inspected for @-galactosidase expression with x-gal cytohistochemistry. however, there were no gfp positive hepatocytes and no gross blue staining of the livers with x-gal in any of the 18 recipient mice. gfp positive cells were only located in sinusoids or associated with larger vessels. they were readily distinguishable from hepatocytes through their morphology and were negative for albumin by immunostaining. in addition, the livers from 4 female animals with gender mismatched bm transplantation were also tested with y chromosome fish analysis, which might be a more sensitive method to detect donor derived cells than transgenic epitope tagging. total of 5 isolated hepatocytes were positive for y chromosome in 4 . 1~1 0~ hepatocytes analyzed, although it still remains to be elucidated whether these cells arise from spontaneous fusion events or from transdifferentiation. conclusions: these results demonstrate that there is little or no contribution of bmcs to the replacement of injured livers in these models. thus, we do not believe that bm derived cells can generally lead to a cure of liver damage. background &aims: mouse embryonic stem (es) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. we previously reported that es cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (hnf-3b). in the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugtlal) in undifferentiated and differentiating hnf-3btransfected es (hnf-3 b-es) cells. materials & methods: hnf3b-es cells were established from a mouse es cell line. undifferentiated hnf-3b-es cells were main-tained in gelatin-coated dishes without feeder cells in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), 0.1 mm of z-mercaptoethanol(2me), 0.1 mm of non-essential amino acids (neaa), 1 mm of sodium pyruvate and 1000 ulml of leukemia inhibitory factor (lif). undifferentiated hnf-3b-es cells were allowed to differentiate in dmem supplemented with 10% fbs, 0.1 mm 2-me, 0.1 mm neaa, 1 mm sodium pyruvate, and 20 nglml of fibroblast growth factor 2 (fgfz) in the absence of leukemia inhibitory factor (lif). differentiating hnf-3 b-es cells were collected for rt-pcr analysis on day 14, and for western blotting on days 14 and 28. results: the expression of organic anion transporting polypeptide 1 (oatpl), multidrug resistance-associated protein 1 (mrpl), mrp2, mrp3, and ugtlal was not seen in the undifferentiated hnf-3 b-es cells by rt-pcr, whereas all were expressed in differentiating hnf-3 b-es cells. protein expression for oatpl, mrpl, mrp2, mrp3, and ugtlal was also observed in the differentiating hnf-3 b-es cells by western blotting. an immunofluorescence examination revealed that oatpl was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with cd26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes. we have used cdna microarray analysis of clonal murine hepatoblasts to identify genes that are significantly and preferentially expressed in undifferentiated hepatoblasts compared to adult mouse liver. unique stem cell genes were identified by overexpression in undifferentiated hepatoblasts compared to adult liver. 61 genes were identified by these criteria and were designated candidate stem cell genes (cscg). 54 cscg were distributed on all chromosomes of the mouse except chromosomes 12,14 and the y chromosome. however, chromosomes 2, 3 and 19 showed a preferential distribution of cscg compared to the total number of genes mapped on these chromosomes. approximately half of our cscg are commonly expressed in other murine stem cell populations including embryonic stem cells, neural stem cells, mesenchymal stem cells and hematopoetic stem cells, suggesting that hepatoblasts share a molecular signature with other stem cell populations. some of the commonly identified stem cell genes include: sc14a2, c-myc, nek2, phosphodiesterase 7, nicastrin, smarca 3 and smarca 5 and pias3. these genes function in a variety of pathways including ion transport, anion exchange, cell cycle control, chromatin organization, dna binding activity and signal transduction. approximately 25 % of cscg are expressed in early endoderm, fetal liver and/or the pancreatic bud. a search for homology to known proteins indicated that some of these genes contain homology to membrane transporters, zinc finger proteins and spindle apparatus components. this class of genes is of particular interest because they may represent new hepatic lineage markers. the results of this analysis leads us to conclude that a network of pathways some of which are in common with other stem cell populations function together to maintain the liver stem cell phenotype. valentina m factor, aranzazu sanchez, ju-seog lee, tanya n hoang, snom' s thorgeirsson, national cancer institute, bethesda, m d rat liver epithelial cells (rle) were isolated from normal adult rat livers and established as a cell line . rle cells were found to express undifferentiated characteristics resembling adult liver stem cells. we have addressed cellular and molecular mechanisms controlling lineage commitment of hepatic stem cells using rle-13. to induce rle differentiation along the hepatocytic lineage, differentiation protocols were developed consisting of a sequential treatment with the demethylating agent 5-aza-cytidine (5ac), fibroblast growth factors 1 and 2 (fgf), oncostatin m (osm) and hepatocyte growth factor (hgf) in the continuous presence of the synthetic glucocorticoid dexamethasone (dex). these treatments resulted in a remarkable enlargement in cell size and organelle complexity as shown by facs and confocal microscopy. morphological maturation of rle was paralleled by a decrease in cell proliferation. more significantly, rt and real time pcr analysis revealed an induction of hepatocyte specific markers such as tat, ttr, glucose-6-phosphatase, and connexin 32. in addition mrnas encoding liver enriched transcription factors hnf lalp, hnf 3ru, hnf 4, and clebp p were notably induced along with hepatocytic differentiation. the pcr results were supported by a cdna microarray analysis. comparison of the gene expression profiles following the treatment protocols reflected an activation of the hepatocytic differentiation program as judged by increased expression of a differentiation-associated gene set (phosphofructokinase, glutathione-s-transferase) and decreased expression of a cell cycle regulated gene set. currently, transplantation studies are performed to identify the potential of the differentiated rle to repopulate and restore the diseased livers. the results indicate that cell lines derived from adult liver stem cells may provide a renewable resource for transplantation. adult liver stem cells (adulis) can be isolated from rodent bone marrow. when cultured under specific conditions in co-culture with isogeneic hepatocytes, adulis fiom 14 days bile duct ligated (bdl) rats are transdifferentiating into a hepatocyte-like lineage and are able to produce urea from ammonia. the aim of the study presented is to describe the hepatocyte specific metabolic capacity of cultured adulis from normal or bdl rats in single or co-culture with isogeneic hepatocytes, with or without interleukin-3 (il-3). methods: adulis were isolated by a two-step immunoisolation procedure (i.e. beta-2-microglobulin negativity and thy-1 positivity) from rat femoral bone marrow (male wistar rats, 250g) and cultured on a matrigel layer in 24-well polystyrene dishes at a cell density of 50'000 cells/cm* in small hepatocyte media. isogeneic hepatocytes were isolated by a two-step collagenase perfusion technique and seeded (100,000 cells/cm2) on an inlay with a collagen-coated ptfe membrane (poresize: 0.4pm) for co-culture experiments. of note, the culture media was supplemented with 5% isogeneic serum and dexamethason m) was administered after culture day 3. il-3 was added in the corresponding experimental groups (10nglml). after removal of the hepatocyte-inlay (in the co-culture groups) adulis cultures were exposed to 1.5mmol nh4ci for 5 hours in dmem and urea formation determined thereafter with a colorimetric assay. cells were harvested in trizol, total rna extracted and after reverse transcription cdna used for real-time pcr determination of 18s(rm~) content to standardize the metabolic signal for cell number. relative ureagenesis values were compared statistically using jandel scientific. the significance level was set at pc0.05. results: the amount of adulis isolated from normal rat femoral bone marrow (n=lo) was 1.4620.75 x lo6 and 1.8321.97 x lo6 in animals (n=6) after seven days of bdl (p=n.s.). relative urea synthesis in cultures from normal animals was 1.0320.42 in single and 1.3820.41 in co-culture at culture days 3, 6, 9, and 12. with addition of il-3 to the culture media urea genesis was determined to be 1.6820.6 in single and 2.6521.0'2 in co-culture. in cell cultures from seven days bdl rats relative urea formation was 1.5821.43 in single and 2.6351.32 in co-culture. with the addition of il-3 to the culture media values were 2.3920.51 in single and 3.1620.81 in co-culture. addition of il-3 to the culture media increased the metabolic signal in all cell cultures from normal animals (anova on ranks, p<0.05) but not in cultures with cells isolated from bdl animals (p=n.s.). co-culture induced stronger ureagenesis under all culture conditions examined (paired t-test: p<0.05). conclusions: to exclude immunological interference from adult immunocompetent bone marrow cells cultures were strictly isogeneic (i.e. adulis, hepatocytes, and serum from the same animal). co-culturing adulis with isogeneic hepatocytes increased ureagenesis in all paired culture experiments. as there is no direct cell contact between hepatocytes and adulis paracrine soluble, so far undetermined, factors must be involved. interestingly by the addition of il-3 transdifferentiation was inducible in cultures of adulis from normal animals but no additional effect was detectable in the cell cultures from bdl animals. in further studies it will be necessairy to determine if cholestatic serum also induces accelerated and more pronounced hepatocyte specific metabolic capacity in adulis from normal animals. factors responsible for this transdifferentiation should then be isolated from cholestatic serum and might be used to support the failing liver in vivo potentially by activation of the adulis pool in the bone marrow and the liver. disclosures: saji, shinji tamura, yuichi yoshida, shinichi kiso, ayuko iizuka, hitoshi matsumoto, takako kawasaki, yoshihiro kamada, yuji matsuzawa, graduate school of medicine, osaka university, suita, osaka, japan background and aims: recently, the evidence that bone marrow cells (bm cells) have trans-differentiating potential into hepatic lineage cells has been described in animal transplantation experiments and human pathology of bone marrow transplantation recipients. however, the molecular mechanism underlying this phenomenon has remained unclear because of lack of effective culture system. to address this issue, we developed a novel in vitro culture system in which murine bm cells are cultured with growth factors and investigated factors required for the transdifferentiation of em cells into hepatic linage cells. methods and results: (1) bm cells were isolated from the femora of 6-to 10-week-old male c57bl/6] mice. they were cultured in three-dimensional culture system using type i collagen gel and stimulated without or with the growth factors (egf, hgf, hb-egf, osm, bmp-4, bfgf, fgf-4; 2ongiml) for 12 day. to investigate the effect of these growth factors, the gene expression of albumin was examined by quantitative rt-pcr. when bm cells were cultured with hgf, osm, bfgf and fgf-4, albumin was induced effectively. among these growth factors, bfgf was found to induce albumin the most effectively in the cultured bm cells. (2) bm cells were cultured in collagen gel with bfgf (2onglml) for 6, 12, 18 days. the gene expression of hepatocyte-specific markers (albumin, cytokeratin 18, alphal-antitrypsin, glucose 6 phosphates and tyrosine aminotransferase), cholangiocyte-specific marker (cytokeratin 19) and liver-enriched transcription factors (hnflalpha, hnf3alpha, hnf3beta, hnf4alpha, gata4, gata6, clebpalpha, and ciebpbeta) were examined by rt-pcr. upon the stimulation of bfgf, bm cells were found to express cytokeratin 18, alphal-anitripsin and glucose 6 phosphates, cytokeratin 19 and liver-enriched transcription factors including hnflalpha, hnfjalpha, hnf3beta, gata4 and gata6. (3) bm cells were cultured in collagen gel with bfgf (20ngiml) for 6 days. we assessed expressions of albumin and ck18 proteins of cultured bm cells by immunohistochemistry. about 5% cells of them were positively stained for both albumin and cytokeratin 18. conclusions: we established a novel in vitro culture system of bm cells and demonstrated that basic fibroblast growth factor could induce the trans-differentiation of bm cells into hepatic lineage cells the most effectively. furthermore, this conversion was associated with induction of liver-enriched transcription factors including hepatocyte nuclear factors and gata family proteins. these results indicate these liver-enriched transcription factors are the major components, which induce this trans-differentiation. disclosures: ayuko iizuka -no relationships to disclose yoshihiro kamada -no relationships to disclose takako kawasaki -no relationships to disclose shinichi kiso -no relationships to disclose hitoshi matsumoto -no relationships to disclose yuji matsuzawa -no relationships to disclose yukiko saji -no relationships to disclose shinji tamura -no relationships to disclose yuichi yoshida -no relationships to disclose crtteil, france; nadine martin, hbpital henri mondor, criteil, france; dominique couchie, anne m preaux, yannick laperche, elie 5; zafrani, inserm u581, cre'teiz, france liver can regenerate from oval precursor cells when the replication of hepatocytes is inhibited. these cells proliferate in the periportal region, migrate in the lobule and differentiate into hepatocytes. stromal-derived factor-1 (sdf-1) is a chemokine which plays a major role during embryogenesis. since sdf-1 and its sole receptor cxcr4 are involved in the differentiation of progenitor cells (e.g. b lymphopoiesis, digestive epithelial cell renewal), the aim of our work was to study the expression of sdf-1 and cxcr4 in a model of hepatic regeneration from precursor cells. hepatic regeneration from oval cells was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. rats were sacrificed the day of surgery (day 0) and at days 1,5,9,14 and 21 after partial hepatectomy. rare oval cells were observed at day 1. their number was moderate at day 5, marked at day 9 and 14 and declined thereafter. oval cells predominated in the periportal region and usually formed canalar structures resembling cholangioles. individual or cholangiole-forming oval cells strongly expressed sdf-i, as demonstrated at the mrna level by in situ hybridization (ish) and at the protein level by irnmunohistochemistry. interlobular bile duct epithelial cells were also positive for sdf-1 protein but negative for its mrna. cxcr4 mrna hepatic levels, as assessed by quantitative rt-pcr, paralleled the degree of oval cell proliferation. ish showed marked cxcr4 mrna expression in individual as well as in cholangiole-forming oval cells. in order to determine which role the sdf-1icxcr4 couple could play in hepatic regeneration from oval cells, rats were treated or not with fucoidan (25 mglkg ip, twice daily), a sulfated polysaccharide known to bind to sdf-1 and to block its biological effects. as compared to untreated animals, oval cell proliferation was markedly decreased in five of the seven fucoidan-treated rats. in conclusion, oval cells express sdf-1 and its receptor cxcr4 during hepatic regeneration from precursor cells. our results strongly suggest that the sdf-1lcxcr4 couple acts in an autocrinelparacrine way to stimulate the proliferation of these precursor cells. disclosures: dominique couchie -no relationships to disclose background: bone marrow cells and embryonic stem cells are being used for cell transplantation. although these cells are known to be multipotent and capable to become hepatocyte, several problems have been pointed out, such as cell fusion and teratoma generation. thus we paid attention to side population (sp) cells, those with a verapamil-sensitive ability to efflux hoechst 33342. sp cells have been identified in several tissues. the bone marrow sp phenotype appears to be a common feature of stem cells, but hepatic and splenic sp cells have been less well characterized. aim: we tried to separate and culture sp cells from non-parenchymal liver cells (npcs) and splenocytes, and examined whether they would obtain hepatic phenotype. methods: 8-12-week-old female transgenic rats that ubiquitously express egfp were used to isolate non-parencymal liver cells by the standard collagenase two-step perfusion method, and splenocytes were isolated by forcing the tissue through sterile mesh, then mononuclear cells were separated by ficoll density gradient centrifugation. they were resuspended at lo6 cellslml in hanks balanced salt solution (hbss) containing 2% fetal calf serum, 1mm hepes (hbss+), and 5uglml hoechst 33342 (hst) w or wlo verapamil and were incubated at 37 degrees centigrade for 90min. cells were washed twice in hbss+ and then resuspended in hbss+ with 2uglml propidium iodide. cells were analyzed and sorted on a dual-laser facstar plus flow cytometer. rt-pcr was performed with rna extracted from freshly isolated sp cells. isolated sp cells were cultured with male rat primary cultured hepatocytes in collagen gel sandwich. cell morphology was monitored under a phase-contrast microscope and a fluorescence microscope. the expression of albumin, cytokeratin 18, and cytokeratin 19 was analyzed by immunohistochemistry. fluorescence in situ hybridization (fish) for the sry gene was performed to exclude ceil fusion. results: the rate of npc-sp was 0.05% of isolated npcs. 89% of npc-sp expressed cd45, while 55% of npc main population (mp) did. freshly isolated sp cells were smaller than mature hepatocytes. they looked like monocytes or lymphocytes. these cells did not express liver specific markers, such as albumin, alpha-fetoprotein, cytokeratin 18, or cytokeratin 19. during one-week culture with hepatocytes using collagen gel sandwich method, we could find no change in gfp-positive sp cells, but after further culture, we found several colonies consisting of gfp-positive sp cells, whose shape became polygonal. after 2-week-culture, most of gfp-positive cells were stained positive for albumin. npc-sp cells died within one week without hepatocyte co-culture. npc-mp cells formed colonies after 2 weeks culture with hepatocytes, but unlike sp cells, they looked like fibroblasts and were negative for albumin. freshly isolated sp cells from splenocytes were also small and monocyte-like, and expressed no liver specific markers. spleen sp cells could culture for more than 2 weeks in the same way as npc-sp cells, and looked like hepatocytes, but did not proliferate. after having been cultured, most of gfp-positive cells were stained positive for albumin and cytekeartin 18, and negative for cytokeartin 19; on the other hand splenic main population cells after 2 weeks' culture expressed those very little. conclusion: we succeeded in culturing liver and spleen sp cells. sp cells which did not express mrna of liver specific markers, such as albumin or cytokeratin 18, became to express those after the culture with hepatocytes. these results suggest that these sp cells have plasticity and obtain hepatic function. thus, tissue stem cells, especially sp cells, are useful for cell transplantation. spleen sp cell might be a candidate for source of cell transplantation for treatment of liver cirrhosis. miura, takashi goto, ken-ichiro mikami, kunio nakane, kazuo yoneyama, hirokazu nagai, kunihiko terada, toshihiro sugiyama, katsuyuki imai, haruki senoo, sumio watanabe, akita university, akita, japan background epimorphin, a mesenchymal cell surface-associated molecule identified as an epithelial morphogen, is detected on stellate cells in the liver. we have reported that a contact with hepatic stellate cells (hscs) promotes differentiation of hepatic stem-like cells (hslcs). here, we show the involvement of epimorphin in that process.materials and methods: hslcs and hscs were isolated from healthy adult rats using two-step collagenase perfusion and percoll gradient centrifugation, and maintained standard medium, dulbecco's modified eagle's medium with 10% fetal bovine serum. hslcs were cultured in stellate cell-conditioned medium, co-cultured with hscs to maintain cellcell contact or in the presence of epimorphin. phenotypical and morphologic changes were investigated by rt-pcr and with an electron microscopy, respectively. results: hslcs are polygonal in shape and assume a cobblestone appearance when cultured in standard medium. transmission electron microscopy showed that hslcs , 20 to 25 micro-m in diameter, have a round to ovalshaped nucleus, a few vacuoles, scant organelles and a high nucleuslcytoplasm ratio compared with normal hepatocytes. the phenotypic properties of hslcs did not change as well as their size and shape during this experiment. hslcs characterized by rt-pcr are follows: positive, c-kit, musashi-1 (a neural stem cell marker), alpha-fetoprotein, cytokeratinl9, connexin43; negative, albumin, transferrin, tyrosine aminotransferase, gamma-glutamyl transpeptidase. hslcs cultured in stellate cell-conditioned medium had no phenotypical and morphological changes. hslcs co-cultured with hscs expressed albumin, transferrin, and tyrosine aminotransferase, which were inhibited by an anti-epimorphin antibody. furthermore, epimorphin induced the markers not only for hepatocytes including albumin, transferrin and tyrosine aminotransferase, but also for cholangiocytes including gammaglutamyl transpeptidase in addition to increased expression of connexin43 and cytokeratinl9, with decreased expression of c-kit and musashi-1. in addition, clebp beta was enhanced, which has been reported to mediate through morphogenesis by epimorphin. hslcs co-cultured with hscs piled up and subsequent development of bile-canalicui-like structures, which was dramatically inhibited by an anti-epimoirphin antibody. hslcs, close to epimorphin, stated piling up, changed their shape from flat to cuboidal, became rich in mitochondria and rough endoplasmic reticulum and formed bile-canalicui-like structures. conclusions: hslcs have a self-renewing capacity and multilineage differentiation potential. epimorphin is involved in differentiation of hslcs though a contact with hscs. disclosures: takashi goto -no relationships to disclose n order to identify new and differentially expressed genes in fetal rat liver that are specific for epithelial stemlprogenitor cells and genes involved in liver progenitor cell differentiation, we used murine cdna microarrays containing 8,976 cdnas, available at the functional genomic facility, aecom. the expression pattern of fetal liver stemlprogenitor cells was studied from embryonic day 13 through birth, 7 days after birth and in adult liver. the driver rnas were isolated from cells adhered to the dish after plating the cell suspension in order to remove the blood cells. reference rna was isolated from the livers of newborn rats. we found that 511 genes present on the cdna microarrays were developmentally regulated. these genes fall in two major hierarchical clusters, according to their pattern of expression. the 281 genes that are down-regulated during fetal liver development were distributed in functional groups and further analyzed. in this study, special attention was paid to genes that were induced in fetal liver but were not expressed (or expressed at a very low level) in adult liver. these genes are of special interest because they can serve as specific markers for identification and for isolation of liver stemlprogenitor cells. in addition, these genes represent links to understanding the fetal liver specific molecular pathways that govern cell proliferation, survival, apoptosis and differentiation. to determine which of the 281 over-expressed genes in 13-14 day fetal liver that are down-regulated in adult liver are progenitor cells specific, we searched in the available databases whether the expression of these genes in adult liver was previously reported. seventy genes were further analyzed: the clones of interest were hybridized to radioactive labeled 32p cdna synthesized from fetal and adult liver rnas. for 48 of the clones, we found that there was little or no expression in adult liver. the expression level of 25 selected clones was analyzed further by quantitative pcr, and they were confirmed as highly induced in fetal hepatoblasts compared to adult liver. half of the 48 clones are ests. the known genes fall in different categories, the major four being: genes related to transcription; signal transduction; morphogenesis, histogenesis and organogenesis; cell adhesion, de-adhesion and migration. some of the known genes over-expressed in fetal liver that are not expressed or expressed at very low level in adult liver are: grblo (aa260248), fh12 (aa023645), tnc (aa270625), peg3 (aa003064), hey1 (aa049474), enah ((aa217593), pkcd (aa276844), lox (w96914), shcbpl (aa265225), magoh (aa254528), manba (aa200473), klf5 (aa432818), gpc3 (aa274932), pcolce2 (aa153907), ppap2c (aa220316), nfkb2 (aa060802), adam19 (aa051790), akapl2 (aa387076), tagln (bc003795. it should be noted, that most of the 48 clones and all those listed here that we have identified as liver progenitor cells specific, are expressed in stem cells of embryonic, hematopoietic, or mesenchymal origin. two of the presented genes encode cell surface proteins: a disintegrin and metalloproteinase domain 19 (adaml9) (meltrin beta), glypican 3. using in situ hybridization, we are currently verifying whether our putative liver progenitor cell specific genes are expressed in hepatoblasts and in rare progenitor cells that remain in the adult liver. identifying and cloning new genes that are expressed uniquely in liver stemlprogenitor cells will allow us to design a method for isolation of these cells and to study their role in liver development, growth control and regeneration. hepatocyte transplantation has been shown to be an effective treatment for liver diseases including fulminant hepatic failure and metabolic defects in liver function. however, the availability of sufficient numbers of hepatocytes with which to conduct clinical studies limits the wide-spread availability of this therapy. previously, we have shown that human placenta derived stem cells (pdsc) differentiate along neural or hepatic lineages depending on the culture conditions and suggested that these stem cells could be a source of cells for clinical transplantation. here we report on a protocol to further optimize hepatic differentiation of pdsc. a three stage culture system was applied to induce hepatic differentiation. ae cells were propagated in dmem based standard media which contains egf 10 nglml for a week (stage i) and subsequently with growth factors such as fgf-1, 2, 4, 7, 8, oncostatin m, hgf, andlor dexamethasone (dex) and insulin-transferrin-selenium (its) for 2 weeks (stage 11). the cells were then changed to media supplemented with different nuclear hormone receptor agonists for 1 week to stimulate hepatic maturation (stage 111). total rna samples were examined for hepatocyte specific gene expressions with real-time quantitative pcr (rtq-pcr). additional studies examined hepatic differentiation on different culture substrates including collagen, gelatin, fibronectin, arg-gly-asp-ser, and matrigel 1%, 20%, 100% were also examined. in the three stage differentiation system, 20% (vlv) matrigel coated plates and dexlits containing media, and phenobarbital (pb) were identified as the best conditions to upregulate liver specific gene expression. the principal human drug-metabolizing enzymes (cytochrome p-450, cyp) were also examined by rtq-pcr. cypla1, 2c8,2d6, and 3a4 were induced with either rifampicin or pb. in parallel, expression of the relevant liver-enriched transcription factors, hnf-4, clebp-a and ciebp-b mrnas were increased under conditions which induced hepatic differentiation. under slightly different conditions, differentiation of pdsc into cells which expressed pax6, pdxl and nkx2.2, insulin and glucagon was observed. flow cytometric analysis showed isolated nayve ae cells contains a population of cells which express the embryonic stem (es) cell markers, ssea-3, 4, tra 1-60, and tra 1-81. furthermore, the placental stem cells formed embryonic body (eb)-like structure when cells were cultured on matrigel. the eb-like structures retained the expression of ssea-3, 4, tra 1-60, and tra 1-81. these data indicate that cells can be isolated from term placenta which express markers of es cells. under appropriate conditions pdsc expand, and differentiate into cells with characteristics of hepatocytes, neurons, or pancreatic cells. we propose that pdsc could provide a new source of cells for clinical transplantation and regenerative medicine. unlike with es cells, there should be no social, ethical or religious objections to the isolation and use of placental-derived stem cells. using the ssh technology, we have identified previously 643 induced clones in 13 day fetal compared to adult liver; 202 of these clones were independent transcripts, 64 of them represented ests and4 appeared to be new sequences (petkov et al., genomics 6 8 197-209,2000) . applying the rapid amplification of 5' and 3' cdna ends to subtracted genes (generace kit, invitrogen), we have obtained the full-length cdnas for two of the subtracted clones. one of them (clone 2g8) encodes a novel putative serine proteasel subtilase highly expressed in fetal liver and comparatively low in adult. the expression of 2c8 mrna was analyzed by northern blots with rna isolated from different rat tissues: fetal and adult liver, isolated 14 day fetal hepatoblasts, brain, bone marrow, kidney, spleen, intestine, colon, stomach and muscle. on northern blot with total rna, a strong signal was detected with fetal liver/ hepatoblasts rna and a weak signal with adult liver rna, which showed that the expression of this mrna is developmentally regulated. to confirm the liver specific expression of 2g8, quantitative rt-pcr was camed out. the results of this analysis confirmed our previous results and revealed also a very low expression of 2g8 mrna in the colon. the 2g8 cdna is 3345 nucleotides in length and codes for a novel protein of 691 amino acids. we were able to identify in the data bases the sequences of a rat contig which contains the complete gene for 2g8. the gene coding for 2g8 comprises 21,920 bp of the rat genome and includes 12 exons and a long 3'-utr segment of 1,000 nucleotides. the initiation of the translation begins with an aug codon, 188 nucleotides downstream from the 5'-end of mrna. using specific primers designed from the contig sequences, we obtained clones corresponding to the upstream promoter region and downstream of the gene in the pcr4 top0 vector (invitrogen). these two upstream and downstream clones were ligated to the 2c8 cdna upstream and downstream sequences, respectively, so that the whole gene with the 5' and 3' regulatory sequences was obtained in the pcr4-top0 vector. analysis of the structure of 2g8 showed that this protein resembles 8 others mammalian subtilases, named convertases described during the last decade: furin, pc1/3, pc2, pc4, pace4, pc516 and pc7ilpcipc8 and ski-1isip. these proteases are synthesized as proproteins and secreted from the cell. 2g8 contains a putative signal sequence for release from the er located between amino acid 30 and 31 (alanine 4 glutamine). however, it does not share the consensus cleavage sequences characteristic for the other convertases: (k,r)-(x)n-(k,r) for processing, release and secretion of the active enzyme form. at present, we do not know the processing site of the proprotein. in situ hybridization experiments showed that this serine protease is expressed in fetal liver hepatoblasts. convertases are secretory proproteins implicated in tissue specific processing of hormones, growth factors, metalloproteinase, extracellular matrix proteins, viral proteins etc. they show tissue specificity and some are implicated in development and differentiation. 2g8 is a novel convertase, it is developmentally regulated and most likely functions in processing and activation of growth factorslcytokines or extracellular matrix proteins implicated in morphogenesis. further studies of its promoter region will reveal the control sequences and transcriptional activators and repressors regulating its expression during development. college, london, uk introductioniaims: bone marrow cells (bmcs) can contribute to regeneration of the chronically damaged liver but in human studies and animal models the magnitude of this axis is highly variable. in a murine model of hepatitis b we examined whether this pathway of regeneration is enhanced by inhibiting endogenous hepatocyte regeneration. methods: 2 month old female mice transgenic for hepatitis b surface antigen (hbs-tg) received lethal irradiation and were then transplanted with c57b1/6j male bmcs by tail vein injection. 6 weeks later half the mice were treated with retrorsine, a pyrrolizidine alkaloid, to irreversibly block proliferation of endogenous hepatocytes. mice were sacrificed at 3 and 6 months following retrorsine injections. y chromosome containing hepatocytes were identified using in situ hybridisation and phenotype markers (positivity for cytokeratins 8/18, cytochrome p450 and glycogen, negative for cd45). results: in the control mice with chronic liver damage there was an increase in y chromosome positive hepatocytes over time, but the proportion remained <5% of the total number of hepatocytes. however, 19.3% (corrected count) of y chromosome containing hepatocytes could be found repopulating the livers of the mice that had received retrorsine. conclusions: bmcs contribute to the regeneration of the chronically damaged liver, but under conditions where regeneration of endogenous hepatocytes is possible this is minor. however, when chronic liver damage occurs and regeneration of the endogenous hepatocytes is inhibited, the contribution to liver regeneration from the bone marrow is significantly enhanced. beaujon, paris, france; thierry poynard, groupe hospitalier pitie-salpetriere, paris, france background portal hypertension depends in part on the development of hepatic fibrosis. thus, since fibrotest (ft) is a potential biochemical marker of fibrosis, this test was prospectively used to evaluate the presence and severity of portal hypertension in patients with different liver diseases. methods: 89 consecutive patients (56 males; 32% chronic viral hepatitis c, 9% hepatitis 8, 33% alcoholic liver disease, 7% transplanted, 19% miscellaneous) with transjugular liver biopsy had same day measurements done for hemodynamic parameters (gradient, free hepatic, wedged pressures), histological parameters (fibrosis scoring system in 4 stages, necroinflammatory activity grades and modification of architecture in 3 classes), and biochemical parameters via blood sample (ft for fibrosis and actitest for activity). the measurements of histological and biochemical parameters were done blind to any other characteristics. sensitivity (se), specificity (sp), predictive values (npv and ppv), spearman correlation (sc), accuracy (ac), kappa (k) and the area under the roc curves (auroc) were assessed. the main endpoint was the prediction of elevated portal pressure (epp), defined by a gradient of 2 5 mm hg. the secondary endpoint was the prediction of highly elevated portal pressure (hepp) of 212 mm hg (hepp). results: the mean ft value was 0.47 (se 0.05) for 15 patients without epp, 0.73 (0.04) in 23 patients with moderate epp and 0.87 (0.03) in 49 patients with hepp (p<o.ol between all groups, dunnett's multicomparison test) (figure 1 ). high aurocs of ft were obtained for the prediction of epp, 0.86 (0.04) and hepp 0.78 (0.05); a cutoff at 0.70 had a 95% ppv for the presence of epp (se=76%, sp=80%) and a 0.45 cutoff had a 100% npv for the exclusion of hepp (se=100%, sp=28%). there was a significant correlation between ft and gradient (sc= 0.50, p<o.ool). the auroc of ft for the histological architectural disturbance (fibrosis or cirrhosis) was very high 0.94, (0.03). there were also excellent ft diagnostic values for stages f2f3f4 ac=87%; k=0.54 (0.11); sc =0.57, p<o.ool; auroc=0.87 (0.04). therefore histological staging did not give higher diagnostic values than ft for epp, auroc=0.91, (0.03) introduction: the prevalence of gastric varices ranges from 20-25% in cirrhotic patients and is associated with high morbidity and mortality rates. conventional endoscopic hemostatic treatment is not effective for gastric variceal bleeding. tips can achieve initial control of bleeding but it is associated with high cost, shunt dysfunction, and increased encephalopathy. cyanoacrylate injection has been shown to be very effective with 90% hemostasis and low rebleeding rates of 0-28%. in our current series of gastic varix patients at the university of virginia, injection with n-butyl-2-cyanoacrylate (histoacryl) has yielded similar results (scaldwell, fda-ide). however, distal embolization after n-butyl-2-cyanoaaylate injection has been reported and probably results from "beading" upon polymerization. whether other cyanoacrylates with different physical-chemical properties have less embolic risk with equal or greater efficacy remains to be determined. &to investigate and compare the efficacy and safety of two different cyanoacrylates in the management of gastric varices. meth-ods:polymerization properties of two cyanoacrylates, n-butyl-2cyanoacrylate (histoacryl) and octyl-cyanoacrylate (dermabond), were compared in vitro by adding a drop of blood to different concentrations of n-butyl-2-cyanoacry1atel:ethiodol and octyl-cyanoacry-1ate:ethiodol (ethiodol, iodinated poppy seed oil, is radio opaque and delays polymerization). the two cyanoacrylates were then compared for "beading" by injecting each cyanoacrylate solution into a flowing circular system of fresh frozen plasma and then collecting the resultant polymer. lastly, cyanoacrylate injection was tested in an animal model using the femoral vein of a pig to simulate a gastric varix. each femoral vein was injected with n-butyl-2-cyanoacrylate and octyl-cyanoacrylate, respectively, and then both veins were ligated and examined for histologic change. results n-butyl-2-cyanoacry1ate:ethiodol (1:l) and octyl-cyanoacrylate alone had similar polymerization properties as far as initiation of polymerization to formation of a "hard substance"these concentrations were used for subsequent testing. preliminary results from the circular flowing system show that n-butyl-2-cyanoacrylate forms a "beaded" and "crystallized" polymer whereas the octyl-cyanoacrylate polymer is more "stringy" and "rubbery". in the animal model, n-butyl-2-cyanoacrylate had formed a large, extended thrombus in the vein with an irregular surface that appeared granular. in contrast, octyl-cyanoacrylate appeared uniform throughout and occluded the vein wall with only a s m d thrombus in the center. conclusion octyl-cyanoacrylate appears to be a viable alternative with possible lower embolic risk. vessel occlusion appears to involve a combination of polymer and thrombus formation, although interaction with the dotting cascade has not been adequately investigated. further studies in an animal model of gastric varices are in development to assess the relative safety and efficacy of these and other cyanoacrylates. disclosures: stephen evgeny farber, doron fisher, rami eliakim, nira beck-razi, ahuva angel, ella veikman, irit chermesh, camal yassin, diana gaitini, michael libas, rambam medical center, ha+, israel; soboh soboh, porria hospital, tiberia, israel; yaacov baruch, rambam medical center, haqa, israel background: current guidelines recommend that patients with liver cirrhosis, undergo egd screening every 1-2 years for the presence of ev. patients with large ev should be treated with beta-blockers and those with small ev should be rescreened. platelet count and us correlate with high risk patients but are not accurate enough to avoid endoscopy in low risk patients. barium swallow is preferred by most patients but data concerning its sensitivity is not sufficient. to evaluate the accuracy of be to diagnose esophageal varices in patients with compensated cirrhosis and portal hypertension at an early stage. patients and methods: 45 patients with liver cirrhosis where evaluated by egd (japanese general criteria), as a gold standard and be for the presence and size (small, large) of varices. x-rays were taken within 3 weeks of endoscopy, with 98% barium sulfate (e-z-hd), as suspension adjusted for double contrast studies and barium paste (e-z, 60%, esophageal cream) for mucous coating. results: 45 patients diagnosed by liver biopsy; 60% postnecrotic, 20% cryptogenic, 10% nash, 10% alcohol. 90% were child-pugh's score a and 10% were child-pugh's r. the correlation between egd and be was very high; r=0.84, p<o.oool. there were only 4 false negative results with be. overall sensitivity of re was 89%. all large varices (f2-f3) were diagnosed by be. sensitivity declined with f1 varices to 73%. the positive predictive value was 86%. the cost of x-rays was one fourth that of egd. conclusions: re can be used as a non invasive procedure for the screening of patients with varices and compensated liver disease at a minimal cost. using be will allow to avoid universal prophylaxis with beta-blockers. studies dealing with the prevention of rebleeding have shown that hemodynamic response to pharmacological treatment of portal hypertension is adequate when the hepatic venous pressure gradient (hvpg) decreases to ?12mmhg or by ?20% from baseline. with beta-blockers these targets are achieved in only around 30% of cases. however, even with such a high rate of poor hemodynamic response, variceal bleeding occurs in few patients when beta-blockers are used for primary prophylaxis (around 15%). the aim of this study was to assess whether the cut-off value to define response in primary prophylaxis may be defined more accurately and to investigate whether the acute response to beta-blockers may predict evolution (avoiding a second study). methods: a total of 101 cirrhotic patients with large varices and without pre-vious bleeding were investigated. a baseline hemodynamic study was performed in all patients. after initial measurements, propranolol was administered in 55 patients (0.15mg/kg i.v.) and measurements were repeated 20 minutes later. subsequently, nadolol was chronically administered to prevent variceal bleeding. a second hemodynamic study was performed 1 to 3 months later in 72 patients. results: during a mean follow-up of 37 months, 17% of patients had a bleeding episode and 19% died. there was a significant correlation between acute and chronic hemodynamic response to beta-blockers (r=0.6, p=o.ool). using roc curve a decrease of hvpg ?lo% from baseline was the better cut-off point to predict bleeding, both for acute and chronic studies. hemodynamic responders (defining response as a decrease of hvpg to ?12mmhg or ?lo%), had a significantly lower probability of bleeding (p?o.ol) and of requiring hospital admission because decompensations of cirrhosis, while survival probability was higher, both after acute and chronic beta-blocker administration. the hvpg decreased ?20% in 38% of cases and decreased ?lo% in 74% (p?o.ool) after acute beta-blocker. these figures were 31% vs 68% (p?o.ool) after a chronic administration. conclusions: our results suggest that: 1) a decrease of hvfg ?lo% from baseline may be the target to identify hemodynamic responders in primary prophylaxis of variceal bleeding; 2) ,4cute hemodynamic response to beta-blockers may provide an accurate prognostic information on long-term risk of variceal bleeding. ut; william m grosse, corneliu sanda, milton taylor, lndiana university, bloomington, in treatment of chronic hepatitis c patients with type 1 interferon (ifn-alpha) and ribavirin leads to a sustained virologic response in -50% of patients. the elimination of serum hcv rna in an individual patient displays biphasic kinetics with the first order attributed to the direct antiviral effects of ifn-alpha and the second order attributed to an immune mediated clearance of infected cells by induction of th1 cytokines, primarily ifngamma (type 2 ifn). we have postulated that the direct antiviral effects as well as the th1 response of current therapies may be enhanced by the addition of type 2 ifn (ifn-gamma). to study this hypothesis, we examined the activity of a type 1 ifn, infergen; (ifn-alfaconl) in combination with a type 2 ifn, actimmune; (ifn-gamma l b ) in preclinical models of hcv. these included an infectious flavivirus system (west nile virus) and the hcv replicon. in addition, global transcriptional profiling was examined utilizing the hg-u133a genechip; (affymeirix, santa clara, california) system. effective concentration (ec) studies of the l effects of ifn-alfaconl, ifn-gamma l b and the combination against the west nile virus and the hcv replicon are shown below: analysis of synergy for the combination of ifn-alfaconl and ifngamma l b in the hcv replicon using the chau-talalay methodology demonstrated very strong synergistic effects for a range of doses (c1<0.1 for all observations). initial statistical analysis (pv<o.ool; fold change 2 1.2) of global transcriptional profiling revealed that treating hcv replicon containing cells with ifnalfaconl up-regulated 156 transcripts, while 61 transcripts were significantly down-regulated. ifn-gamma l b treated cells had 109 transcripts up-regulated and 32 down-regulated transcripts. most significant was the finding that several transcripts were up-regu-lated by the combination of ifn-alfaconl and ifn-gamma l b that were not up-regulated by either ifn alone. these transcripts were involved in cell-cycle regulation, antigen presentation, apoptosis and immuno-activation. in addition, several known interferon stimulated genes were highly elevated by the combination when compared to the monotherapy treatments. these results demonstrate synergistic effects of the combination of type 1 and type 2 ifns for the treatment of chronic hepatitis c both on a cellular and molecular level. further study in hcv infected patients is warranted. hcv is leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. clearance of hcv is associated with strong t cell immunity to hcv immunogens including the non-structural ns3 protein (ns3). dendritic cells (dcs) play a pivotal role in priming rare antigen specific t cells to initiate protective hcv immunity. therefore, dcs are an important target to elicit broader and more robust immune responses against hcv. to target immunogens to dcs, 12-mer peptides were screened from a phage display peptide library for specific binding to human dcs. a candidate 12-mer peptide (dc-peptide# 3) was identified that bound human monocyte-derived dcs. dc-peptides bind to ligands that are strongly involved in receptor mediated endocytosis. dc-peptides could be linked to hcv ns3 biochemically or fused genetically with preservation of dc targeting specificity and immunogenicity. dcs pulsed with ns3 that has been genetically fused to dc peptide augmented t ceil proliferation >3 fold better thanns3 alone, and skewed t cells towards thl polarization resulting in elevated levels of ifn-.)i (higher than with ns3 alone) but not il-4 or il-10. t cells also acquired an activated phenotype (cd69+). to test in viva efficacy of ns3-dc peptide fusion protein, nod-scid mice were xenotransplanted with hcv-nake t cells, and vaccinated with three separate weekly injections of autolo-gous dcs (106) pulsed with nothing, ns3 alone, or the ns3-dc pep-tide#3 fusion construct. vaccination using dcs pulsed with ns3-dc peptide# fusion protein significantly increased ns3-specific t cell proliferationlactivation and enhanced the expression of ifn-y, and tnf-a compared to ns3 alone but no detectable levels of il-10 or il-4 were observed. these data indicate that targeting hcv subunits to dcs using specific dc-peptides represents a novel vaccine approach that may facilitate targeting of immunogenic antigens to dcs to elicit specific t cell immune responses against ns3 of hcv. this immunotherapeutic strategy can be implemented against the broad range of immunogenic antigens of various pathogens. it has recently been reported that the administration of eta in the setting of chronic hcv infection enhances ifn-induced viral clearance (1). the mechanism of these synergistic effects remains to be understood. our hypothesis was that eta enhances antiviral effects of ifn by increasing t cell reactivity to antigens. methods: peripheral blood mononuclear cells (pbmcs) were isolated in cpt tubes from blood of two healthy volunteers. cells were washed and then cultured in complete media 24-well plates in the absence of any antigen (negative control) and in the presence of immobilized anti-cd3 antibody (i-cd3, 150 nglml, positive control) as a nonspecific t cell stimulant. i-cd3 stimulated cells were treated immediately with peg ifn alpha-2a at two different concentrations consistent with physiologic doses in humans (10 nglml or 100 nglml) with or without the addition of eta at two different concentrations that were also consistent with physiologic doses in humans (1.65 pglml or 3.30 pglml). supernatants from each of the previous conditions were collected and assessed by elisa for ifn-7 secretion as a marker of t cell activation. results: our findings are displayed in the figure below. there was no spontaneous ifn-7 detected in cells that were not stimulated. ifn-y was detected at a modest level after exposure to i-cd3 alone, a response that became greater after exposure of cells to peg ifn alone or eta alone. production of ifn-7 was substantially enhanced in cells that were exposed to a combination of peg ifn and eta compared to those exposed to peg ifn alone or eta alone. conclusion: our findings suggest that eta has a synergistic effect to that of peg ifn in promoting in vitro activation of pbmcs and ifn-7 production in healthy individuals. increased t cell activation with eta may also suggest that tnfa suppress t cell function and may contribute to refractoriness to inf therapy in hcv patients. the influence of absolute dose and dose in mg per kg body weight of ribavirin on sustained virologic response (svr) in interferonbased combination treatment of chronic hepatitis c as well as the influence of dose reduction is still controversial. here, we address this problem by reanalyzing data of 343 previously untreated patients with chronic hepatitis c from a multicenter trial who were treated with interferon alfa-2a plus ribavirin and amantadine or interferon alfa-2a plus ribavirin (berg et al, hepatology 2003 ,37,1359 -1367 and completed therapy. thereby, we used a multivariate approach to account for correlations of the dose of ribavirin with body weight and body mass index (bmi) and known predictor variables from this data set which are low baseline hcv rna, high platelet counts, high pretreatment alt, and low y glutamyl transpeptidase (ggt) as well as hcv genotype non-1. because per protocol dosage of ribavirin was weight-adjusted (1000 mg for body weight below 75 kg and 1200 mg for body weight 75 kg or more) and body weight was positively correlated with ggt and negatively correlated with platelet counts (spearman rank correlation, p<o.ol% and p=0.2'x), respectively, we used a stratification with respect to genotype (gt 1 vs. non-1), ggt, and platelet counts in the analysis of ribavirin dose. when comparing body weight, bmi, the absolute intention to treat (itt) ribavirin dose as well as the itt dose of ribavirin measured in mg per kg body weight and the respective ribavirin doses at the end of treatment (eot), a significant association with svr was found for the eot ribavirin dosage (mg per kg body weight) and bmi (p=1.8% and p=3.0%, respectively). for predicting svr, a weight-based ribavirin dosage threshold of 13.75 mg/kg was calculated using receiver operating characteristics (roc) curves. the svr rate was 66% (1121169) in patients with a ribarivin dose of more than 13.75 mglkg as compared with 46% (79l172) in patients receiving equal or less than 13.75 mglkg ribavirin at eot (odds ratio 2.3; 95% ci 1.5-3.6; p=o.l%). in conclusion, the analysis of ribavirin dosage motivates new considerations of weight-adjustments of the ribavirin dosage to further increase svr in hcv-infected patients treated with combination therapy. effective and approved for treatment of patients with chronic hepatitis c virus (hcv) infection, the medications have many side effects and frequently require dose reduction or discontinuation. improved adherence to the medical regimen increases sustained response rates. data currently available regarding dose reduction, discontinuation and svr are largely derived from registration trials with experienced hepatologists at academic centers. anecdotal information suggests that dose reduction and discontinuation rates are higher and svr lower in community practice, where the majority of hcv patients are treated. actual dose reduction and discontinuation rates and svr in community practice are unknown. aim: we sought to determine dose reduction and discontinuation rates and svr in community practice in comparison with academic centers within the context of a prospective, randomized, controlled, multi-center trial. methods: patients with chronic hcv (+hcv rna) were evaluated at academic centers and community sites within the context of a randomized trial comparing peg ifn a (1.0 pglkglwk) + ribavirin (800-1400 mgld) vs. peg ifn a (1.5 pglkglwk) + ribavirin (800-1400 mg/d). demographic, serological (alt), virological (hcv rna level and genotype) and histological data were obtained. eligible patients were randomized to one treatment group. medications were administered for 24 weeks (hcv gt non-1) or 48 weeks (hcv gt 1). tolerability (dose reduction rates, adverse events and discontinuation rates) through week 24 of therapy was determined and comparisons were made between academic (ac) and community-based centers (cc). results: 294 patients have been enrolled, 74 in ac and 220 in cc. 6 patients in ac and 80 in cc remain on therapy within the initial 24 weeks and are not included in this report. genotype, histology, gender and age profiles were similar in the 2 groups. * designates a significant difference p<0.05. by week 24,7168 (10 %) in ac discontinued medications in comparison to 29/140 (21%) in cc". 3/68 (4%) discontinuations were due to aefsae in ac in comparison to 101140 (7%) in cc. 2155 (4%) patients in ac who did not discontinue by wk 24 received peg ifn dose reduction in comparison to 23/108 (21%) in cc". 14/55 (25%) patients in ac who did not discontinue by week 24 received ribavirin dose reduction in comparison to 31/108 ( the clinical outcome in response to combination therapy for treatment of chronic hepatitis c virus (hcv) infection appears to be different for caucasian versus african american patients. combination therapy of ribavirin and interferon alpha-2b has generally been found to be more effective for eliminating hcv in caucasians. the present study has been undertaken to further define this difference in response to therapy between caucasians and african americans and to determine whether the difference may be related to a difference in t cell-dependent immune responses to hcv in the two patient populations. to this end peripheral blood mononuclear cells (pbmc) were collected from patients prior to the initiation of combination therapy with ribavirin and pegylated interferon alpha-2b, week 0, and at 28 weeks and 44 weeks after initiation of therapy. the pbmc were stimulated in vitro with recombinant hcv-superoxide dismutase (sod) fusion proteins for ns3 (sod-c33c), core (sod-c22-3), ns4 (sod-c100-3), ns5 (sod-nss), and the relevant recombinant sod controls from yeast or bacteria (all generous gifts from dr. michael houghton, chiron). after four days, 3h-tymidine was added to the cultures and incubation continued for 16 hours to measure proliferation. the cultures were harvested on day five. supernatants from identical cultures were collected for assay of th1, il-2 and interferon gamma (infy), and t h~, il-10 and il-4, cytokine production. t cell responses to tetanus toxoid and the mitogen phytohemagglutinin (pha) were used as positive controls for t cell response potential. to date 68 patients have entered the study. week 0 data has been collected from 64 patients and 9 normal controls. data have been collected from only 25 patients that have completed the study through week 44. the results indicate that generally all patients' t cells were stimulated by pha and to lesser extent tetanus toxoid. the major cytokine stimulated by both pha and tetanus toxoid was infy. few of the patients had significant t cell responses to hcv proteins at time 0 measured either as proliferation or cytokine production. over the period of combination therapy, most patients in which serum virus titers were reduced to non detectable levels had significant increases in t cell responses to hcv proteins. although t cell proliferation was varied among the patients, there was generally a marked increase in infy production but little production of other cytokines. responses to hcv proteins were less consistent in those patients that either did not clear the virus or in which there was resurgence of virus after initial clearance. hepatitis c virus (hcv) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer (hcc). the high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for hcv. the hcv ns3 serine protease is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. intracellular biological processes can be specifically manipulated by anti-viral antibodies. we have recently presented the modulation of hcv ns3 serine protease transforming activity by non-neutralizing recombinant intracellular antibodies (intrabodies). we herein report the development of a novel bacterial genetic screen for inhibitors of ns3 serine protease catalysis and its application for the isolation of single-chain antibody-inhibitors. our screen is based on the concerted co-expression of a reporter gene, of recombinant ns3 and of stabilized single-chain antibodies (scfvs) in e. coli. the reporter system had been constructed by inserting a short peptide corresponding to the ns5aib cleavage site of ns3 into a permissive site of the enzyme p-galactosidase. the resulting cleavable lac2 gene is carried on a low copy plasmid that also carries the ns3 coding sequence. the resultant p-galactosidase enzyme when active, conferring a lac' phenotype (blue colonies on indicative x-gal plates) while co-expression of active ns3 results in loss of / 3 -galactosidase activity (transparent colonies on x-gal plates). the identification of inhibitors, as shown here by isolating ns3 inhibiting single-chain antibodies is based on the appearance of blue colonies (ns3 inhibited) on the background of colorless colonies (ns3 active). our source of inhibitory scfvs was a scfv library we prepared from spleens of ns3-immunized mice. once isolated, the inhibitors were validated as genuine and specific ns3 binders by an elisa and as bone fide ns3 serine protease inhibitors by an in vitro catalysis assay. these antibodies are also analyzed for intracellular immunization against hcv ns3 serine protease, as inhibiting intracellular antibodies. single chain ns3 neutralizing antibodies may emerge as useful clinical reagents for the treatment of infectious diseases and cancer. our genetic screen, although applied here for the isolation of antibody-based inhibitors, should be applicable for the identification of candidate inhibitors from other sources. 1ntroduction:genetic heterogeneity is an important feature of hepatitis c virus (hcv) genomes with six known genotypes and more than 50 subtypes. genotypes 1,2 and 3 are broadly distributed in patients around the world, whereas the other types are more geographically restricted. the peptidomimetic inhibitor biln 2061, optimized to have a high affinity for genotype 1 ns3 protease, has been reported to reduce viral load in genotype 1 hcv infected individuals. in this study, the ability of biln 2061 to inhibit the ns3 proteases from the other widespread genotypes 2 and 3 was assessed. methods: the ns3 protease domains and the ns3-ns4a proteins of genotypes la, lb, 2ac, 2b and 3a were cloned, expressed in e. coli and purified. protease activity was evaluated using fluorogenic depsipeptide substrates derived from the amino acid sequence at the ns4a-ns4b and ns5a-ns5b junctions. results: the activity of the ns3 protease domains revealed no major differences among the various genotypes. for the ns3-ns4a proteins, the catalytic efficiencies of the non-genotype 1 enzymes, although higher than the ones observed for the corresponding protease domains, were similar to that observed for genotype 1 (within 3-fold). differences in activity observed among genotypes were mainly related to changes in k,,, values. binding constant (k,) values for biln 2061 were similar among non-genotype 1 proteases with k,'s ranging from 80-90 nm for the ns3-ns4a proteins, up to a 60-fold reduction in affinity when compared to genotype 1. hepatitis-c-infection (hcv) is associated with an increased incidence of the chronic fatigue syndrome and depressive mood changes. beside immunological changes the modulation of the serotonergic system might be related to these psychiatric syndromes. serotonin concentration, 5-ht uptake and the activity of the enzyme mao-b in platelets are useful peripheral parameters to monitor serotonergic activity and function in patients with a chronic hepatitis c (chc). method in a prospective controlled study biochemical measures included assays of 5-ht concentration and 5-ht uptake activity at a low physiological substrate concentration in platelets as well as mao-b activity of 64 patients with a chronic hepatitis c were compared to 21 age and gender-matched healthy subjects. furthermore the effect of interferon-alpha treatment on serotonergic parameters was evaluated in 30 patients before and during treatment. results: the mean serotonin platelet concentration did not differ between controls and hcv-infected patients. however, 5-htuptake and the mao-b activity were significant lower in patients with hcv-infection (p<o.ool respectively, t-test, two-tailed). interferon-treatment led to an increase in 53it reuptake (p=o.ol), to an increase of mao-b activity (p<o.ool) and to a decrease of serotonin concentration (p<o.ol). thus, during hcv-infection serotonin concentration was stabilized by a decrease in re-uptake and reduced metabolism by lowering mao-b activity. treatment with interferon-alpha led to a decompensation of those mechanisms by increasing the mao-b activity and 5-ht-reuptake, followed by a decreased serotonin concentration. conclusion: our data support the hypothesis of a tryptophan depletion and serotonergic deficit in patients with chronic hcvinfection and interferon-alpha treatment. furthermore the results give evidence for a useful role of serotonin reuptake inhibitors for the treatment of the serotonergic deficiency during chronic hcvinfection and interferon-alpha treatment. chronic background: recent large prospective trials demonstrated that the combination therapy of interferon (1fn)-alphalribavirin significantly increased the ratio of a sustained virological response in patients with chronic hepatitis c in comparison with ifn monotherapy, especially in patients with high hcv-rna titer and genotype lb. however, the mechanism of beneficial effect of ribavirin is still unknown. on the other hand, interleukin 18 (il-m), originally termed ifn-y inducing factor, is one of proinflammatory cytokines, which acts on th-1 cells and strongly induces intrahepatic nk or nkt cells as well as activated t cells to produce ifn-y. to clarify the effect of ribavirin in combination with ifn, special emphasis on il-18, we examined the correlation between serum il-18 level and anti-viral effect, and compared it with ifn monotherapy in patients with chronic hepatitis c. patients and methods: all patients in this study are biopsy proven chronic hepatitis c with high hcv-rna titer (hcv-rna>loo kcopieslml before therapy) and serotype 1 (genotype l a or lb). forty-two patients were treated with ifn alpha-2b (6 mu) in combination with ribavirin (600-800 mg) daily for 2 weeks, following 22 weeks with ifn alpha-2b (6 mu) 3 times a week (the combination group). another 40 patients who were treated with ifn alpha-2b alone between 1998 and 1999 (before introduction of ribavirin in japan) with the same schedule are used as control (the monotherapy group). serum samples were taken before, 2 weeks after administration and 12 weeks after cessation of therapy. serum il-18 are examined by enzyme linked immuno-sorbent assay (elisa), using human il-18 elisa kit (mbl, nagoya, japan). the il-18 ratio is defined as serum il-18 level before administration divided by serum il-18 level 2 weeks after administration. hcv-rnas are quantitatively examined before and 2 weeks after administration. sustained viral responses are confirmed 12 weeks after cessation of administration by rt-pcr. results: there are no differences in the background of patients between the combination group and the monotherapy group. in the combination group, the decline of hcv-rna level highly correlates with the il-18 ratio in patients with higher viral titer (hcv-rna>500 kcopieslml before therapy) despite no correlation is observed in patients with lower viral titer (hcv-rna<soo before therapy). similarly, the hcv-rna level 2 weeks after administration controversially correlates with the il-18 ratio in the higher titer group despite no correlation in the lower titer group. interestingly, serum il-18 level itself does not correlate with the decline of hcv-rna level in both the higher and lower hcv-rna titer group. on the contrary, in the monotherapy group, the level of up-regulation of serum il-18 is lower than that of the combination group. furthermore, although the decline of hcv-rna also correlates with the il-18 ratio in the higher titer group, the level of a correlation coefficient is lower than that of the combination group. however, the sustained viral clearance 12 weeks after cessation of treatment is not correlated with the il-18 ratio. it suggests that ribavirin enhances the up-regulation of serum il-18 level in combination with ifn therapy and the effect of ribavirin is critical for the early anti-viral response during therapy, not for sustained viral response. objective: treatment with pegylated interferon alfa and ribavirin has an efficacy in chronic hepatitis c patients with sustained response rates of about 50%. however, response in genotype 1 patients with high viral load is considerably lower and is not significantly improved by pegylation of interferon. methods: in the present study, the efficacy of cifn daily dosing and induction therapy followed by combination with ribavirin in 200 naive patients with chronic hepatitis c and genotype 1 was evaluated. au patients had histologically proven hepatitis, elevated alt values and were viremic, the average weight was 80 kg. patients were treated with cifn dosages of 27 or 18 ug qd for 4 weeks, followed by 18 or 9 ug qd for 8 weeks. treatment was continued with cifn at 9 ug qd with ribavirin (7.5 or 15 mglkgld) for another 36 weeks. results: at 48 weeks therapy an undetectable hcv-rna was observed in 79 % and 72 % in the cifn 27/18 and 18/9 ug groups, respectively. data regarding sustained response rates showed 64 '10 and 58 % for cifn 27/18 and 18/9 ug groups, respectively. due to side effects cifn had to be dose reduced in 11% and discontinued in 6% of patients. addition of ribavirin potentiated the effect of consensus interferon in a dose-dependent manner as has been shown for other interferons previously. when viral response rates were related to the initial viral load, in the genotype 1 i low viral load group, sr rates of 71 and 74% were obtained for the cifn 27/18 and 18/9 ug groups (diff. n.s.). in contrast, genotype 1 / high viral load patients showed sr rates for the cifn 27/18 and cifn 18/9 ug groups of 41 and 49% (diff. sig.), respectively. the viral response rates obtained with daily dosing of cifn were significantly higher than an independently run control arm using cifn at 9 ug tiw with ribavirin irrespective of the initial viral load. four patients (2%) experienced grade i11 thrombocytopenias, while no grade iv neutropenias or thrombocytopenias were observed. the overall tolerability in the cifn 18/9 ug qd arm was comparable to standard therapy with pegylated interferon a2b and ribavirin, while the cifn 2711819 ug arm was less tolerable during the high dosing period. however, the withdrawal rates were not sigruficantly affected by the higher dose of cifn. conclusions: cifn daily dosing l induction therapy in combination with ribavirin thus shows significant response rates with the highest response rates in genotype 1 patients seen to-date. the sustained response rates for genotype 1 i high viral load patients are higher than the ones demonstrated in the peg interferon and ribavirin registration trials. these data suggest that for difficultto-treat genotype 1 / high viral load patients cifn with daily and higher initial dosing may be a worthwhile alternative to standard combination therapy with pegylated interferons. introduction: hcv-induced cirrhosis or hepatocellular carcinoma account for at least a quarter of all patients undergoing liver transplantation. almost 100% of hcv related liver transplant patients are re-infected with hcv after liver transplantation, often within days. in most of these cases, the recurrent infection leads to chronic hepatitis. these patients are in need of preventive therapy against re-infection of the transplanted liver. antibodies may be used as a stand-alone prophylactic therapy to prevent re-infection following liver transplantation or in cases of accidental exposure (e.g. needle stick injury). in addition, it may be feasible to use monoclonal antibodies in combination with other antivirals to treat chronic hcv patients. purpose: to develop an effective therapy to prevent hcv infection in liver transplant patients. methods: several human monoclonal antibodies were generated from peripheral blood b cells isolated from individuals infected with hcv genotype lb. these antibodies are directed against the hcv envelope protein (e2) of the virus and have high affinity and broad reactivity against different genotypes. the antibodies were shown to neutralize hcv in vitro in a cell-based infection assay and in vivo using the hcv-trimera mouse model. hepex-c, a single human monoclonal antibody, was further developed and studied clinically for safety, tolerability, and potential antiviral activity in phase l a and l b clinical studies in chronic hcv patients. results: in a phase l a study, single doses of 0.25, 1.0, 2.5, 10, and 40 mg of hepex-c were administered to a total of 15 chronic hcv patients. these patients had varying levels of hcv-rna (2.6 x lo3 to 1 x lo6 iulml) and endogenous anti-e2 antibodies (5 to 550 pglml). doses of hepex-c were well tolerated and no moderate or serious adverse events (sae) were reported. in 8 out of 15 patients, hcv-rna levels were reduced at least by half immediately following infusion and then gradually returned to initial levels. a multidose phase l b study of 10,20,40,80, and 120 mg of hepex-c in 25 hcv chronic patients was conducted. doses were administered weekly for 3 weeks and then 3 times a week during the fourth week. the patients were followed for an additional 3 months. multiple doses were well tolerated; no drug related saes were reported and no specific pattern of ae noted. 9 out of 20 patients from the 10 through 80 mg cohorts had at least 1 log infusion related reduction in hcv-rna from pre-treatment levels following administration of hepex-c. data from the 120 mg cohort are pending. conclusions: the good safety and efficacy data from this trial provided a basis for a phase 2a pilot study of the safety and efficacy of hepex-c for the prevention of recurrence in hcvinfected patients who have received a hepatic allograft for endstage liver disease. a multicenter, blinded, placebo controlled study is currently underway in 19 liver transplant patients receiving 20, 40, 80, and 120 mg doses of hepex-c. logic response rates to interferon (ifn) therapy in aa patients w i t h chronic hcv infection have been reported to be lower than that for caucasians (ca), but the effect of therapy on hepatic histology in aa patients remains to be assessed. objective: to evaluate histologic progression of disease in conjunction with anti-viral efficacy and safety in non-hispanic aa hcv genotype 1 patients and non-hispanic ca, hcv genotype 1 patients treated with peginterferon alfa-2a (40kd) in combination with ribavirin (rbv). methods an open-label, multicenter study was conducted to evaluate the efficacy and safety of the combination of peginterferon alfa-2a (4okd) and rbv in aa patients and in ca patients. all patients were required to have previously untreated chronic hcv genotype 1 infection, elevated alt levels and. a liver biopsy obtained within the 24 months prior to enrollment demonstrating chronic hepatitis but without cirrhosis. patients received peginterferon alfa-2a (4okd) 180 pg sc once weekly plus rbv 1ooo or 1200 mg orally based on body weight (<75 kg or =75 kg) for 48 weeks, with 24 weeks of treatment-free follow-up. histologic responses based on the knodell hai score were determined from liver biopsies obtained prior to treatment and within 4 weeks of completion of the 24-week untreated follow-up period. hai activity scores range from 0-18 and fibrosis scores range from 0-4. the histology outcome was evaluated for patients with paired biopsies only. improvement in necrointlanunatory activity was defined as 2 2 point decrease, and improvement in fibrosis as 2 1 point decrease. the primary efficacy endpoint was sustained virologic response (svr) with undetectable hcv rna (<50 iulml) at 72 weeks. results: the intent-to-treat population included 106 patients. paired biopsies were available for 53 of the 78 aa patients and 16 of the 28 ca patients. these patients were representative of all patients with respect to knodell hai scores. mean baseline hai activity scores were 7.2 0.32 for aa and 6.9 & 0.69 for ca patients; mean baseline fibrosis scores were 1.8 + 0.14 and 1.9 + 0.3, respectively. the svr rate for all aa patients was 20l78 (26%) and for all ca patients, 11/28 (39%). for those patients with paired biopsies, svr rates were 17/53 (32%) in aa patients and loll6 (63%) in ca patients. the majority of patients in both groups exhibited improvement in activity scores: 64% for the aa group and 69% for the ca group. more importantly, the table beiow shows that the proportion of patients with worsening fibrosis was similar in both groups, while 13/53 (25%) of aa patients and only 1/ 16 (6%) of ca patients showed fibrosis improvement. mean changes in fibrosis scores were -0.4 +-0.14 for aa and -0.1 t 0.14 for ca patients. among aa patients who had paired biopsies, 5/17 (29%) patients who achieved svr had fibrosis improvement; and 8/36 (22.2%) viral nonresponders exhibited fibrosis improvement. the only patient who had fibrosis improvement among ca was a viral non-responder. conclusions: therapy with peginterferon alfa-2a (4okd) plus rbv produced improvement in necroinflanunation and fibrosis in a substantial group of aa patients in this study. although the svr of 26% in aa with genotype 1 hcv is the highest response to combination therapy yet reported in this population, even patients without an svr achieved a benefit with regard to hepatic histology. higher sustained response rates (svr). however, modification of interferon-alfa by pegylation also reduces biologic potency. delivery of a bio-optimized alpha interferon in an unmodified form (consensus interferon, infergen(r)) by continuous infusion can be expected to provide sustained and constant levels of a fuuy potent protein. we hypothesize that such an approach may potentidy result in greater antiviral activity and improved tolerability by avoiding wide swings in interferon levels. methods:we conducted a phase 2 pilot study to assess the safety, tolerability and viral kinetics of mergen (12 fg/day) by continuous infusion using a subcutaneous infusion device (medtronic minimed(r) pump) in combination with ribavirin (loo0 mg or 1200 mg daily). the minimed model 508 micro infusion pump is cleared for use in the treatment of diabetes. hcv rna viral levels were measured at days 1, 3,7,10,14,21,28 and then at weeks 6 and 12. results: among 10 nonresponders treated to date with infergen by continuous infusion with available early viral kinetics data, 7 showed a substantial reduction in hcv viral levels (at least 1 loglo reduction in 4, and 2 loglo reduction in 3). all 7 of these patients were genotype 1 and 6 had less than 1.0 loglo decrease with prior treatment while 1 had a 1.5 loglo decrease at similar time-points with other combination therapy [either pegylated interferonlrbv (4 pts) or interferon-alfa/rbv (3 pts)]. no serious adverse events were reported and 3 patients discontinued (2 due to moderate interferon-related side effects and 1 due to inability to acclimate to the pump). other patients continuing on study experienced mild adverse events that have been tolerable. conclusions: consensus interferon administered by continuous delivery via the medtronic minimed pump shows early substantial reduction of hcv rna levels among nonresponder patients and shows a good safety and tolerability profile. updated data on a total of 22 patients (including 10 na'ive genotype 1 patients) will be presented. background side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in a given hcv patient population. furthermore, it has been suggested that higher ribavirin doses and longer treatment duration are required to achieve a sustained response rate in patients with genotype 1. coupling of ribavirin to hemoglobin offers the potential of a therapeutic with improved safety and efficacy by targeting the delivery of ribavirin to key tissues infected by viral infections such as hcv. these tissues include liver parenchymal cells and macrophages, both of which possess receptors for binding and internalization of hemoglobin-haptoglobin complexes as part of the natural clearance pathway for cell-free hemoglobin. hemoglobin thus serves as a natural drug carrier for targeted therapy of such receptor-bearing tissues. aim: to evaluate the effect of a ribavirinhemoelobin coniueate (rbv-hb. hrc 203) in a mouse model of viral hepatitis induced by the coronavirus, mhv-3. methods: ribavirin was covalently coupled to highly purified hemoglobin hbao at a mean molar drug ratio of 8 1 (ribavirin:hemoglobin). the rbv-hb was evaluated for retention of haptoglobin binding and cellular uptake in vitro, as well as the ability to recover bioactive ribavirin following enzymatic cleavage from rbv-hb. the rbv-hb was complexed to haptoglobin prior to administration to mhv-3 infected balblc mice, and carried one-third of the drug dose that was used for the ribavirin group. the rbv-hbtreated group was compared to control infected untreated and ribavirin-treated groups for survival, clinical behavior, viral titer, biochemistry and liver histopathology. results: rbv-hb was specifically internalized by cultured human and mouse hepatic cells but not by control non-hepatic cells. ribavirin enzymatically cleaved from rbv-hb retained in vitro bioactivity similar to unmodified ribavirin. untreated and mhv-3 infected mice all developed clinical and biochemical signs of acute viral hepatitis and died by day 4 post infection (altmax 7000 iuil). livers recovered from untreated and infected mice showed greater than 90% necrosis. in contrast, survival was enhanced in both ribavirin and rbv-hb treated and mhv-3 infected mice with a marked reduction in biochemical (alt 1000 iuil) and histologic evidence of hepatic necrosis ( 4 0 % ) . clinically, rbv-hb treated mice behaved normally at all time points, in contrast to ribavirin treated mice which developed lethargy and abnormal fur texture compatible with drug toxicity or ongoing viral infection. conclusions: in this study, a beneficial effect of rbv-hb was shown on the course of mhv-3 infection as demonstrated by prolonged sunival, improved clinical behavior, and a reduction in biochemical and histologic disease. the study provides a rationale for additional studies to examine the mechanism for the beneficial effect. ( introduction: the teravic-4 study targets patients with chronic hepatitis c who do not show a very early viral response at week 4 of therapy, as defined by detectable serum hcv rna (>50 kjlml, pcr methodology). after 48 weeks of therapy, these patients are expected to achieve a lower rate of sustained viral response (svr) than patients with undetectable hcv rna at week 4 (vr4). models based on viral dynamics and data from pilot studies with conventional interferon (ifn) suggest that a treatment period longer than 48 weeks may lead to a higher svr rate in these difficult-to-treat patients. this trial compares 72 versus 48 weeks of treatment with pegasys(') plus copegus('i in treatmentna'ive patients without vr4. methods: this phase 111, randomized, parallel group, multicenter study is being conducted in spain in accordance with the principles of good clinical practice.(l) after signing the informed consent form, all patients received the study treatment (peginterferon alfa-2a [40kd] [pegasys(']] 180 pg once weekly and ribavirin [copegus(')] 800 mg daily 1400 mg bid]) for 4 weeks. at this time, viral response (vr) was assessed using the cobas amplicor hcv(') test v2.0 (detection limit hcv rna 50 iulml). patients achieving vr4 continued therapy for an additional 20-or 44-week period, according to their hcv genotype and viral load. patients without vr4 were randomized 1:l to continue the study treatment for either an additional 44 weeks (total active treatment duration 48 weeks or an additional 68 weeks (total active treatment duration 72 weeks ). a 24-week follow-up period is ongoing to determine the svr rate. results: the mean age of the patients was 42 years, the mean weight was 73.5 kg, and 37% were female. the prevalence of hcv genotype 1 or 4 was 78%. vr4 occurred in 36% out of 511 patients, (23% of those with hcv genotype 1 or 4,83% of those with other hcv genotypes). hcv genotype distribution, viral load, age, and body weight were similar in the randomized study groups (group-48 and group-72). among patients without vr4, hcv rna was undetectable in 62"h in group-48 and in 69% in group-72 at week 24. the biochemical response rate (br) at week 24, defined as normal alt levels, was similar as well: 81% in group-48 and 78% in group-72. the dropout rate after 24 weeks was 9% in group-48 and 6% in group-72. at the end of therapy (eot), hcv rna was undetectable in 93% of patients with vr4. the eot vr rate in group-48 was 74% versus 81y0 in group-72. the rate of br in patients with vr at eot was also higher in group-72 (85%) than in group-48 (77%). dropout rates and reasons for dropping out by the eot visit were different between groups: 17% in group-48 and 36% in group-72. therapy was well-tolerated and no unexpected adverse events were observed. the prolongation of therapy from 48 to 72 weeks did not cause an increase in the frequency of neutropenia or thrombocytopenia. conclusions: pegasys(') 3 80 p g weekly plus copegus(') 400 mg bid administered for 72 weeks offers higher rates of vr and br at eot than 48 weeks of treatment in patients with detectable hcv rna four weeks after treatment initiation. the 800 mglday dose of ribavirin used in the trial has been proven to be optimal in patients infected with hcv genotype 2 or 3, but not in those with genotype 1 who require 100011200 mglday. this evidence was not established when the trial was initiated. "the teravic-4 study group also included v. ripollks background isis 14803 is an antisense phosphorothioate oligodeoxynucleotide inhibitor of hepatitis c virus (hcv). in a previous phase i study, plasma hcv rna reductions 2 1.0 log were observed in 3 chronic hcv patients treated with isis 14803 for 4 weeks. aims: the goals for this phase i1 study were to evaluate the safety, antiviral efficacy, and pharmacokinetics of isis 14803 given for 12 weeks. methods: the study was conducted in noncirrhotic chronic hcv patients. following 2 weeks of thrice weekly intravenous dosing at 2.5 mglkg ideal body weight (ibw), patients were treated for an additional 10 weeks at higher doses given either once (group a) or twice weekly (group b). the two doses studied were 4 (initial 3 patientslgroup) and 6 mglkg ibw. results: of 43 patients enrolled into the study, 41 had genotype 1 hcv, 39 had plasma hcv rna levels 2 2 x lo6 copieslml, and all but two had previously failed interferon-based therapies. two of the 16 group a and 5 of the 18 group b patients treated at 6 mglkg ibw had plasma hcv rna reductions 2 1.0 log. three in group b had reductions of 3.2-3.8 log. hcv levels remained 2.5 log reduced at 110 days after the last isis 14803 dose in one of these patients. seventeen patients had transient elevations in alt including all those with hcv rna reductions. peak alt levels ranged 5-30 times the upper limit of the normal range (x uln). for those with hcv rna reductions, the alt flares were temporally associated with the reductions. the alt flares were not associated with any clinical signs, symptoms, or sequelae. there were no changes in prothrombin time or albumin level. minor alkaline phosphatase (5 2.2 x uln) and bilirubin (5 1.5 x uln) elevations occurred in 2 patients. dosing with isis 14803 was continued during 7 alt flares, all of which resolved to baseline levels despite continued dosing. three flares, ranging from 9-29 x uln, occurred 3-8 weeks after the last dose of isis 14803, when presumably little or no drug remained in the liver. similar alt elevations have not been observed in the clinical studies of other phosphorothioate oligodeoxynucleotides. aside from the alt flares, common adverse events in this study were mild to moderate headache, fever, chills, fatigue, nausea, myalgia and other flu-like symptoms. the fever and chills usually occurred several hours after the end of the 6 mglkg infusions and resolved within 24 hours. two serious adverse events that were considered possibly related to isis 14803 occurred in the study: one patient had cryoglobulinemic glomerulonephritis and one was hospitalized for precautionary monitoring of an altlastlbilirubin flare. the former event was possibly due to an exacerbation of a pre-existing condition. the isis 14803 plasma pharmacokinetics observed in this study were consistent with those seen in the previous phase i study. conclusions: isis 14803 appears to have significant antiviral activity in some chronic hcv patients. the plasma hcv rna reductions (up to 3.8 log) achieved in this study were in difficult-to-treat patients (i.e., genotype 1, high virus level, andlor nonresponders to interferon-based therapy). although, alt flares were observed in some patients, the results suggest the flares may not be due to a direct hepatotoxic effect of the drug. other than the alt flares, isis 14803 treatment was generally well tolerated. this study established the dose of 6 mglkg ibw twice weekly for further clinical evaluation. a study of isis 14803 in combination with peginterferon and ribavirin for patients not achieving an early virologic response during standard peginterferon and ribavirin therapy is in progress. with the long serum half-life of albumin. the objectives of this phase 1/2, open-label, dose escalation study were to evaluate the pharmacokinetics, safety, tolerability, immunogenicity, and pharmacodynamics of albuferon in subjects with hepatitis c virus (hcv) who had previously failed ifna containing regimens. methods: subjects were initially enrolled in 4 sequential dose groups (10-12 subjects per each group at 7 pg, 20 pg, 40 pg, and 80 pg). within each dose group a minimum of 5 and a maximum of 6 subjects per cohort were enrolled sequentially to receive 1 or 2 subcutaneous (sc) doses of albuferon administered 14 days apart. dose escalation beyond 80 pg included single injections of 120 pg, 180 pg, 240 p g 320 pg and 400 pg. plasma albuferon concentrations and antibody to albuferon were measured by elisa. hcv rna was measured using the amplicor hcv monitor kit (roche). 2' 5' oligoadenylate synthetase (oas1) mrna levels in whole blood was measured by a research-based taqman pcr assay. results: of the 63 subjects currently enrolled, 96% were infected with hcv genotype 1 with a mean baseline viral burden of 2 million copies/ml. 78% of subjects enrolled in these cohorts had previously failed pegylated ifna containing regimens. albuferon pharmacokinetics showed linear increases in auco.8 and c, , , with mean terminal half-lives of 139-158 hours with doses of 80 pg and higher. t, , , occurred between days 4 and 5. there is an approximately 40% increase in auc after the second injection in the double injection cohort at 80 pg. albuferon was well tolerated and there were no discontinuations. no subjects developed detectable anti-albuferon antibodies. adverse events were transient and most were mild to moderate. the most common adverse events were injection site erythema (39%), headache (30%), fatigue (26%), mylagia (21%) and arthralgia (19%). the mean reductions in the nadir neutrophil counts in the higher single dose cohorts ranged from 40-60%. there was induction of oasl mrna expression in all cohorts, with approximately 9-fold increase in median values at day 7. in the single dose cohorts (=80 pg), 52% of subjects showed a maximal 0.55 log or greater reduction in hcv viral load during the first two weeks. also, 20% or greater reductions in alt levels were observed in 36% of subjects in these single dose cohorts. conclusions: in the ongoing phase 112 study, albuferon demonstrated a favorable safety and immunogenicity profile. the pharmacokinetic profile supports dosing every 2-4 wks given its reduced clearance and extended half-life of up to 158 hours. all cohorts showed evidence of biological activity, as demonstrated by oasl induction, with anti-viral activity evident in the higher single dose cohorts. disclosures: v balan -human genome sciences, inc.: investigator t bambury -human genome sciences, inc.: investigator g everson -human genome sciences, inc.: investigator w freimuth -no relationships to disclose h mesghali -no relationships to disclose j murray -no relationships to disclose d nelson -human genome sciences, inc.: investigator l novello -no relationships to disclose b osborn -no relationships to disclose j recta -no relationships to disclose g subramanian -no relationships to disclose m sulkowski -human genome sciences, inc.: investigator j zhong -no relationships to disclose introduction pirfenidone (pfd) is an orally bioavailable pyridone derivative that affects a variety of cytokines, including inhibition of tgfbeta, tnf-alpha, pdgf, and egf. pfd has been shown in clinical studies to improve physiologic parameters in patients with pulmonary fibrosis. we have previously shown that pfd decreased hepatic fibrosis in two different rat models of cirrhosis of hepatology 37: [797] [798] [799] [800] [801] [802] [803] [804] [805] 2002) . our objective in this pilot clinical study was to evaluate the safety and preliminary activity of pfd in patients with cirrhosis of varying etiologies. methods all patients had histologic andlor clinical evidence of cirrhosis. pfd was given orally at a dose of 400 mg tid for 12 months. physical examination and labs including alt, ast, bilirubin, albumin and prothrombin time, platelet count were assessed at baseline and on monthly basis. hcv rna levels were measured in patients with chronic hepatitis c (cobas amplicor hcv monitor v2.0). liver biopsies were obtained at baseline and after 12 months of treatment and were read independently by two hepatopathologists who were blinded to the biopsy sequence. modified histological activity (hai) index of knodell and ishak fibrosis stage were used to assess changes in necroinflammatory scores and fibrosis stage, respectively. change in steatosis was also assessed. results a total of 26 patients with cirrhosis due to hepatitis c (15), ethanol (s), amyloidosis (l), autoimmune disease (1) and budd-chiari syndrome (1) were included. the mean age was 57 years (range, 29-75) with 13 males. liver biopsies at end of therapy showed a 2-point or greater reduction in the ha1 necroinflammatory score in 41% of the patients. steatosis decreased in 33% of the patients, was unchanged in 42% and worsened in 25%. while there was no significant reduction in the ishak fibrosis stage, improvement in interstitial fibrosis was noted. evidence of cell regeneration was seen in some patients. hcv rna levels were measured in 15 patients with chronic hepatitis c. at 6 months, 9 patients had a decrease in viral load, 2 patients remained unchanged and 4 patients displayed an increase in viral load compared to baseline. no patient had a sustained virologic response. 4 out of 15 (27%) hcv patients had normalization of alt, 7 out of 15 (47%) had decreased alt values, 1 did not change (7%) and 3 patients showed a modest increase in alt (20%). pfd was well tolerated with the predominant drugrelated adverse events being nausea, photosensitivity rash, and itching occurring in 15% of the patients and which improved after 2 to 3 months of therapy. conclusions: in this pilot study, treatment of cirrhotic patients with pirfenidone for one year was well tolerated. a significant reduction in necroinflammation ( 2 2-point reduction in ha1 grade) and steatosis was observed in a substantial proportion of patients. a subset of patients with chronic hcv infection showed on-treatment reduction in hcv rna levels. longer treatment duration or a less cirrhotic patient population may be needed to demonstrate effects on fibrosis. these data support conducting clinical studies to evaluate a potential role of i'fd in the treatment of steatosis, or in combination therapy for chronic hepatitis c. this work was supported by grants of marnac, inc and intermune, inc. disclosures: arnulfo alvarez -no relationships to disclose gil arechiga -no relationships to disclose juan armendariz-borunda -no relationships to disclose background: viral kinetic modeling of hcv response to interferon-based therapy provides important insights into factors associated with treatment outcomes. hcv/hiv coinfected patients appear to have lower overall response rates than that observed in monoinfected subjects, but the reason for this is not clear. we speculated that kinetic responses in key parameters would be decreased in coinfected subjects. methods: hcvlhiv coinfected patients and hcv monoinfected patients prospectively matched for treatment, genotype, age (k 5 yrs), gender, race and histology were evaluated. coinfected patients were randomized within the context of a large us. multicenter trial (actg 5071) to receive pegylated interferon alfa-2a + ribavirin vs. interferon alfa-2a + ribavirin. quantitative hcv rna (roche cobas amplicor) kinetic testing was performed at 0, 6, 12, 24, 48, and 72 hours and at days 7, 14. non-linear regression and linear models were evaluated in an effort to best predict response and identify prognostic factors. results: twenty-seven subjects underwent viral kinetic sampling and evaluation. these included twelve hcvlhiv case subjects (10 men, 2 women) and 15 matched hcv controls (some patients double-matched). the mean age of coinfected subjects was 46.1 years (range, 38-60). among hiv+ subjects the mean cd4+ count was 325 cells/mm3 (range, 175-855). 11/12 had baseline hiv rna 1400 copieslml. mean hcv viral load was 6.6 log iulml among coinfected vs. 6.2 log iulml in controls. 75% of coinfected subjects had hcv genotype 1. the remainder were genotype 2.25% of coinfected patients had no detectable virus 24 weeks after completion of 48 weeks of therapy (svr). overall svr in control subjects was 46.6%. efficiency ( e ) of phase 1 response (a) slope at 72 hours, lambda2 (slope of phase 2 decline) were calculated. efficiency of clearance ( e ) at 72 hours was highly associated with actual viral clearance across all groups (p=o.ool) but a2 was not. regression analysis failed to demonstrate a relationship between e and baseline cd4+ count, hcv viral load or genotype. in contrast, #955;2 was significantly associated with genotype. viral kinetic parameters were not predictive of svr. conclusion: viral kinetic modeling demonstrated that the efficiency of clearance at 72 hours was significantly associated with viral clearance during treatment. coinfection status did not affect key kinetic parameters. peg-ifn was superior to standard interferon in terms of viral clearance efficiency, particularly in the coinfected group. diminished svr in coinfected patients may be related to immune factors that are operative after reduction of viral loads to undetectable levels. the prevalence of chronic hepatitis c (hcv) infection is greater in african-americans (aa) than in caucasian-americans (ca). however, small studies and post-hoc analyses of larger clinical trials have not found racial differences in disease severity at study entry, though indicate that interferon and ribavirin combination therapy is not as efficacious in aa as in ca. it is important to identify disease and patient characteristics that differ between aa and ca in a large study designed to ascertain whether the apparent racial difference in response to therapy can be explained. aim: the study of viral resistance to antiviral therapy in chronic hepatitis c (virahep-c) is investigating reasons for the lack of sustained viral response (svr) to pegylated interferon and ribavirin therapy in previously untreated patients chronically infected with hcv genotype 1. a key objective is to determine whether previously reported response rate differences between aa and ca exist in a large multi-center cohort. methods: virahep-c will recruit 200 aa and 200 ca from 8 clinical centers in the united states. hypothesizing that aa and ca patients differ by factors that influence svr which could explain racial differences in svr, we compare demographic, clinical, histological, virological and health-related quality of life variables (hrqol) before treatment begins. liver biopsies performed within 18 months of study entry were assessed without knowledge of patient race by a central pathologist. results: based on the first 155 participants recruited, age and sex distributions are similar in the two racial groups with patients averaging 48 years of age and 2/3 being male more than 80% of the cohort has at least a high school education, but aa are more likely to have less than a high school education. body mass indices tend to be higher among aa than among ca (p=.06), though waistto-hip ratios are similar (mean 0.9 in each group). there are no racial differences in mode of hcv transmission, estimated duration of hcv infection, or baseline serum hcv rna levels. aa are more likely to have sub-genotype l b (p=.004).the ishak fibrosis score does not differ by race, but severe lobular inflammation is greater in aa patients (p=.03). diabetes and hypertension are more common in aa. the sf-36 physical component score in ca is similar to the general population and higher than among aa (p=.002). the sf-36 mental component score is similar in the two racial groups and comparable to the general population. summa-wlconclusion: with few exceptions, pretreatment virological, clinical and liver biopsy findings were generally similar in the two racial groups. differences in response are unlikely to be explained by these factors. lshak flbrosls score 58 (%) severa lobular influnmation (%) hypertension (%) diabetes ( bar-llan university, ramat-gan, israel background: combination therapy with peginterferon alfa and ribavirin for 48 weeks achieves sustained virologic response rates (svr) of 54-61%0 in patients with chronic hepatitis c. however, the svr rates are highly variable according to baseline (hcv genotype) and on-treatment parameters (initial viral decline). thus, it must be anticipated that current standard therapy recommendations lead to under-treatment in some and over-treatment in other individual patients. objectives: comparison between a dynamically individualized treatment schedule according to the early virologic response versus the standard of care combination therapy with peginterferon alfa-2a (40kd) (pegasys @) (peg-ifn) (180 pg qw) plus ribavirin (copegus @) (rbv) (1000-1200 mg qd) for 48 weeks. the primary aim of the study was to increase svr, while optimizing the available drugs, treatment duration, quality of life and the socio-economical burden of therapy. the secondary aim of the study was to enable a comprehensive analysis of virallhostlimmune correlates of response to treatment (ongoing). methods: all patients (n=273) were initially treated with peg-ifnlrbv for 6 weeks and initial viral kinetics were defined according to centralized serum hcv rna quantifications (cobas amplicor@ hcv monitor v2, roche molecular systems) on baseline and days 0,1,4,7,8,15,22 and 29 . after classification into viral response categories at 6 weeks, patients were randomized (n=270) to individualized therapy (arms al, a2, b1, b2, c, d) or continuation of standard therapy (std arm). treatment tailoring included: in rapid viral responders (rvr)discontinuation of rbv (al) or shortening of treatment duration to 24 weeks (a2); in slow partial responders (spr)addition of histamine (bl) or prolongation of treatment to 72 weeks (b2); in flat partial responders (fpr)addition of histamine (c); in null responders (nur)retreatment with high-dose of peg-ifn (360 pg qw) plus rbv (d). svr was defined as undetectable serum hcv rna (< 50 iulml) at the end of 24 weeks of untreated follow-up. results: demographic and baseline virologic parameters were similar in the standard and the individualized treatment groups. according to the initial viral decline patients were categorized as rvr (51% for genotype 1 and 94% for genotype 2-3), spr (35% and 5% accordingly), fpr (5% and 0% accordingly), nur (10% and 1% accordingly) or unclassified (1%). the overall svr rates for genotype 1 patients were 49% for the individualized and 56% for the standard treatment arm (ns), and for genotype 2-3 patients 90% and 87% respectively (ns). svr rates (by itt analysis) according to hcv genotype and within each initial viral response category and arm are given in the table. conclusion: the overall sustained virologic response rates of 53% in genotype 1 and 88% in genotype 2-3 patients with chronic hepatitis c treated with peginterferon alfa-2a (40kd) in combination with ribavirin support previously presented data. individualized treatment according to initial viral kinetics appears to be clinically feasible, but did not improve the sustained virologic response rate with the drugs and dosages usable at the time of this study. nevertheless, the possibility that in rapid viral responders discontinuation of ribavirin does not decrease the sustained virologic response rate warrants future prospective trials. supported by the european community (qlk2-2000-00836), hoffmann la-roche and maxim pharmaceuticals. 10% 10% (hepatology 2000; 32(suppl) :844a). the rectally administered ifn is transferred not into blood but into regional lymphatic system and reaches to the thoracic duct via intraperitoneal lymphatics (pharmaceut. res. 1986; 3,116) . therefore, the ifn suppository appears to have different antiviral mechanism from the conventional ifn injection. in this study, we performed a combination of the ifn suppository and ifn injection. the antiviral effect and immune-related markers were examined comparing with the controls (ifn injection therapy). (patients and methods) fourteen patients with chronic hepatitis c were given an ifn suppository and ifn alpha (6-10m units) injection daily for 4 weeks. after that, only ifn injection was continued three times a week for 20 weeks. the control group contains 9 patients with chronic hepatitis c, who received ifn alpha (6-10m units) injection daily for 4 weeks and were followed by the ifn injection three times a week for 20 weeks. the viral loads were 1.52-2400kiulml (median 280kiulml) in the ifn suppository group and 18.7-616kiulml (median 97kiulml) in the ifn injection group. the genotype population (lb/2a,zb) was 5/9 and 3l6, respectively. there is no significant difference in the clinical background in the two groups. serum hcv rna was tested every 4 weeks. peripheral blood nk cell activity and cd4lcd8 ratio were investigated before and 4 weeks after the beginning of the therapy. the ifn suppository contains low dose ifn alpha (ball-1). (results) serum hcv rna turned negative in 73.3% of the ifn suppository group and in 66.7% of the ifn injection group (n.s.) after 4 weeks (end point of ifn suppository administration). the hcv rna seronegative rates of the two groups were 80.0% and 77.6% (n.s.) after 24 weeks, respectively. however, while the nk cell activity was decreased in 78% of the ifn injection-treated patients after 4 weeks, only 36% of the ifn suppository patients had decreased nk cell activity (p=0.049, chi-square test). furthermore, whereas serum alt levels were significantly decreased after 4 weeks in the ifn injection group (83.5+47.9 iull vs. 53.3+30.1 iull, p=0.026, paired t test), no change was seen in the ifn suppository group (80.1iu/1+63.9 vs. 80.5+53.7, n.s.). (con-clusion) the conventional ifn therapy decreased nk cell activity and serum alt levels. however, the ifn suppository combination therapy maintained augmented nk cell activity and elevated alt levels. these findings suggest that the ifn suppository continuously activates the hosts' immunity during ifn treatment. we used the ifn suppository only for 4 weeks; however, longer administration may lead to more frequent elimination of hcv the aim of the study was to evaluate the efficacy and tolerability of induction dose pegylated-interferon and ribavirin vs pegylatedinterferon and ribavirin as treatment strategies in relapsers to standard interferon + ribavirin in chronic hepatitis c patients. methods: 110 patients, virological relapsers after a first treatment with standard interferon and ribavirin for at least 6 months, were randomized to receive either pegylayed-interferon alpha-2b 2 fglkg qw plus ribavirin 800-1000mg qd during 8 weeks then peg-interferon alpha-2b 1 pglkg qw plus ribavirin 800-1000mg qd during 40 weeks (n= 52 pts) or peg-interferon alpha-2b 1.5 pglkg qw plus ribavirin 800 -1000mg qd during 48 weeks (n= 58 pts). efficacy assessments consisted of serum hcv rna level by pcr and serum alt at the end of treatment and after 24 weeks of follow up (week 72) and liver fibrosis with metavir score before treatment and at week 72. safety evaluations included adverse events and laboratory tests. results: patients were not different for baseline characteristics in induction and non induction groups including sex (male 58 % and 71% ), mean age ( . liver fibrosis metavir score decreased in 41% of patients in non-induced group and in 19% of patients in induced group (p=0,16) whatever the response (34% in sustained responders and 25% in non responders or relapsers). conclusion 1) combination therapy with pegylated-interferon is efficient in half of relapsers to standard combination therapy. 2) the response is similar to the response observed in naive patients. 3) 8 weeks induction dose does not increase the virological response. 4) we observed a regression of liver fibrosis in 30% of patients whatever treatment regimen and virological response. backgroundanemia has been shown to be an important factor in impairing hrql in cancer patients receiving chemotherapy, with changes in hb directly correlated with changes in hrql (gabrilove et al., j clin oncol2001). since 29-36% of patients with hcv develop anemia as a side effect of combination ifnlrbv therapy (rebetron@ package insert), it is important to assess the relationship between hb and hrql in this population. the objectives of this analysis were to (1) descriptively correlate changes in hb to changes in hrql in an anemic hcv-infected population; and (2) analyze the independent relationship of hb to hrql. methods: hrql scores and hb values were obtained from clinical trial data of 185 anemic hcv-infected patients (hb 10.8 t-0.9 gldl) who had been receiving ifnlrbv therapy for 12-14 weeks (afdhal et al., ddw 2003) . during the 8-week double-blind phase, patients were randomized to receive either epoetin alfa 40,000 u once weekly or placebo. hrql was measured using the short form-36 health survey (sf-36), an accepted and validated tool that measures 8 domains of hrql (table l) , and the linear analog scale assessment (lasa), which measures constructs of overall quality of life, energy, and activity. patients were categorized into the following groups based on their change in hb levels between randomization and week 9: (1) decrease in hb; (2) hb increase from 0 to <2 gldl; and (3) hb increase 2 2 gldl. changes in hrql (by domain of each instrument) corresponding to the aforementioned hb categories were summarized (table 1) . regression analyses were conducted to evaluate the independent impact of hb change on hrql improvement. the factors included in the regression model (in addition to hb change) were: age, gender, baseline hb, baseline hrql domain, fibrosis status, rbv dose change, duration of hcv therapy, and hcv rna. results: changes in hb values were directly related to changes in hrql for all 8 domains of the sf-36 and the lasa (table 1) . hrql changes were seen in an hb-dependent manner; patients whose hb increased 2 2 g/dl had higher improvements than patients whose hb increased between 0 to <2 gldl. similarly, patients whose hb decreased from randomization to week 9 had mean decreases in hrql for most of the domains (table 1) and minimal increases for others. regression analysis substantiated that the hb change was a significant independent predictor (p=.006 to r.001) of hrql change in all subscales of the lasa and the sf-36, with the exception of bodily pain and general health. conclusions: similar to cancer patients receiving chemotherapy, improvement in hb is a strong independent predictor of improvement in the hrql of hcv-infected patients. patients on combination ifnlrbv therapy should be monitored and considered for anemia treatment to improve hrql and potentially enhance their adherence to hcv therapy. virological response. the current trial was designed to see if better results could be achieved by retreating with higher doses of peginterferon alfa-2b (peg2b) + weight-based ribavirin. aim to compare the efficacy, safety and tolerability of three different doses of peg2b + weight-based ribavirin among interferonlribavirin non-responders. methods: patients were randomized to 1 yr of treatment with peg2b 0.5, 1.5 or 3.0 mcglkglwk, plus ribavirin 12-15 mglkglday. treatment assignment was stratified for sex, race, hcv genotype and histologic fibrosis. treatment was stopped at 24 wk if pcr(+). doses were reduced by 33% for toxicity; growth factors were not allowed. results: patients: enrollment took place between february 2001 and november 2002, with 963 patients recruited from 100 centers; data forms have been received on 794 thus far. enrollment was stopped in the low-dose group after fda approval of higher doses of peg2b. the study population is 31% female, 16% african-american, 93% genotype l, and 63% f2/3/4. efficacy: on-treatment virological response rates were dose-related at 24 wk but less so at 48 wk (see table) . this was partly due to a higher rate of discontinuation after a satisfactory response at 24 wk on the higher dose. on-treatment response rates were lower among african-americans and patients with more advanced fibrosis. sustained response data will be available for most patients by october 2003. 'tolerability: the rate of dose reduction was 33% on peg2b 1.5 mcglkglwk, vs. 45% on 3.0. rates of discontinuation were the same for peg2b 1.5 and 3.0 mcglkglwk: 25% vs. 24% overall, and 13% vs. 14% for adverse events. the frequencies of subjective adverse events were similar between the two groups. some degree of iieutropenia was observed in 27% on 1.5 mcglkglwk, vs. 32% on 3.0; however, only 14 patients in the study discontinued treatment for neutropenia overall. conclusions: 1) thirty to 40 percent of interferon/ of high-dose peginterferon alfa-pb + ribavirin m s l d abstracts 313a ribavirin non-responders achieved initial clearance of viremia on peginterferon alfa-2b + ribavirin. 2) doubling the peg2b dose to 3.0 mcglkglwk resulted in a higher viral clearance rate at 24 wk but a similar rate at 48 wk. 3) the higher dose of peg2b 3.0 mcglkglwk was well tolerated, with a higher rate of dose reduction but an identical rate of discontinuation. beliefs about therapy and psychosocial factors may influence the course of treatment and therefore could be important for developing comprehensive medical care plans that improve treatment adherence and optimize response to therapy. objectives: (1) to describe baseline indices of social support, depression, and self-efficacy (i.e., perceived confidence in the ability to perform health behaviors necessary for successful treatment) of patients with hcv genotype 1 participating in a treatment trial of combination peginterferon and ribavirin therapy. (2) to assess the degree to which baseline psychosocial measures are interrelated. methods: the sample consisted of the first 155 patients enrolled in the virahep-c study, a multicenter, collaborative clinical trial, designed to examine reasons for non-response to hcv therapy in african american and caucasian patients. using a touch-screen computer, patients completed the 21-item medical outcomes study social support survey (mos-sss); the 20-item centers for epidemiological studies depression scale (cesd); 24 questions about self-efficacy, and the sf-36 quality of life instrument. questionnaires were completed during an 8-week period prior to initiation of therapy. mean scores were calculated for each of the 4 subscales of the mos-sss (l=supported none of the time, 5=all the time): a) tangible support (material aid, behavioral assistance); b) affectionate (expressions of lovelaffection); c) emotional-informational (expressions of understandinglencouragement); and d) social interaction (others to have an enjoyable time with). similarly, mean scores were calculated for 5 subscales of the selfefficacy instrument (o=no confidence, 10= high): a) obtaining help; b) communicating with physicians; and managing c) symptoms, d) depression, e) medications. correlation analysis was used to compare each subscale with the cesd and sf-36. results: of the participants in the analysis, 43% were african american and 33% were female. on average, perceived social support and self-efficacy beliefs were high at the initiation of treatment. mean scores for the tangible, affectionate, emotional, and social interaction subscales were 4.2 (sd=0.9), 4.4 (sd=0.8), 4.4 (sd=0.7) and 4.2 (sd=0.8), respectively. for self-efficacy, the mean scores were 8.0 (sd=2.0) for obtaining help; 9.2 (sd=1.5) for communicating with physicians; 7.3 (sd=2.2) for managing symptoms; 8.3 (sd=1.6) for managing depression; and 9.4 (sd=1.3) for managing medications. the distribution of social support and self-efficacy scores were similar for both racial groups and genders. depressive symptoms were common in the sample at baseline with 55% having a score of at least 16 on the cesd and 7% having a score of at least 28. in general, the social support and self-efficacy subscales were unrelated to the cesd and to the subscales of the sf-36. conclusion: african american and caucasian study participants rate their social support and self-efficacy beliefs high in the weeks prior to initiating combination antiviral therapy for hcv, although depressive symptoms are common. social support and self-efficacy instruments may measure different psychosocial constructs as compared with other instruments used in hcv research like the sf-36 and cesd. as a result, social support and selfefficacy may represent new and important predictors of adherence and sustained virologic response (svr) in hcv treatment. baseline data from virahep-c will be used to evaluate factors associated with adherence and svr to determine if racial differences exist among these measures. if so, this information can be used to develop new initiatives for educating patients and families prior to initiating hcv therapy. background and aims: interferon(1fn) a-2blribavirin therapy is an effective anti-viral therapy for the patients with hepatitis c virus (hcv) genotype 1. however, the response to this therapy is not satisfactory because only about 40-50% of the patients become sustained responder(sr)s. further, the patients receiving ifn a-2blribavirin therapy have been frequently withdrawn because of severe adverse effects. the efficacy of ifn therapy is mediated by both genomic type and viral load and to immunological response of the hosts. reportedly, some amino acids were identified to modulate host's immunological response. in order to achieve more higher sr ratio after ifn a -2blribavirin therapy and to decrease the number of withdrawn patients, it seems important to improve the nutritional condition and to upregurate potential immunological response by modifying the balance of amino acid. in the present study, we investigated the dynamics of a panel of 41 amino acids during ifna-2blribavirin therapy, then analyzed the effects of nutritional condition on the clearance of hcv. materials and methods: seventy patients with chronic hcv infection were included after informed consent. six-million unit of ifna-2b in combination with ribavirin (600-800mglday) were given during 6 months. during the first 2 weeks of the therapy, ifn a -2b was given daily. blood samples were obtained after overnight fasting at zweeks, 8weeks, and 24weeks of the therapy and served for the analysis of amino acids. at the same time, peripheral blood mononuclear cells (pbmc) were collected and the number of ifny-and il4-producing cells were measured by facs analysis. thllth2 ratio was calculated as following; thll results: hcv rna was undetectable in 22%, 52%, and 78% of the patients at 2, 8, and 24 week of the therapy, respectively. thllth2 ratio decreased gradually during the therapy. thllth2 ratio at the end of therapy (24 week of the therapy) was significantly smaller than that at the beginning of the therapy (17.8210.9 vs 13.558.0, p=o.o11). however, there was no significant correlation of thll th2 level with the rate of hcv eradication. total amino acids (taas) and branched chain amino acids (bcaas) decreased gradually during the therapy. even at 8week of the therapy, there was a significant decrease in taa and bcaa (p=0.017 and p<o.oool, respectively). fischer ratio (fr) also showed drastic change. it changed from 2.83 (at the beginning of the therapy) to 2.19 (at 8 week of the therapy), indicating that the balance of amino acid rapidly altered to the similar condition to the patients with liver cirrhosis. of interest, the levels of taa and bcaa at the beginning of the therapy were significantly higher in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (taa: 3243+.275 vs 28772252 nmol/ml; p=0.004, bcaa: 470292 vs 377265 nmollml; p=0.024). although we could not find a significant difference, fr at the beginning of the therapy was larger in hcv rna-negative group than in hcv rna-positive group at the end of the therapy (2.8120.51 vs 2.4750.68, p=0.15). analysis of each amino acid showed that there were significant differences in the concentration of citrulline and ornithine, both amino acid are involved in urea cycle, between hcv rna-negative and hcv rna-positive group at the end of the therapy. conclusions: interferon a-zblribavirin therapy induces malnutrition at early stage of the therapy. successful elimination of hcv rna seemed to depend much on nutrition rather than the ratio of thl/th2 during the therapy, suggesting that better response in interferon a-zblribavirin therapy might be possible by improving the condition of nutrition at the beginning of the therapy. in addition, adequate nutrition support would contribute to the improvement of the prevalence of hcv infection in egypt varies between 9% and 24% and approximately 90% are infected with hcv genotype 4.to date limited data exist on the effect of antiviral therapy on genotype 4-infected subjects. the purpose of this study was to assess the effect of interferon (standard or pegylated) in combination with ribavirin in na'ive-to-treatment subjects with chronic hepatitis c who reside in egypt. methods: in a prospective, open-label, randomized clinical trial, 200 egyptian subjects with chronic hepatitis c documented by liver biopsy and detectable hcv rna in serum received interferon alfa 2b (ifn-a2b) 3 mu tiw or pegylated ifn-a 2b (100 mcglweek) in combination with weight-based oral ribavirin (800 or 1000 mg for both groups). the study design was that if hcv rna was detectable at week 24, the treatment was stopped but if hcv rna was undetectable at week 24, treatment was extended for a total of 48 weeks. subjects were observed for an additional 24 weeks and hcv rna status at week 72 determined the response to antiviral therapy. safety laboratory tests were done at regular intervals. the study received irb approval. the hcv rna was done by a pcr technique with a detectability cutoff of 50-copieslmb results: both randomized groups were similar at baseline without statistical significant difference, 190 (90%) were infected with genotype 4, mean age was 39.5 years ? 8.7 years, 158 were men (79%), mean bmi was 27.8 2 4 kglm2, mean inflammatory ha1 score was 7/18 2 2 and fibrosis stage 2.716 t 1.3. of the 190 genotype-4 infected individuals, 156 have finished therapy (week 48) and 78 had completed the 24week follow-up (week 72). the study will be completed by october 2003. the table shows the intention to treat rates of undetectable hcv rna in genotype-4 infected subjects. serious adverse events that led to discontinuation of medications were observed in 6 subjects in the standard ifni'rbv group and in llsubjects in the peg ifnirbv, one death was observed in the latter group. expected adverse events were observed in similar rates in both groups. laboratory adverse events were observed between 2"h and 10% of subjects receiving either treatment regimen depending on the week of therapy. no growth factors were used in this population. in summary: subjects with chronic hepatitis c and genotype-4 infection have a similar antiviral response to standard or pegylated interferon in combination with ribavirin. the safety and tolerability of the medications is similar to what has been observed for other hcv genotypes. patients infected with hcv genotype 4 respond effectively to therapy and should be encouraged to undergo antiviral treatment. daily for 4 weeks, followed by 3 times a week for 20 weeks (week 24) and followed up for 24 weeks after cessation of therapy (week 48). the expression of socs-1 protein in the liver specimen obtained before the treatment was investigated by immunohistochemistry for socs-1. results: patients with the expression of socs-1 protein in the liver were defined as "socs-1 positive group". all other patients without staining were classified as "socs-1 negative group". sixteen patients were socs-1 negative and 61 patients were socs-1 positive. we compared baseline characteristics among two groups. socs-1 positive group had a significant lower rate of svr to ifn therapy than socs-1 negative group (p=0.0014). socs-1 positive group had significantly higher hcv-rna titer than socs-1 negative group (p=0.015). we analyzed factors with ifn svr in univariate logistic regression analysis. not only hcv genotype (p=o.oool) and hcv-rna titer (p<0.0001), but also the expression of socs-1 were found to be significant predictors of ifn svr (p =0.004 background: interferon(1fn) and ribavirin combination therapy is now a standard regimen for chronic hepatitis c infection and achieves a higher sustained virological response than interferon monotherapy worldwide. but the response rate to patient with genotype l b and high viral load is still not satisfactory. some virological factors have been shown to influence the effect of interferon monotherapy in previous studies. however little is known about those influencing the outcome of combination therapy. to examine the genetic basis of the genotype l b virus resistant to combination therapy, we analyzed full-length nucleotide and deduced amino acid sequences of hcv rna. method: among patients infected with genotype l b and high viral load, 6 patients treated with monotherapy : (group a-3 nullresponders, 3 relapsers) and 10 patients treated with combination therapy: (group b-3 nullresponders, 7 relapsers) were studied. the group a consists of 6 naive patients and group b consists of 5 naive patients and 5 patients who failed to previous ifn monotherapy. the group a patients were treated with 6-18miu of ifn, there times a week for 6 months. the group b patients were treated with 6miu-1omiu of ifn three times a week and ribavirin 600mg-800mg per day for 6 months.sera were obtained from the patients at three points: 6 months before the treatment, just before the treatment, and after or during the treatment. complementary dna was reverse-transcribed from total rna extracted from these sera, amplified by polymerase chain reaction and directly sequenced. the mutation rates during natural course of infection and during the treatment were calculated by comparing the nucleotide sequences obtained from the samples just before the treatment and 6 months before the treatment, and just before the treatment and after or during the treatment. the deduced amino acid sequences in different region were also analyzed in each case. result: the mutation rates of natural evolution analyzed in 14 patients were 1.89~10-~nt isitelyear. the mutation rates of hcv rna from nullresponders during the treatment were almost identical with those before the treatment in both groups a and bcu (3.3052.06 vs 3.0421.22, p=ns and 0.681-0.69 vs 0.6420.78, p=ns, respectively).in contrast, those from relapsers during the treatment were higher than those before the treatment in both groups ( table. no major differences were observed in the frequency or intensity of saes and aes. conclusion: the combination of peginterferon alfa-2a (40kd) (pe-gasysb) and ribavirin (copegusb) was safe and effective in patients with hcv genotype 1. despite the use of a dose of ribavirin (800 mglday) that is lower than the recommended dose for patients infected with g1 (100011200 mglday), approximately half of the patients in the study achieved an svr. because the study could not be blinded, a bias might have influenced the outcome of the study as suggested by the unequal discontinuation rate. these preliminary data do not support the hypothesis that higher svr rates can be achieved in patients with hcv g l by extending treatment duration. final data will be presented. *itt population, defined as all patients who took at least one dose of study medication and had at least one hcv rna evaluation postbaseline. **safety population, defined as all patients who took at least one dose of the study medication elusive goal, particularly in previously treated patients who failed or relapsed following prior therapy. we hypothesize that response rates in these patients who are re-treated with pegylated interferon and ribavirin will be improved compared to standard interferon preparations with or without ribavirin. the present prospective study is designed to determine the efficacy and safety of treatment with peg-intron (pegylated interferon alfa-2b) and ribavirin in a group of patients who failed prior therapy with interferon with or without ribavirin. methods: 462 aatients are uresentlv enrolled in this multi-center study. the first250 enrolled patie& were treated with 1.5 mg/kg o~p e g -intron sc q week, and ribavirin 800 mg po qd, which was standard at the time. the intended duration of treatment is 48 weeks. 225 patients have been trcated for at least 48 weeks and week 72 data is available in 141 patients. adverse events: dose reduction was required in 22% of pts most commonly due to leukopenia and anemia. permanent discontinuation of therapy was required in 8% most often due to: depression or anxiety serious adverse events: (1.8% of pts) included one patient each with neutropenialmrsa sepsis, optic neuritis with monocular blindness, perirectal abscess, cellulitis and multi-organ failure, cutaneous and pulmonary sarcoidosis, pneumonia, homicidal ideation, and suicide. conclusions: 1) overall etr was 50% and svr was 41%. 2) svr was significant in genotype non-1 patients and patients who failed or ;elapsed after previous monotherapy 3) 18% a multi-centre, randomised controlled study comparing the combination of interferon and ribavirin with no treatment was undertaken. patients were eligible if they had biopsy proven mild hcv (defined as necro inflammatory scores less than 4 and fibrosis scores less than 3 using the ishak scoring system. interferon was administered at a dose of 3 mu thrice weekly for 48 weeks regardless of viral genotype. ribavirin was administered according to the patient's body weight ( 5 75kg 1000mg/day, > 75kg 1200mgl day in two divided doses). randomisation was stratified according to viral genotype (1 and non-1). the primary end point was the virological outcome of treatment with sustained response defined as an undetectable hcv rna pcr 6 months post cessation of therapy. in addition there were a number of secondary aims: 1. viral kinetics. serum was collected at baseline, days 3,7,10 14 and week 12 for quantitative rt pcr to determine hcv viral load. the ability of early phase kinetics and the presence or absence of a 2 log drop at week 12 to predict eventual outcome is assessed in the context of mild disease. 2. health economics. the collection of resource use data in hcv infected patients who have mild, moderate and cirrhotic disease was performed as an integral part of this study. this data has been incorporated into a markov chain based model to assess likely future costs of the disease and their possible avoidance by treating mild cases. response rates from this trial will form the basis of the model. 3. quality of life assessments. sf36 and euroquol data were collected at baseline, weeks 1 2 2 4 and 48 during treatment and at post treatment weeks 12 and 24 in order to compare quality of life between treated patients and controls before during and after therapy. results. 98 patients received treatment and were matched with 98 controls (no treatment). sustained viral response rates will be available at the end of july when the follow up period of the trial is complete and will be included in the presented results. end of treatment results are shown in table 1. 30% of patients infected with genotype 1 and 61% of patients infected with genotype non-1 achieved end of treatment responses. sustained viral response rates to date are shown in table 2 .20% of patients infected with genotype 1 and 53% of patients infected with genotype non-1 have achieved sustained viral responses so far. 25% of patients in the treatment group were unable to complete at least 6 months of therapy due to side effects of medications. there were 4 hospitalisations in the treatment group, 3 of which were related to adverse events. patients with mild hcv have lower response rates to those with more severe histological change on liver biopsy. side effects are common and frequently result in treatment discontinuation, lowering response rates. the decision to treat patients with mild histological change must be weighed against the side effects. deferring treatment until later in the disease process does not prejudice response to therapy and may increase likelihood of success. the hcv ns5b polymerase is essential for hcv viral replication and infectivity as demonstrated in a chimpanzee model. the enzyme adopts a unique molecular structure that resembles a "thumb-palm-finger" which has some features different from other known dna and rna polymerases, highlighting the attractiveness for this enzyme as a target of antiviral therapy. from an in-house high throughput screening campaign and the subsequent lead optimisation, we have identified a novel chemical series of substituted thiophene-2-carboxylic acids that have good potency against hcv polymerase in vitro. x-ray crystallography studies revealed that these compounds bind to an allosteric site that is -35 angstrom away from the active site. further studies on selected members of the series confirmed the ability of these compounds to inhibit the sub-genomic replication of hcv in huh-7 cells. moreover, a representative compound was also found to have good in vitro safety index and favourable in vitro and in vivo metabolic and pharmacokinetic properties. in an open and prospective trial we investigated, if a pre-treatment with citalopram as an ssri can reduce the frequency of ifn-associated depression in hepatitis-c-infected psychiatric risk patients during methadone substitution. 36 patients with a chronic hepatitis c were treated with pegylated interferonalpha 2b and ribavirin according to body weight. 11 patients without any psychiatric history (group a) were compared to 25 patients during methadone substitution. the methadone substituted patients were separated into two groups: the first group (group b, n = l l ) were only treated with ifn-alpha and ribavirin and the second group (group c, n=14) received a two week pre-treatment with citalopram (20mglday) before combination treatment with ifn-alpa and ribavirin was started. antidepressant treatment was continued over the study period of four months. the hamilton depression rating scale (hamd, 17-item) was used and major depression defined as >20 points. patients were followed over the first 4 treatment months. group differences were calculated with anova for parametric and chi2-test for non-parametric scales. results: hamd scores at baseline were significantly higher in the psychiatric groups as compared to the controls (p=o.olo dallas, t x background: peg plus rbv is the mainstay of chronic hepatitis c treatment. however, an upper limit in terms of safety and efficacy for dosing with peg has not been evaluated. methods: we studied a group of nake patients using either conventional weight-based dosing of peg; (1.5 pglkg) or double the dose (3.0 pglkg) plus rbv 13+2mg/kg/day for 48 weeks. genotype 1 patients were randomized 1:l and to receive medication accordingly in unblinded fashion, with intent to enroll a total of 1,100 nayve patients n, an additional 300 previous nr or r to any type of interferon i rbv in a non randomized arm, using only the 3.0 pglkg peg dose. values for hcv rna at week 12 were compared to those obtained at baseline. results: to date, 573 patients 306 n, 193 nr, 74 r have enrolled in the study. 175 patients 106 n, 48 nr, 21 r have reached week 12. hcv rna results at week 12 are shown in the table below. treatment was well tolerated in most patients. dose reductions were required in 20% of n 1.5pglkg peg and 29% of n 3.0pglkg peg patients. side effects were equivalent between the two groups as shown in the table below. serious adverse events (sae's) were less than 5% and equally observed in both arms of the n patients. none were directly attributed to study drug. there were 25 patients in the wk 12 analysis that discontinued therapy. the reasons these patients were discontinued after reaching wk 12 were side effects (36%), +hcv rna (32%). it is interesting to note that 40% of the patients that were discontinued had 2 2 log drop or negative hcv-rna at week 12 and a positive hcv rna was not a reason for their discontinuation. conclusion: 3.op.glkg peg dosing does not appear to improve 12 wk viral response, but we await 24 wk results and sustained viral response rates. increased side effects as a result of doubling the interferon dose were not observed. more data is needed to verify the long-term efficacy and safety of this dosing regimen for patients with chronic hepatitis c . this study was supported by a grant from integrated therapeutics group a subsidiary of schering plough. background and aim: one of the major adverse effects of the combination therapy for chronic hepatitis c is ribavirin-induced hemolyhc anemia. however, little is known about the mechanism of this anemia. oxidative stress has been suggested as potentially important pathological mechanism in hepatitis c. this burden may cause peroxidation of erythrocyte membrane phospholipid in conspiracy with the accumulation of ribavirin triphosphate in the erythrocyte, which potentially attenuates the mobility of erythrocyte membrane. the aim of this study was to examine the change of fatty acid composition in erythrocyte membrane and the effects of vitamin e and vitamin c on fatty acid composition in combination therapy. methods: thirty-nine patients with chronic hepatitis c were enrolled in this prospective study. they were randomized to receive daily oral vitamin (500 mg of vitamin e plus 750 mg of vitamin c) (vitamin group) or none (control group), in addition to injections of 6 million units of interferon-a-2b daily for two weeks, followed by thrice-weekly for 24 additional weeks, plus daily oral ribavirin (600 or 800 mg) for 26 weeks. blood samples were obtained at 0, 2, 4, 8, and 26 weeks after initiation of therapy. phospholipid was separated by one-dimensional thin-layer chromatography after extraction of total lipid from the erythrocyte ghosts. following transmethylation, fatty acid methyl esters were quantified by gas chromatograph. a-tocophenol concentrations in erythrocyte were determined by high performance liquid chromatography. plasma thiobarbituric acid reactive substances (tbars) were measured by spectrofluorometer. results seven patients were obliged to suspend or cease receiving therapy because of adverse effects including anemia by 8 weeks after initiation of therapy. twenty-one patients have completed the assigned therapy up to now. among the 26 kinds of fatty acid analyzed, the mean content of 205n-3 polyunsaturated fatty acid (pufa) significantly decreased at 8 (0.96 5 0.12 mol% vs. in a cross-sectional study we investigated mxa expression in patients with chronic hcv infection(n = 60 ) and in patients with chronic hcv infection receiving ifn-alpha therapy (n = 23) as well as in healthy controls (n = 22). in a prospective study with 16 chronically infected patients (hcv genotype 1) known to be ifn non-responders, we followed mxa gene expression during combination therapy with pegylated ifn-alpha2b and ribavirin. results: patients with chronic hcv infection showed higher mxa gene expression levels than healthy controls, indicating that hepatitis c virus induces ifn production. however there was no correlation between mxa gene expression and clinical parameters (viral load, alt, liver histology). patients with chronic hcv infection receiving ifn-alpha had significantly higher mxa levels than patients without treatment or healthy controls. further we addressed the question whether mxa expression in pbmcs during therapy in previous non-responders could be a predictive marker of therapy outcome. mxa expression was clearly induced in all but one patient compared to pretreatment values (median: 10.2 fold). importantly, the level and kinetics of mxa expression did not correlate with the response to antiviral therapy. surprisingly, one patient showed an unusually low pre-treatment level of mxa expression and a total lack of mxa induction during ifn therapy, which was associated with complete non-response to therapy. in a neutralization assay, we could detect neutralizing antibodies to ifn-alpha2b in the serum of this patient whereas no neutralizing antibodies were found in all others patients. hepatitis c virus (hcv) infection is a common cause of transfusion acquired hepatitis and is today a major health problem throughout the world. the present study reports the prevalence of hepatitis c virus (hcv) infection in general population and in various high-risk groups from the city of hyderabad in south india. a total of 308 out of 3589 (8.6%) people (both general and risk groups) tested positive for hcv rna by rt-pcr, while anti-hcv antibody positivity, as determined by third generation eia, was found to be 5.8% (209/3589). this suggests that a number of cases go unreported, as screening of blood and blood products is done primarily by elisa. among 124 chronic renal failure (crf) patients with history of either renal transplant or hemodialysis, 46% were infected with hcv alone, 6.5% hbv alone while 37.1% were found to be co-infected with bothe hcv and hbv. our findings implicate these viruses as the major cause of post transplant hepatitis in indian patients with crf and indicate the necessity for immediate implementation of stringent screening procedures for these viral infections. additionally we report here that tattooing and slashing (a cultural practice among one sect of muslims) increase the mode of transmission of hcv infection. in addition, we also report a high incidence of hcv infection ( background in japan, long-term treatment with glycyrrhizin, given as snmc, is used to reduce alt and the risk of hepatocel-glycyrrhizin given as stronger neo-lular carcinoma in patients with chronic hepatitis c. phase 1/11 studies confirmed the alt lowering effect of glycyrrhizin therapy in western patients with chronic hepatitis c. the treatment duration in these studies was limited to 4 weeks. alt response was lost during follow-up. aim 1) to evaluate which dose frequency is required beyond week 4 to maintain the initial alt response. 2) to evaluate the effect of snmc treatment on liver histology. methods: hcv-rna positive patients with alt >2x uln, fibrosis stage 2 3 or necro-inflammation score 2 6 (ishak's score) who were not eligible for interferon therapy (prior non response, absolute contraindications) could enter this multi-center, randomized, open phase i1 clinical trial. all patients were treated 4 weeks with six infusions weekly of 100 ml snmc (minophagen pharmaceutical co. ltd., japan) containing 200 mg glycyrrhizin. patients with an alt response at week 4 (defined by a decrease of more than 50 percent of the baseline value or alt 51.5 uln) were randomized to continue treatment in three dose frequency groups for a total of 26 weeks. snmc was given 6 times weekly (group l), objectives: it is important to maintain reduced serum alanine aminotransferase (alt) levels in cases with chronic hepatitis c (ch-c) that do not respond to interferon (ifn) and in those with no indication of ifn therapy. we reported previously that dietary restriction of iron intake reduces serum alt levels in such patients. we evaluated ch-c patients treated with iron-restricted diet for two or more consecutive years, mainly focusing on the balance of energy intake, physical examination, and changes in hematological indices of nutrition. methods: twenty-two patients with ch-c (males, 18; females, 4; mean age, 56 year-old) that consulted our outpatient department were enrolled in this study. the inclusion criteria were as follows: 1) elevation of alt levels above the upper normal limit for 3 months or more; 2) positive tests for hcv-antibody and hcv-rna; 3) absence of other causes of ch (alcoholic liver disease, drug-induced liver injury, hemochromatosis) and negativity for hepatitis b surface antigen and for serum anti-nuclear and anti-mitochondria1 autoantibodies. twenty cases had received ifn therapy for more than 12 months before the beginning of the study; none of them responded to ifn therapy. dietary prescriptions included iron intake 7 mglday or less, energy intake 30 kcallkglday, protein intake 1.1-1.2 glkglday, and a fat energy fraction of 20%. nutritional balance was evaluated based on meal records, and instructions was given when necessary. results: the average energy intake before dietary prescription was 2184 kcal(36.7 kcallkg)lday, and it was significantly reduced to 1655 kcal(28.5 kca1lkg)lday (p < om), and then maintained stable at 30 kcallkglday. the average protein intake before dietary prescription was 85.7 g (1.45 glkg)lday and it was reduced to 1.1-1.2 glkglday after the prescription. the average fat intake of 66.5 g (1.1 glkg)lday and the average fat energy fraction of 27% before the dietary prescription were significantly decreased to 30.8 g (0.52 glkg)lday; p < 0.01 and 16% (p < 0.001), respectively, after dietary instructions. the fat energy fraction was maintained at a level of 20% or less. carbohydrate intake did not change remarkably during the observation period, although the carbohydrate energy fraction significantly (p < 0.001) increased. the average iron intake decreased significantly (p < 0.001) from 9.6 (before) to 6.1,5.2, 5.1,5.2, and 5.1 mglday 6, 12, 18, and 24 months after die* prescription, respectively. body mass index (bh4i) before diet prescription was 23.9 on average; bmi had no significant change throughout the course. the body fat percentage was 24.6% on average before the diet instructions, and it significantly decreased after the diet. the average values of aspartate aminotransferase and alt before diet prescription were 65 iull and 66 lull, respectively, and they were significantly reduced to 48 iull and 49 iull, respectively, after 24 months (p < 0.01). serum iron levels significantly decreased after 18 (p < 0.01) and 24 (p < 0.05) months, while unsaturated iron binding capacity tended to increase. the average serum ferritin levels were 376, 210, 189, 189, 141 nglml before and 6,12, 38, and 24 months after diet, respectively; there was a significant reduction (p < 0.01) in the values measured before and after the diet instructions. the average levels of hemoglobin, albumin and cholinesterase did not change significantly during the follow-up period. conclusions: restriction of iron intake is safe and well tolerated for a long period. the results of our present study suggest that decreased dietary intake of iron may constitute an important adjuvant therapy in patients with ch-c. chru, nice, france; c henquell, chru, clermont ferrand, france; c dizrcha, s ughetto, p dechelotte, hotel-dieu, clemont ferrand, france; h lafeuille, chru, clermont fewand, france; g bommelaer, hotel-dieu, clemont fewand, france peginterferon (pegifn) background: hcv co-infection is common among patients with hiv disease. it has been documented that hiv co-infection accelerates the course of liver disease in patients with hcv, and that liver failure is higher in co-infected patients. at this moment, there is no effective treatment for hcv non-responders to interferon therapy. mono-therapy and interferon-ribavirin combination is only effective in 2-3% and 5-10% interferon resistant patients respectively. treatment strategies to slow the progression to cirrhosis and to prevent liver failure in this population are needed. objective: this is a report of a study that examines the efficacy of peg ifn alfa 2-a (pegasys) vs pegasyslrbv 800mg in co-infected hcvlhiv patients that have not responded to a previous 6-12 months course ifn-alfa. the study also examines the histological benefit of treatment in this population. patients and methods: 76 patients were randomized 1:l to receive either pegasys 180mcg weekly x 24 weeks (group 1) or pegasys 180mcg weekly plus 800mg rbv for 24 weeks (group 2). patients that have hcv non-detectable or 210g decrease from baseline at week 24 (groupl), were added rbv 800mg. all similar responders (group 2) continue treatment for a total of 48 weeks. baseline demographics were similar between both groups. more than 70% of patients were nonresponders to ifn monotherapy.most patients in both groups (80%) were genotype l. the mean hiv baseline log was 2.92 (sd.64) group 1, vs 2.85 (sd.57) group 2 and most patients had baseline cd4 levels>400 cells, and were in stable antiretroviral therapy. the majority of patients (81%) are non cirrhotic, with mean grading group 1 6.63 (sd 3.24), group 2 7 (sd 3.64), and mean staging, group 1 3.51 (sd 2.2), and group 2 3.48 (sd 1.70 conclusions: this study is completed and pending some results, that will be available for presentation. sustained virological response(svr) (intention to treat), will be of the order of 5-20%. (11 group 2 results are pending). in this study, 10 patients were discontinued because adverse events and 10 were lost to follow up, before week 24th.svr in patients that completed at least 24 weeks of therapy will be of the order of 7-26%.a significant number of end of treatment responders are relapsing at week 72th. histology analysis show improvement in both groups of the mean grading and staging after treatment. in responders and relapsers the fpr becomes static or regressive.these results show that pegasys lrbv therapy is effective in this population.the study also suggests that longer duration of therapy ,and higher doses of rbv should be studied in coinfected nonresponders. * -pending results, will be available for presentation. disclosures: josi. rodriguez-orengo -no relationships to disclose maribel rodriguez-torres -roche laboratories: investigator; other: grant to perform study. adverse events by week 12 of therapy were grade 2 to 3 in severity and were therapy related. see table 2 . sixteen patients who continued therapy to week 24, three dropped out due to adverse events (1 anemia, 1 hyperbilirubinemia and 1 related death due to self-overdose). this patient had a history of hypothyroidism and mild untreated depression. at 48 weeks of therapy, 1 patient discontinued treatment due to suicidal thoughts. there was no difference between the ethnic groups regarding the drop out rate due to adverse events. hivfhcv coinfected population had a poor tolerance to pegylated interferon and ribaiirin. this was markedly increased in the first 12 weeks of therapy. the use of high dose pegylated interferon and ribavirin led to a significant drop out rate in comparison to the low dose pegylated interferon. background during liver injury, poorly characterized factors activate quiescent hepatic stellate cells (hsc) to become proliferative, matrix-synthesizing myofibroblasts. activated hsc are the major source of liver collagen and thus, play a key role in the fibrotic response to liver injury.the sns appears to promote fibrosis in injured livers because hepatic fibrosis is increased in the spontaneously hypertensive rat, which has an overactive sns. conversely, prazosin, an adrenoceptor antagonist, inhibits fibrosis in toxin-damaged rat livers. hsc express several neuronal proteins, including glial fibrillary acidic protein (gfap). they also contain synaptic vesicles and receive autonomic fibers. therefore, our hypothesis is that hsc are hepatic neuroglial cells that produce and respond to neurotransmitters in order to become activated during liver injury. methods: hsc were isolated from normal mice and dbh-lmice that cannot produce norepinephrine (ne) due to targeted disruption of the dopamine p-hydroxylase (dbh) gene. lysates of culture-activated hsc were analysed by rt-pcr, immunoblot and hplc to determine if they express adrenoceptors, catecholamine biosynthetic enzymes andlor produce ne. the effect of adrenoceptor antagonists and ne on hsc growth in vitro was also assessed. then in vivo activation of hsc by hepatotoxic diets was evaluated in control and dbh-lmice by comparing numbers of a-smooth muscle actin (asma)+ cells with immunohistochemistry and the induction of tgfp-1 and collagen a-1 gene expression by ribonuclease protection analysis of whole liver rna. results: hsc express a-and p-adrenoceptors, tyrosine hydroxylase and dbh. hsc from control, but not dbh-i-, mice release ne. endogenous ne is an autocrine growth factor for hsc because a-and p-adrenoceptor antagonists inhibit proliferation of hsc cultured from control mice. moreover, hsc from dbh-lmice, which cannot make ne, grow poorly in culture and are rescued by addition of ne. exogenous ne also promotes hsc proliferation. inhibitor studies demonstrate that the latter effect is mediated via g-protein coupled adrenoceptors that activate mitogen activated kinases and phosphatidylinositol 3 kinase pathways. hsc activation in response to diet-induced liver injury is inhibited in dbh-imice, as evidenced by reduced hepatic accumulation of asma (+) hsc and inhibited hepatic induction of background & aims: hepatic cirrhosis is six times more prevalent in obese individuals than in the general population, and obesity is one of the risk factors for liver fibrosis in which plasma adiponectin levels are decreased. adiponectin is an adipocytokine, which we previously identified by screening adipose-specific genes in the human cdna project. hepatic stellate cells (hscs) play central roles in liver fibrosis. when they are activated, they undergo transformation to myofibroblast-like cells, then proliferate, migrate, produce transforming growth factor-pl (tgf-pl), and various extracellular matrix proteins, and express a-smooth muscle actin (a-sma). we previously reported that adiponectin suppresses not only the proliferation and migration of hscs, but also the tgf-pl-induced fibrogenic gene expression in hscs. adiponectin could have biological significances in liver fibrosis. in this study, in order to clarify the effect of adiponectin on liver fibrosis in vivo, we tested the role of adiponectin on liver fibrosis using adiponectin-knockout (ko) mice and adenovirus mediated adiponectin expression system. methods: (1) to investigate the anti-fibrogenic effects of physiological concentrations of adiponectin, male wild type (wt) mice and ko mice were used. mice were each injected with a dose of carbon-tetrachloride (ccl4) (300 pllkglbw) intraperitoneally twice a week for 12 weeks to induce liver fibrosis. (2) to investigate the anti-fibrogenic effects of excessive concentrations of adiponectin, male wt mice were used. mice were injected cc14 (1000 pl/kg/bw) intraperitoneally twice a week for 12 weeks. mice were divided into 6 groups. control, received an injection of corn oil only; gr.1, mice treated with cc14 for 12 weeks; gr.2, mice treated with cc4 for 12 weeks after infusion of adenovirus producing adiponectin (adadn); gr.3, mice treated with cc4 for 12 weeks after infusion of adenovirus producing p-galactosidase (adlacz); gr.4, mice treated with cc14 for 12 weeks with adadn infusion at 6 week; gr.5, mice treated with cclb for 12 weeks with adlacz infusion at 6 week. results: (1) ko mice showed extensive liver fibrosis with an enhanced expression of tgf-p1 and connective tissue growth factor (ctgf) compared to wild type (wt) mice (p<0.05). the hydroxyproline content and the numbers of a-sma positive cells in mice liver significantly increased in ko mice. (2) the fibrosis areas were significantly decreased in gr.2 and gr.4 compared to those in gr.3 and gr.5, respectively. the hydroxyproline content in mice liver significantly decreased in gr.2 and gr.4 compared to that in gr.3 and gr.5, respectively. moreover, in gr.4, the hydroxyproline content was significantly decreased compared to that in 6-weeks of c c 4 treatment even though ccl, was given for an additional 6-weeks (total 12 weeks). conclusions: the findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention. disclosures: (ttg) is observed in mature scars and might promote wound repair by protecting the neomatrix from degradation by matrix metalloproteinases (mmps).however, such ecm crosslinking has the potential to hinder the matrix remodelling required for resolution of liver fibrosis. we have explored this hypothesis by examining expression of ttg and its crosslink product epsilon-(gamma-glutamyl) lysine in livers of: a) rats administered cc14 for 6 weeks (6wk c c 4 cohort)after which liver fibrosis resolves spontaneously after 28 days; b) rats administered cc14 for 12 weeks (12wk cc14 cohort) which develop micronodular cirrhosis; c) 12 wk ccl4 treated rats allowed to recover for 365 days after last cc14 dose (12 wk+365d cohort) which show partial resolution of fibrosis but with persistence of a macronodular cirrhosis. although ttg was identified by immunohistochemistry within and around fibrotic bands in 6 wk ccld cohort, no crosslinks were detectable. however, both ttg and crosslinks were detected in and around mature fibrotic bands in the 12wk cc14 cohort and in persisting fibrotic bands in the 12wk+365d cohort. western blotting of liver homogenates from these two cohorts using antibody against the crosslink revealed a major product of 160 kd which was degradable to lower molecular weight products following incubation of homogenates with bacterial collagenase but was refractory to mmp-2. to evaluate if ecm crosslinking in livers correlated with its resistance to mmps, 10 micron cryostat sections of livers from the three cohorts were incubated with purified active forms of mmp-1 or mmp-2 for 8 hr at 37 c and any residual ecm was stained with sirius red. ecm of 6 wk cc14 cohort was effectively degraded by these mmps but that of 12wk c c 4 and 12 wk+365d cohorts was only minimally degraded. hsc freshly isolated from normal rat or human liver showed no ttg expression whilst hsc activated for 7-15 days on plastic substrate expressed ttg protein (by western blotting) and mrna (by rt-pcr). there was ttg in cell lysates and in conditioned media of activated hsc. pure rat tail type i collagen incubated with conditioned media of activated hsc incorporated crosslink antigen, confirming that hsc secreted functional enzyme. however, no crosslink was found in type i collagen incubated with media from hsc which had ttg gene silenced by cell transfection with small inhibitory rna. cultured hsc which had ttg gene silenced had enhanced apoptosis (by acridine orange staining) following serum deprivation in culture. this suggests ttg or ttg activity promotes survival of these cells. we conclude that in persistent, incompletely resolving liver fibrosis there is evidence of ttg mediated crosslinking of the liver ecm and resistance to exogenous mmps. both features are lacking in ecm of fully resolving fibrosis. ecm crosslinking by ttg might limit resolution of cirrhosis. activated hsc are a potential source of ttg following liver injury and, as this protein supports their survival, it might contribute to their persistence in cirrhotic liver. we recently defined multifunctional biochemical properties of the long and frizzled variants and hypothesized that they may be inherent in the 192-aa-long-specific and the 235-aa-fz-specific modules, respectively. we thus transfected pcdnas.l-v5-his vectors containing variant-specific sequencer3 in mhat3fls315 hepatoma cells. this cell line stably expresses a truncated hnf3 protein turning off endogenous expression of liver-specific genes, such as albumin or c18. consistently, mhat3fls315 hepatoma cells transfected with an empty vector showed no endogenous c18 expression. by immunohistochemistry, using variant-specific and tag-specific antibodies and after analysis by confocal microscopy, long was localized in large supranuclear vacuolae suggesting protein storage/maturation in golgi structures or in beaded perinuclear vacuolae suggesting rer cysternae. in contrast, fz was strongly detected in close proximity to the plasma membrane of single cells. clusters of fz-transfected cells showed a strong and dense signal at sites of cell-cell contact, outlining single cells. these findings were confirmed by transfection of prep-7 vectors containing full-length long or fz forms of human c18 and confocal microscopy analysis after immunohistochemistry using an antibody directed against an epitope common to all c18 forms. immunoblot analysis of conditioned media showed that the fulllength long form was secreted into the medium but not fz. the latter was detected as a heavily glycosylated protein (> 300 kd) in the cell layers, predominantly in the triton-x-100 insoluble pellet after sonication and 10% sds solubilization, suggesting binding to extracellular matrix proteins. finally, glycosydase analysis of fz and long n-terminal modules showed an important increase in mobility after pngase f plus sialidase a digestion. these data show that, in addition to the previously described plasma (long) and tissue (short) forms of cl8, the fz form constitutes the pericellular matrix form, suggesting that the specific modules of c18 regulate tissue targetting through protein-protein interactions. backgr0und:during liver fibrosis, hepatic stellate cells (hsc) acquire an activated phenotype, proliferate and produce an excess of collagens. mycophenolic acid (mpa) is a known immunosuppressive drug. which inhibits the proliferation of b-and t-lymphocytes by inosine mono phosphate dehydrogenase (impdh) inhibition, causing an intracellular guanosine depletion. in addition, mpa has shown to inhibit growth of mesangium cells in the kidney and of skin-and tenon fibroblasts. therefore, we hypothesize that the proliferation of hsc may also be influenced by mpa. in this study we explored whether mpa is able to inhibit the proliferation of primary isolated rat hsc in vitro. furthermore, we studied the in vitro effects, mechanism of cellular uptake, and in vivo pharmacokinetics of mpa coupled to mannose-6-phosphate modified human serum albumin (m6p-hsa). m6p-hsa is a newly developed, hsc-specific drug carrier, homing towards the upregulated m6pligf-i1 receptor on activated hsc. in this way we hope to achieve cell-specific delivery of mpa to the hsc avoiding undesired effects of mpa on the immune system. meth-odslresults: in primary cultures of hsc a dose dependent reduction in the number of brdu-positive nuclei was observed after 24h incubation with 750,3000 and 6000 nm mpa, as assessed immunohistochemically. coupling of mpa to m6p-hsa via a biodegradable ester linker resulted in a conjugate with a maximum drug: carrier ratio of 1.2:l. analysis of the synthesized constructs was performed by hplc, fplc and ms. this conjugate was able to inhibit 3t3-fibroblast growth, as detemined by brdu-incorporation (elisa) in a dose dependent manner, reducing proliferation to 38.0 2 14.5%, 30.0 t 18.0% and 18.0 2 16.8% of control at 120, 240 and 480 uglml of conjugate, respectively. when cells were co-incubated with an excess of m6p-hsa, a competitor for receptor mediated uptake of the conjugate, the anti-proliferative effect was reduced by 69 yo. the organ distribution of the conjugate, 1251 labeled, was evaluated by iv injection of a tracer dose in rats 3 weeks after bile duct ligation (bdl-3). 47.3 t-4.1 % of the injected dose accumulated in the liver after 10 min of injection. in contrast, spleen and thymic gland accumulated 1.47 f 2.65% of the injected dose. intra-hepatic distribution was assessed in bdl-3 rats by immuno-histochemical double staining for hsa and specific markers for kupffer cells (ed-2), hsc (desminelgfap) or endothelial cells (his52). m6p-hsa-mpa showed a non-parenchymal distribution and co-localization with hsc and kupffer cells. conclusions: mpa is able to inhibit the proliferation of primary isolated rat hsc. this suggests that stellate cells are dependent on intracellular impdh activity to proliferate. coupling of mpa to mcip-hsa, a stellate cell specific drug carrier, resulted in a pharmacologically active construct, able to inhibit fibroblast proliferation in vitro after specific uptake via the m6p/igf-ii receptor. the major part of the injected conjugate accumulates in the hsc and kupffer cells of the liver, and avoiding uptake in the major resident organs for band t-lymphocytes. future studies will assess the advantage of this first hsc-selective compound in animals with liver fibrosis. background: platelet-derived growth factor (pdgf) is the most potent stimulator of migration and proliferation of mesenchymal cells. the expression of pdgf-p-receptor is increased during liver fibrosis. our aim was to investigate the effect of p3-integrin subunit blockade using the specific non-peptidic inhibitor emd409915 on migration and proliferation of hepatic stellate cells (hsc) and human foreskin fibroblast (hff). materials and methods: cell migration of human hsc and hff was assessed using a scratch assay. scratches of 600-700 pm width were made in confluent cell monolayers of cells following 24 h starvation in serum-free medium. after wounding. cells were stimulated with pdgf-bb (10 nglml) with or without emd409915 (merck, darmstadt, germany) at increasing concentrations (10-10m -10-6m). cell proliferation was measured as dna synthesis by brdu-incorporation. mitogen-activated protein kinase (erk112, p38, sapkljnk and akt) phosphorylation was evaluated by phospho-mapk-specific western blotting of cell lysates after 10 min of stimulation. results: stimulation of human hsc cells with pdgf-bb at 10 nglml in the absence of other growth factors resulted in pronounced stimulation of cell migration. pdgf-stimulated migration of human hsc was inhibited dose-dependently between 10-9 and 10-6 m by emd409915, with complete abrogation of migration at 10-6m. surprisingly, human skin fibroblast migration was not affected by b3-blockage. there was no effect of p3 integrin inhibition on cell proliferation as measured by brdu-incorporation, neither in human hsc-nor in hff cells. pre-incubation of hsc cells with the p3 integrin inhibitor at 10-6m did not affect the activation of p38 mapk, p44l42 mapk or sapkljnk. conclusions: 1. pdgf-bbinduced migration is strongly b3integrin dependent in human hsc, but not in human skin fibroblasts. 2. in contrast to cell migration, hsc and hff proliferation is p3-integrin-independent. 3. p38, p44l42, sapkljnk and akt mitogen-activated protein kinase signalling pathways are not modified by p3-integrin inhibition. 4. p3-integrin antagonist may be an adjuvant approach to treat hepatic fibrosis. epithelial to mesenchymal transition (emt) is defined as a process, in which epithelial cells loose their epithelial characteristics and acquire typical characteristics of fibroblasts. epithelial cells are tightly attached to their neighboring cells via cell adhesion molecules and they adhere with their basal side to their underlying basement membrane matrices, whereas the apical side faces a lumen. in contrast to immobile epithelium, mesenchymal fibroblasts are specifically designed to invade extracellular matrix (ecm). this is reflected by their prominent mesenchymal cytoskeleton and by their lack of a typical polarity. in the adult organism, emt occurs in epithelia in response to injury, potentially as a means to replenish fibroblasts, which are required for repair of injury. in organ fibrosis however, enhanced conversion of epithelium into fibroblasts is considered to contribute to progression of disease, as parenchymal epithelial cells acquire phenotypic and functional properties of activated fibroblasts. recent studies provided increasing evidence that parenchymal epithelium can potentially contribute to activated fibroblasts by emt, as in mouse models of kidney fibrosis 36% of activated fibroblasts were found to be of tubular epithelium origin. in liver fibrosis, stellate cells are considered the principal source of activated fibroblasts, whereas the role of hepatocytes is considered minor, even though they constitute for more than 80% of the liver mass. here we provide evidence that the pro-fibrotic growth factors tgf-beta1 and egf induce expression of fibroblast-markers fibroblast specific protein-1 (fspl), alpha-smooth muscle actin (alpha-sma) and type i collagen in primary mouse hepatocytes in vitro. furthermore, tgf-beta1 and egf induce acquisition of fibroblast characteristics, migratory capacity and release of mmp-2, suggesting emt of hepatocytes in vitro. additionally, progression of liver fibrosis in a mouse model of tetracarbon chloride-induced liver disease is associated with appearance of fspl positive hepatocytes, indicating emt, which suggests a role of emt in liver fibrosis. in conclusion, our results for the first time provide evidence for an active role of hepatocytes in liver fibrosis by undergoing emt. human studies -serum mcp-1 levels were significantly elevated in both cfld (929t67 pglml; p<o.oool) and ba (1,2902220 pglml; p<o.oool) vs controls (602240 pglml). in celd subjects serum mcp-1 was negatively correlated with the stage of fibrosis (r= conclusion: these results provide strong evidence for a role for mcp-1 in early fibrogenic events in cholestatic liver injury, ie., ductular proliferation and procollagen q(i) mrna expression, and suggest that serum mcp-1 may be a very good marker of the onset of cholestatic liver disease. this is the first study to clearly demonstrate elevated excretion of mcp-1 into bile in obstructive cholestasis, implying a potential role for mcp-1 in hsc and inflammatory cell recruitment to the biliary tree in cholestatic liver injury. [background and aim] angiotensin i1 (ang 11) is considered to play an important role in hepatic fibrogenesis. however, the molecular mechanism of renin-angiotensin system in hepatic fibrogenesis has not been clarified. we previously reported that ang i1 up-regulated mrna expression of pro a (i) collagen and tgf-p in isolated rat hepatic stellate cells and an angiotensin converting enzyme inhibitor, lisinopril, reduced carbontetrachroride-induced rat hepatic fibrosis. ang i1 type i receptor blocker (arb) have shown a good clinical response in patients with hypertension, and a promising preventive effect has been described in rat hepatic fibrosis. recently, the rholrho-kinase has been demonstrated to be involved in the signal transduction pathway of the ang i1 type i receptor signaling downstream. thus,the aim of this study was to evaluate the role of rholrho-kinase pathway in hepatic fibrogenesis in rat. to that effect, a rat hepatic fibrosis model was produced by chronic administration with cholinedeficient, l-amino acid-defined (cdaa) diet and the effect of an arb and a rho-kinase inhibitor (y27632) in this model was investigated. methods: wistar rats (male, 6 weeks-old) were continuously fed the cdaa diet or the control choline supplemented, l-amino-defined (csaa) diet. the rats were treated with 1 mglkg candesartan cilexetil, an arb (a kind gift by takeda chemical induustries, ltd., osaka, japan), or 1 mglkg y27632, a rho-kinase inhibitor (a generous gift by mitsubishi welpharma co., tokyo, japan), once daily through stomach tube. twelve weeks after the start of administration, the liver was excised and subjected to histological examination for fatty change and fibrosis by hematoxylin and eosin staining. serum alanine aminotransferase (alt) values and hyaluronic acid (ha) levels were also measured. since cdaa diet causes hepatic steatosis and fibrosis via production of free radicals, we also studied the oxidative membrane damage by immunostaining of 4-hydroxynoneal (hne) adduct. results: histologic examinations showed severe steatosis and fibrosis in the liver of cdaa-fed rats and no significant change in csaa-fed rats. the liver weight of cdaa-fed rats was significantly increased than that of csaa-fed rats. both candesartan cilexetil and y27632 remarkably reduced not only fibrosis, but also severe steatosis and the liver weight of the treated rats was significantly reduced in both treated groups. serum ha levels but not alt values significantly elevated in cdaa-fed rats compared to csaa diet fed group. the increased levels of serum ha were significantly reduced by the treatment with candesartan cilexetil or y27632. the number of hne-positive hepatocytes increased by cdaa diet and this increase was attenuated in the candesartan cilexetil-or y27632-treated groups. conclusion: these results suggests that an arb, candesartan cilexetil, and a rho-kinase inhibitor, y27632, show the same inhibitory effect on hepatic steatosis and fibrosis induced by chronic cdaa diet, and this free radicalinduced hepatic injury is a good therapeutic target of arbs or rho-kinase inhibitors. the rholrho-kinase may be a key enzyme system in the ang i1 signaling involved in the hepatic fibrosis. in liver cirrhosis, malnutrion and portal congestive enteropathy facilitate bacterial translocation from the intestine to mesenteric lymph nodes and the development of endotoxemia and spontaneous bacterial peritonitis in ascitic patients. igf-i is an anabolic hormone synthesized in the liver, whose levels are reduced in hepatic cirrhosis. igf-i possesses hepatoprotective properties and displays trophic effects on the intestine. here we investigated whether igf-i therapy could in-fluence portal pressure and bacterial translocation in experimental cirrhosis. methods: liver cirrhosis was induced in sprague-dawley rats (n=30) by oral administration of carbon tetrachloride during 8 weeks. after this time, rats were randomized in two groups to receive subcutaneous injection of recombinant human igf-i (2 microgllo0 g body weight in two separated doses per day) (ci-igf, n=15) or vehicle (ci, n=15), for 21 days. eight healthy rats, receiving vehicle were studied in parallel. at the end of treatment period, animals underwent laparotomy to measure portal pressure and to collect blood and tissue samples. endotoxin quantification in blood samples was carried out with a colorimetric limulus amebocyte lysate test. liver and ileum histopathological changes were assessed in tissue sections stained with masson's trichrome, using a numerical scoring system. bacterial translocation was evaluated by culturing blood samples and mesenteric lymph nodes in appropriated culture media. results: treatment with igf-i significantly improved biochemical markers of liver damage, being the alt values significantly lower in the ci-igf group than in ci rats (p<0.05). in addition we found that igf-i administration to cirrhotic rats reduced both the hepatic histopathological score ( the rate of progression to cirrhosis varies among individuals infected with hepatitis c virus (hcv) cholesteryl ester transfer protein (cetp) is a plasma glycoprotein involved in lipid metabolism which transfers cholesterol esters from hdl to vldl and ldl. obesity and steatosis are emerging as key factors in the pathogenesis of liver fibrosis in hcv. variability in function or levels of cetp may affect lipid transport and therefore steatosis. the uncommon vallval genotype results in decreased cetp plasma activity and may protect against myocardial infarction. we tested for a relationship between possession of cetp ile405val polymorphisms and rate of fibrosis in 327 white european patients chronically infected with hcv. rate of fibrosis was calculated by dividing the fibrosis score (ishak modification of knodell) by infection duration. genotyping was by reverse line blot hybridisation. a significant association was seen between rate of fibrosis and the ile405val polymorphism in cetp (p =0.002 (anova)) (figure 1) . disease association showed patients possessing the val allele (which is associated with lower cetp activity) were more likely to be slow fibrosers (no cirrhosis for >30 years) genotype frequency p=0.0017, allele frequency p=0.0007, odds ratio=1.8. (table 1) . possession of the val allele is associated with slow rate of disease progression in hcv. the functionally less active forms of cetp may result in decreased liver steatosis as a result of altered composition or increased levels of hdl, and therefore less fibrosis when a second insult such as infection with hcv is sustained. backgroudslaims: hepatitis c virus (hcv) is a major cause of chronic liver disease worldwide. however, little is known about exact mechanism of fibrogenesis by specific hcv gene or protein because the study of hcv-induced liver fibrosis has been mainly studied in human or animal model. moreover, the dynamically molecular study of hcv role in relation to liver fibrogenesis or immune response has been hampered due to lack of efficient in vitro hcv culture system. in the present study, we investigated whether hcv core protein directly influence on the liver fibrogenesis through stimulation of hepatic stellate cell(hsc) in in vitro or not. methods human and rat hscs were isolated using collagenase perfusion system and density gradient method, and other human hsc line (l190) was purchased from atcc. we established co-culture system of primary hsc and hepg2-core stable cell line which hcv-core gene was transfected into hepg2 cell. co-culture system was divided into two way, mixed co-culture and separated co-culture system. immunocytochemical staining was performed to identify the cytokines such as transfoming growth factor pl (tgf-p1) and a-smooth muscle actin(a-sma) overexpressed from hsc in the liver fibrogenesis. a-sma, tgfpl, transforming growth factorp receptor i1 (tgforii), collagen type i were also quantitated by western blot analysis. the expression of mmp-2 and collagen type i in the culture media was measured and analyzed by each zymogram and elisa. results the expression of tgf-p1 and a-sma was significantly higher in mixed co-culture of hepg2-core plus hsc than in hepg2 plus hsc as negative control. also the markers related fibrosis such as a-sma, tgf-pl, collagen type i and tgfrii, mmp-2 and collagen type i were highly expressed in separated co-culture of hepg2core and hsc conclusions in conclusion, hcv core protein may play a direct role in the fibrogenesis via upregulation of a-sma, tgf-pl, collagen type i and tgfrii, mmp-2 and collagen type i. further study is needed to clarify exact signal transduction of fibrogenesis by hcv core protein in the co-culture system. background: to current knowledge, transforming growth factor beta (tgf-beta) signaling is mandatory to produce liver fibrosis. various molecular interventions designed to affect the tgf-beta system were successfully used to inhibit fibrogenesis. activated hepatic stellate cells have been considered as a major producer of extracellular matrix proteins in liver injury and fibrosis. in the present study, we wondered whether follistatin, activin inhibitory protein, reduce apoptosis and prevent liver fibrosis and whether activin plays a key role in liver fibrogenesis. methods: wistar male rats weighing around 380g were injected intraperitoneally with dimethylnitrosamine (dmn) (lopg/g body weight) three times a week for three weeks. either follistatin or saline were also injected intravenously three times a week. on the 22nd day, blood was collected and biochemical parameters were measured using the standard methods. the liver was either fixed with 4% bufferedparaformaldehyde for histological examination, or frozen immediately in liquid nitrogen for the rna analysis. tissue sections were either stained with hematoxyline-eosin, masson-trichrome, or subjected to immunohistostaining using antibodies against collagen type iv, alpha-smooth muscle actin (sma), fibronectin, tgf-beta. the mrna expression of activin, tgf-beta, timp were measured by rt-pcr. apoptosis was analyzed by tunel methods. wistar male rats were injected with dmn (10kglg body weight) once and liver tissues were obtained and fixed with 4% buffered-paraformaldehyde 0, 12, 24, 36, 48, 60, 72 hours and 7 days after injection for immunohistochemical staining. hepatocytes and hepatic stellate cells were isolated by collagenase perfusion method and by collagenase-pronase perfusion method and analyzed activin and tgf-beta mrna expression by rt-pcr. results: 50% of control rats died, whereas none of follistatintreated rats died within 22 days. the serum level of hyaluronic acid in follistatin-treated rats were significantly reduced. ast and alp levels were also decreased significantly. apoptosis was reduced by follistatin dose-dependently. the expression of tgfbeta, timp, collagen lv and alpha-sma expression were also decreased in follistatin treated rats. we then examined activin expression in liver after dmn treatment. activin expression was observed at the maximum level in hepatocytes 12 hours after dmn treatment. tgf-beta expression was not detectable 12 hours after dmn administration, and it was sfxikingly increased in stellate cells 48 hours after dmn administration but it was not detectable in hepatocytes. activin appeared in hepatic stellate cells as well as in hepatocytes 72 hours after dh4n administration. inducible nitric oxide synthase (inos) has been reported to play pivotal roles in the development of various types of liver injury and inos expression may be important in the process of fibrogenesis of nonalcoholic steatohepatitis in obesity. no-induced active substance was shown to activate matrix metalloproteinase. the inos mrna and protein activity have been found to be induce in rats on a high-fat diet. objectives we investigated whether 1) induction of nos occurs in liver of mice fed high-fat diet, and 2) nos increases matrix metalloproteinase activity and reduces collagen content, thus attenuates liver fibrosis. we compared fatty and fibrotic changes of hepatic tissues in inos-knockout (inos-'-) and wild-type (inos+/+) mice which were fed with high-fat diet for 12 weeks starting from 8 weeks old. marked induction of inos mrna occurred in inos+/' mice, but not in inos-/-mice. immunohistochemically, nitrotyrosine staining which is a footprint of no-induced active substance, showed positive in inos+'+ mice, and negative in inos-'-mice. in histopathologically showed fatty metamorphosis, but did not have any distinction between groups inos+'+ and inos-/-. the extracellular collagen content with azan staining in inos+/+ mice was markedly decreased compared with that in inos-'-mice. in gelatin zymography of matrix metalloproteinases we found that prommp-2 and prommp-9 were increased both in inos+'+ and inos-'-mice, but their active form was found only in inos+'+ mice. similar results were obtained from in situ zymography of hepatic tissue. the stronger gelatinolytic activity was found diffusely in hepatic tissues of inos+/+ mice than inos-/-mice. (129xc57bl16) were subjected to either sham operation or bile-duct ligation. animals were sacrified two weeks after sur-gery and blood and liver samples obtained. bile duct ligationinduced elevation of serum liver enzymes was similar between wt and atla (-/-) mice. however, the liverlbody weight ratio was greater in wt than in atla (-/-) mice. bile duct ligated wt mice showed severe septa1 fibrosis, as assessed by trichromic masson and sirius red staining. in contrast, atla (-/-) mice showed minor fibrotic lesions, which were mainly located in peribiliary areas. collagen accumulation, as assessed by morphometric analysis of sirius red staining and hepatic hydroxyproline content, was markedly lower in atla (-/-) mice compared to wt mice. positive sirius red stained area in bile duct ligated wt and atla (-/-) mice were 8.150.9 and 4.651.0%, respectively (p<0.05). the increase in hepatic concentration of bioactive tgfb and proinflammatory cytokines (tnfa and illb) was attenuated in atla (-/-) mice compared to wt mice, as assessed by elisa. moreover, immunohistochemistry analysis revealed decreased lipid peroxidation products as well as decreased phosphorylation of c-jun and p42-44 mapk in atla (-/-) mice compared to at1 (+/+) mice. chronic liver failure stimulates the onset of interstitial liver fibrosis, which eventually results in "capillarization" of the sinusoid, impeding clearance of toxic substances by hepatocyte cells. the goal of this work was to search for the therapeutical effect of adjuvant gene therapy using adenoviral vectors containing cd-nas for human urokinase plasminogen activator and human matrix metalloproteinase-8 (ad-hupa plus ad mmp-8) on cirrhosis and its relationship with manganese brain accumulation, striatum dopamine and its metabolites in the rat. mn+2 is a wellknown neurotoxic metal which has been found accumulated in brain and blood of cirrhotic patients with hepatic encephalopathy. mnt2 elimination takes place via the hepatobiliary route. methods 200 gr wistar rats underwent bile-duct ligation (bdl) for 4 weeks and concomitantly treated with 1 mglml of mncl2 in drinking water (bdl/md2). after this point, five animals were sacrificed and serum, liver and brain tissue (striatum) were obtained. of the remaining bdl/mn+2-cirrhotic animals (n=10), 5 were injected with ad-hupa plus ad-mmp-8 (3x10" + 1.5~10~' vp/kg respectively) and 5 rats injected with (4.5~10" vp/kg of ad-p-gal). this treatment lasted 10 days. then, biological samples were recollected as before. an additional experimental model represented by cirrhotic rats injured chronically for 7 weeks with cclb were also monitored for their response to this therapy. results seven wk-cc4-cirrhotic animals treated with ad-hupa plus ad-mmp-8 (total 4.5x101'vp/kg) were monitored at 2,4,6,8, 10 and 12 days after combined gene therapy treatment (n=18). these animals showed improvement in liver fibrosis of up to 55% as compared with their ad-p-gal (n=18) treated cirrhotic counterparts. these results correlated with hydroxyproline detemiinations. furthermore, survival was determinated in 28 additional cirrhotic animals. 14 cirrhotic rats treated with ad-hupa plus ad-mmp-8 had a significantly higher probability of survival at 60 days after beginning of treatment as compared with 14 ad-@-gal cirrhotic rats. bdl/mn+2 injured rats displayed tremors, rigidity, and gait abnormalities. ten days after treatment with combined therapeutic gene therapy, these symptoms decreased. also, liver fibrosis was evidently less (25%) as compared with ad-p-gal treatment rats. brain tissue (striatum) was recollected 10 days after bdl/mnf2 animals were injected with either ad-hupa plus ad-mmp-8 or ad-p-gal alone. dopamine (3.04 mglgr) was decreased in 30% in ad-p-gal treated animals, as compared with 4.79 mglgr of dopamine found in ad-hupa plus ad-mmp-%treated animals. dyhydroxyphenylacetic acid (dopac a main dopamine metabolite) was as high as in ad-hupa plus ad-mmp-%treated animals (13.56 mglgr striatum) indicating a higher dopamine turnover in nontherapeutically treated cirrhotic animals. moreover, animals treated with ad-hupa+ad-mmp-8 showed a 50% decrease in ascitis and gastric varices as compared with ad-p-gal-treated animals. (gastroenterology 1221924,2002) . while ppar-a was reported to be involved in induction of antioxidant enzymes expression and activities (life sci 63:135,1998). furthermore, in respect of hepatic fibrosis, oxidative stress induces hepatic fibrosis and many reports have demonstrated that several antioxidants can inhibit hepatic fibrosis. therefore, in the present study, we examined whether ppar-a ligands, i.e. wy-14,643 and fenofibrates, can suppress hepatic fibrosis by attenuating oxidative stress in experimental rat model and possess a possibility of therapeutic candidate for hepatic fibrosis. methods: fibrosis was induced in male wistar rats by intraperitoneal administration of thioacetamide (taa) (200 mglkg twice weekly for 6 weeks). in the treated groups, ppar-a ligands, wy-14,643 (wy) and fenofibrates, were fed a diet containing 0.1% (wlw), and ppar-y ligands, pioglitazone (pio), were fed containing 0.01% (wlw) all through the experiment. control cirrhotic rats received saline injections for 6 weeks. after killing all rats, histological examinations (he, azan staining, immunohistochemistry of a-sma), serum value of alt and hyaluronic acid, mrna expression of ppars, acyl-coa oxidase (aco), a1 (i) procollagen, and antioxidant enzymes, such as superoxide dismutase (sod) and catalase. we also determined lipid peroxidation (lpo), glutathione levels, and activities of sod and catalase in the perfused liver. results: semi-quantitative analyses of fibrotic area revealed that wy co-administration with taa reduced to only 19% of the area of taa-treated rats. there was no significant difference in azan staining of the liver between taatreated rats and taa-treated rats. wy administration could not lower serum alt value in chronic and acute taa injury. these observations suggest that wy fails to modulate the hepatotoxicity of taa. an increased expression of ppar-cy in taatreated rats were observed, but the expression of ppar-a was abolished in taa-and taa-treated rats. wy intensively enhanced mrna levels of aco which was regulated by ppar-a, increase nearly 50-fold than controls, but the expression of aco diminished in taa-and taa-ppar-a activators, wy-14,643 and fenofibrates, treated rats as well as ppar-a. the mrna levels of a1 (i) procollagen and tgf-/3l were strongly increased in taa-and taa-administrated rats than in controls, while they were dramatically suppressed in taa-treated rats. the catalase mrna was reduced to 15% of the controls in taa-and taa-treated rats, however, taatreatment prevented this decrease to 70% of the control levels. catalase activity increased 2-fold in taatreated rats than in controls, while it decreased 60% of the controls in taa-and taa-treated rats. in taa-and taa-treated rats an increase of lpo was observed, but not in ta-atreated rats. we c o n h e d that fenofibrates treatment could reduce hepatic fibrosis to approximately 16% of taa treatment for 6 weeks by semi-quantitative analysis of fibrotic area. conclusion: our data indicate that war-a activators, not ppar-y, can markedly inhibit hepatic fibrosis in the experimental taa-induced rat cirrhotic model. we suggest that ppar-a and catalase are important in the development of hepatic fibrosis. therefore, we conclude that suppression of hepatic fibrosis by ppar-a activators is probably due to their antioxidant effects via increased activitiy of catalase which reduces hydrogen peroxide, and that fibrates, such as fenofibrates and bezafibrates, might be new candidates for the treatment of hepatic fibrosis. fibrosis is a common end-point in clinical trials of chronic hepatitis c. liver biopsy remains the gold standard for fibrosis evaluation. however, variability in fibrosis distribution within the liver (sampling variability) is a potential limit to liver biopsy. in order to assess the influence of sampling variability on the accuracy of liver fibrosis assessment with biopsy, we measured liver fibrosis on virtual biopsies of increasing length reconstituted from digitalized images of large liver sections using two different methods of fibrosis assessment. method large sections (3cmz) were performed from 17 surgical liver samples with chronic hepatitis c and various degree of fibrosis. measurement of fibrosis on the whole section was considered as the reference value. from the digitalized image of the whole section, virtual biopsies of increasing length (2.5 to 200mm) were reconstituted. fibrosis was assessed independently on each individual virtual biopsy using both image analysis and meta-vir score. results were compared to the reference value. results: a total of 10.659 virtual biopsies were studied. using image analysis, a strong dispersion of area of fibrosis was observed for virtual biopsies smaller than 4omm. only 22% of 1,5 mm length virtual biopsies had a measured of area of fibrosis equal to reference value 2 10%. this percentage increased progressively with increasing size (30%, 50%, 69% for biopsies of 25mm, 4omm, loomm length, respectively). using metavir scoring system, 65% of biopsies of 1,5 cm length were correctly categorized according to the score assessed on the whole large section. it increases to 75% for a 2,5 cm size without any substantial benefit for longer biopsies. a same trend was observed whatever the stage of fibrosis. conclusion: sampling variability of fibrosis is a significant limit for fibrosis assessment with liver biopsy. this study suggests that a length of at least 25mm is necessary to valuably evaluate fibrosis with semiquantitative score. sampling variability become a major limitation for more accurate method such as automated image analysis. (msi-1) , a rna-binding protein, is highly observed in developing central nervous system and thought to be a mammalian neural stemlprogenitor cell marker. we have reported that cells positive for musashi-1 (msi-1) protein appeared during spontaneous resolution of rat liver cirrhosis and that some msi-1-postive cells were also positive for matrix metalloproteinase (mmp)-13, possibly implicating active participation of stem cells in the resolution of collagen. though msi-1 antigen is expected to be expressed during very early stage of differentiation in the cellular lineage of liver, the type of cells appeared in the damaged liver and expressed msi-1 remain to be elucidated. the present study is designed to characterize the msi-1-positive cells in cirrhotic liver from the aspect of differentiation, significance and expected function of stemlprogenitor cells in the liver, especially fibrolytic function. methods: rat liver fibrosislcirrhosis was established by intraperioneal injection of cc14 twice a week. liver samples were obtained 2, 5 and 7 days after the last injection of 12-week cc14 intoxication. liver tissue samples were immunohistochemically stained for msi-1, c-kit, nestin, a-smooth muscle actin (a-sma), ck-19 and mmp-13. gene expression of msi-1 was also observed by reverse-transcription polymerase chain reaction (rt-pcr). results: neural stemlprogenitor cells, as defined by their expression of msi-1 andlor nestin, were not observed either in normal rat liver or after 4 weeks of c c 4 administration. rt-pcr analysis revealed very slight signals of msi-1 mrna at 2 days after discontinuance of 8-week ccld treatment. on the contrary, remarkable increase of msi-1 mrna was observed at 2 days after the 12 weeks ccl4 treatment, then the signals decreased gradually. immunohistochemical analysis showed that a considerable number of cells positive for msi-1 appeared in the early recovery stage from advanced liver fibrosis, especially at day 5 after the last injection of 12 weeks ccl4 treatment. expression of msi-1 in liver tissue was proceeded by and partly overlapped with that of c-kit, a marker of hematopoietic stem cells. some msi-1-positive cells observed around perivenular regions were as very small as oval cells, while the size of msi-1-positive cells present along the resolving fibrous bands varied and some of them expressed mmp-13. some ductal cells were positive for msi-1 but not for ck-19 examined in serial sections. msi-1 positive cells did not express hepatocytes markers such as albumin or afp. cells positive for a-sma and msi-1 were observed in some portions, but most of msi-1 expressing cells did not show positive staining for a-sma or desmin. at day 7, a few msi-1-positive cells were observed around the remaining fibrous bands. s-adenosyl-l-methionine represses the fibrosis, constitute a good model for studying the mechanisms responsible for antifibrotic therapy. to test whether administration of s-adenosyl-l-methionine, a precursor of glutathione and an agent shown to prevent liver toxicity in a variety of settings, could repress the activity of the mouse pro-alpha 2(i) collagen gene in hepatic stellate cells, homozygous transgenic mice harboring the -17 kb to +54 bp of the proximal promoter of the mouse alpha 2(i) collagen gene cloned upstream of the escherichia coli beta-galactosidase reporter gene (lacz) were used. chronic liver injury was induced by injecting intraperitoneally 5 mllkg of body weight of cc4 (25% vlv in mineral oil) three times per week for 4 weeks. s-adenosyl-l-methionine was administered intraperitoneally at a dose of 10 mglkg of body weight every day for 4 weeks. control groups were given either mineral oil or s-adenosyl-lmethionine alone. hepatocellular damage and protection by sadenosyl-l-methionine was confirmed by measuring serum levels of transaminases; s-adenosyl-l-methionine lowered alt levels in the ccl4-treated mice from 143 to 28 ull and ast from 171 to 66 ull (control values in the absence of cc14 were 26 and 58 ull for alt and ast, respectively). hematoxylin and eosin staining in the cc14-treated mice revealed the presence of mallory bodies, lymphocyte infiltration, centrilobular steatosis, and perivenular and pericellular fibrosis, and s-adenosyl-l-methionine minimized the pathology score. masson's trichrome staining showed less collagen deposition in mice treated with cc14 plus s-adenosyl-lmethionine than in the cc4-treated mice. histochemical analysis using x-gal staining allowed for the precise identification of the cell type in which the beta-galactosidase gene was active as driven by the pro alpha 2(i) collagen promoter. results indicated activation of the pro alpha 2(i) collagen promoter in mice treated with ccl, and repression of such activation in mice co-treated with s-adenosyl-l-methionine. immunofluorescence analysis of mice injected with cc14 revealed colocaliiation of alpha-smooth muscle and beta-galactosidase positive cells, suggesting that the activation of the promoter occurred only in hepatic stellate cells. these results suggest that one mechanism by which administration of s-adenosyl-l-methionine, a precursor of glutathione synthesis, could ameliorate liver fibrosis is by decreasing the responsiveness of the promoter of the alpha 2(i) collagen gene to a profibrogenic stimuli such as ccb. these transgenic mice should prove to be useful in further studies on how s-adenosyl-l-methionine exerts this repression of the alpha 2(i) collagen gene and perhaps to studies with other liver toxins such as alcohol. background endothelin-1 (et-i), the most powerful constrictor of the liver vasculature and stimulator of the synthesis, by kupffer cells, of a potent systemic vasodilator, platelet-activating factor, is also implicated in the fibrosis of lung, kidney and liver. thus increased hepatic concentrations of et-1 and its receptors in human and experimental cirrhosis suggest its major role in the pathology of chronic liver disease and its complications (fibrosis, portal hypertension and systemic hypotension). we investigated whether et-1 receptor antagonism, after the development of fibrosis and cirrhosis, arrestslreverses the progression of chronic liver disease. methods: chronic liver injury was induced in rats by cc14 treatment ( of the development of cirrhosis in group 1 (histopathology score of 3.7 ? 0.41 vs 4.8 2 0.16, p< 0.01) and reversal of cirrhosis in group 2 (histopathology score of 3.6 2 0.41 vs 5 2 0, p<o.ol) rats. this was accompanied by reduced a-smooth muscle actin-positive cells (activated stellate cellslmyofibroblasts) in the tak-044treated versus saline-treated rats receiving cc4. tak-044 treatment caused significant amelioration of portal hypertension (p<0.05 in both groups), systemic hypotension (p<0.05 in both groups) and liver injury (reduced activities of serum ast, alt and ldh), and improved hepatic synthetic capacity (increased serum albumin concentration) in both groups of rats relative to the vehicle-treated rats. tak-044 treatment reduced collagen synthesis as evidenced by decreased hepatic hydroxyproline, collagen-a type i mrna expression, and mrna and protein expression of a potent fibrogenic cytokine tgf-p1. conclusions: the results emphasize the role of et-1 in the development of cirrhosis and strongly suggest that blockade of its actions can be a rational therapy for chronic liver disease and its complications. subunits which are reported to be stimulatory. however p50 and p65 can form homodimers which have been described as inhibitors and stimulators of gene expression respectively. the aim of the study was to determine the role of the p50 subunit of nfkb in the progression of fibrosis. methods -chronic liver fibrosis was induced in p50 knockout and wild type control mice by administering twice weekly ccl4 injections for 12 weeks. livers and blood samples were harvested at day 1, 3, 5,7 and 14 after the last ccl4 injection. results -p50 knockout mice developed more severe liver disease as measured by serum alt's and peak fibrosis scores. the increased liver injury in p50 knockout mice was associated with a massive infiltrate of inflammatory cells into the liver compared to the control wild type mice. the majority of the inflammation was neutrophilic as measured by immunohistochemistry, however we also observed an increase in cd3 positive t cells. taqman quantitative rt-pcr analysis was used to measure collagen i, alpha-smooth muscle actin (asma) and timpl gene expression in the p50 knockout and wild type mice. all three genes were elevated in the mice laking p50 subunit of nfkb. immunostaining confirmed an increase in asma positive cells in the livers of p50 knockout mice compared to wild type controls. conclusion: the p50 subunit of nfkb may play an important role in protecting against the development of liver fibrosis and reducing liver inflammation in chronic liver injury. gnainsky, volcani center, bet dagan, israel; orna halevy, hebrew university of jerusalem, rehovot, israel; gadi spira, technion -israel institute of technology, haifa, israel; rafael bruck, wolfson medical center, holon, israel; arnon nagler, sheba medical center, tel hashomer, israel; mark pines, volcani center, bet dagan, lsrael introduction halofuginone is an inhibitor of hepatic fibrosis as demonstrated by prevention of dimethylnitrosamine (dmn) and thioacetamide (taa)-induced fibrosis. halofuginone is also responsible for the resolution of advanced fibrosis and increase in cirrhotic liver regeneration. methodslaims in this study atlas microarray technique was applied in vivo in an effort to identify genes involved in the halofuginone-dependent prevention of liver fibrosis. to that end we compared cdna array hybridization of taa-treated rat liver biopsies to biopsies of livers from rats treated with both taa and halofuginone. results from the 588 genes of the array, 13 were differentially expressed. one of the genes up-regulated by halofuginone was identified as protein tyrosine phosphatase of regeneration liver -1 (prl-l), an immediate early gene known to be induced during liver regeneration. the atlas microarray results were confirmed by northern blot analysis and by in situ hybridization. hepg2 cells in culture expressed high levels of prl-1 gene that was down-regulated upon serum starvation. halofuginone increased the prl-1 gene expression in the serum-starved cells in a dose and time-dependent manner. on the protein level, halofuginone caused translocation of prl-1 from hepg2 and huh-7 nucleus outer membranes to the cytosol as demonstrated by immunofluorescence using prl-1 antibodies. prl-1 translocation is known to be associated with alterations in cell cycle. halofuginone caused accumulation of hepg2 cells in the gym phase as analyzed by flow cytometry suggesting a putative g2im arrest. conclusions these results demonstrate the involvement of halofuginone in the hepatocytes cell cycle through prl-1 gene expression and its cellular translocation. nathalie senant, universitk victor segalen bordeaux 2, bordeaux, france; genevieve freyburger, hdpital pellegrin, bordeaux, france; toulouse, france; alexis desmouliire, universitk victor segalen bordeaux 2, bordeaux, france several lines of evidence incriminate the hemostasis system in liver fibrogenesis. first, experimental and human pathological data suggest a role for micro-thrombosis in fibrosis progression. secondly, several hemostasis proteins, such as thrombin, can regulate the expression of fibrogenic mediators through signaling by cell-surface receptors. the aim of this study was thus to evaluate the effect of direct thrombin inhibition on experimental liver fibrosis. fibrosis was induced in rats by administration of cc14 in olive oil orally 3 times a week for either 3 or 7 weeks. the thrombin antagonist ssr 182289 (sanofi-synthelabo research) was given daily orally at 30 mglkg (a dose that has been shown to significantly inhibit thrombin in vivo) starting 1 week after the beginning of cc14 intoxication. control groups received either the respective solvents or ssr 182289 alone. nine animals were used in each group. fibrosis was quantified with histomorphometrical analysis of sirius red-stained liver sections and expressed as a percentage of the surface of the section. we also measured the level of fibrogenic cell activation with quantitation of the area occupied by alpha-smooth muscle actin (asma)-positive cells. the levels of tissue inhibitor of matrix metalloproteinase-1 (timp-i) transcripts were analyzed by northern blot on total liver rna and expressed as timp-l/gapdh ratios following quantitative measurement with an instant imager. after 3 weeks of cc14, the area of fibrosis increased from 0.285 % 0.08 in controls to 2.62 i 0.68 % and was slightly but non significantly decreased by ssr 182289 (2.26 lr 0.62 %, ns). the area of asma-positive cells was however significantly decreased by ssr 182289 from 3.83 ? 1.04 to 2.86 i 0.79 % (p =0.03). in animals receiving cc14 for 7 weeks, treatment with ssr 182289 resulted in a decrease in fibrosis area from 7.01 t 0.24 to 4.62 t 0.77 % (p =0.05). similarly, the asma-positive area decreased from 11.57 5 5.57 to 7.71 i 3.89 % (p =0.05). the timp-i/gapdh ratio was decreased by ssr 182289 at 3 weeks (from 0.118 2 0.018 to 0.087 2 0.014, p =om). ssr 182289 alone had no effect on the measured parameters at both time points. additionally, it did not alleviate the acute toxicity of cc14 as shown by the levels of serum transaminases and the area of necrosis that were not significantly modified. altogether, these data provide evidence that thrombin antagonism can reduce the extent of liver fibrosis in this model. ongoing experiments should clarify whether this results from an anticoagulant effect or an inhibition of thrombin signaling effects. center, tel aviv, israel background and aims: proliferation and "activation" of hepatic stellate cells (hscs) and production of collagen are involved in the pathogenesis of liver inflammation and fibrogenesis. ligands of nuclear hormone receptors, such as triiodothyronine (t3) and peroxisome proliferator activator receptor y (ppary) ligands have been shown to affect fibrogenesis. levels of ppary are reduced in activated stellate cells and addition of the iigand increases ppary receptor expression and ameliorates fibrosis. in contrast, t3 has the opposite effect on liver fibrosis. low circulating t3 levels reduces liver fibrosis and cirrhosis. in this study, we investigated the in vitro effects of t3, ppary ligand and their combination on proliferation and activation of a rat hsc cell line. methods: transforming growth factor (tgf-pi) was used to induce myofibroblast-like phenotype in rat hscs (hsc-t6). activation of thyroid hormone and ppary receptors was induced with t3 and 15-deoxy-prostaglandinj(2)(pj2), respectively. signal transduction markers and activation marker a-smooth muscle actin were determined using western blotting. collagen i expression was measured by northern blotting. results: both t3 and pj (2) inhibit hsc proliferation induced by platelet-derived growth factor and by tgf-p. t3 inhibits cell proliferation by inducing apoptosis, while pj(2) reduces expression of proliferation marker cyclin d1. both "i3 and pj(2) decrease expression of activation marker a-smooth muscle actin. in addition, t3 strongly increases both basal and tgfeinduced collagen i expression. by contrast, pj(2) decreases collagen expression and inhibits t3-induced collagen production in tgfp-activated hscs. conclusion: both t3 and pj(2) reduce mitogen-induced proliferation of rat hscs, but have different effects on collagen i expression. these results suggest that prolierationlactivation and collagen i expression are two differently regulated processes in kato, sadakazu aiso, hiromasa lshii, keio university, tokyo, japan connective tissue growth factor (ctgf) is a profibrogenic molecule involved in several human fibrotic disorders including liver fibrosis. in human as well as experimental liver fibrosis, ctgf overexpression, through upregulation of ctgf mrna in hepatic stellate cells (hscs), is associated with the degree of fibrosis. it has been known ctgf is produced in many cell types in the live and exogenous ctgf modulates even the activation of hscs. although alcohol is the major cause of liver fibrosis, the role of ctgf in alcoholic liver fibrogenesis, has been poorly understood. cytochrome p-4502e1 (cyp2el)-mediated oxidative stress, as a result of chronic and excessive ethanol consumption, has been implicated as a crucial factor in alcoholic liver fibrogenesis. the goal of a series of our studies was to clarify the effect of cyp2e1mediated ethanol oxidation on ctgf expression. we previously reported the results of the assay using the well-established hepg2 cell line constitutively expressing cypzei, which was kindly provided by dr. a. cederbaum ( mt. sinai school of medicine, ny). ctgf mrna quantitation assay by a real-time reverse polymerase chain reaction (rt-pcr) showed a significant increase in ctgf mrna levels in ethanol-treated e9 cells in a time-dependent manner up to 36 hours after initial ethanol treatment. there was nearly a 2-fold increase in ctgf mrna levels when ethanoltreated e9 cells were coincubated with phorbol myristate acetate (pma), a reagent which enhances expression of cyp2e1. ctgf mrna levels were significantly reduced when ethanol-treated e9 cells were co-incubated with 4-methylpyrazole(4mp), a cyp2el inhibitor, or antioxidants, n-acetylcysteine and trolox. in the present study, we visualized the state of oxidative stress in ethanol-treated e9 cells by using mab5f6, a newly-established marker for oxidative stress. the mab5f6, which was generously provided by dr. k uchida (nagoya university, japan), was raised against the acrolein-modified keyhole limpet hemocyanin to verify the protein-bound acrolein in vivo. the staining, procedure was detailed elsewhere. immunohistochemical detection of proteinbound acrolein revealed e9 cells were stained when treated with ethanol. without ethanol treatment, no staining was observed in ethanol upregulates pro-fibrotic connective cells via cytochrome p-4502e1-mediated e9 cells even at a 24 -hour incubation period. moderate or no staining was observed when ethanol-treated e9 cells were coincubated with antioxidants. staining intensity by mab5f6 is associated with ctgf mrna levels in this cell line. collectively, these data suggest ctgf mrna upregulation in e9 cells seems to be mediated by the state of oxidative stress. our present study first linked cyp2el-mediated oxidative stress with ctgf gene regulation, thus suggesting a possible involvement of ctgf in alcoholic liver fiberogenesis. disclosures: sadakazu aiso -no relationships to disclose hiromasa ishii -no relationships to disclose mikio kajihara -no relationships to disclose (2) human livers with biliary fibrosis. methods: changes in ntp-dase2 expression in rat liver were determined in normal and 2-wk bile duct ligated (bdl) or 6-wk c c 4 rat livers and pflpm using confocal immunofluorescence, immunoblot, and northern blot. changes in ntpdase2 expression in human liver were determined in de-paraffinized liver biopsy specimens using confocal immunofluorescence. results: as was previously reported, ntp-dase2 was expressed in pf surrounding intrahepatic bile ducts in normal and sham bdl controls. in bdl rat livers, however, nt-pdase2 fluorescence was completely lost. no decrease in ntp-dase2 fluorescence was noted in cc14 rat livers within portal areas, although an increase in ntpdase2 fluorescence was seen in central areas. pf from sham bdl control animals expressed nt-pdase2 and the intermediate filament vimentin. "pf" from bdl animals did not express vimentin but were positive for a-smooth muscle actin (sma), suggesting that they were in fact pmf. most bdl pmf did not express ntpdase2; however, occasional cells were seen with sma and ntpdase2 in a pen-nuclear compartment. immunoblot analysis of pf revealed a marked decrease in ntpdase2 detection in bdl cells relative to sham bdl. no change was noted in ccll or c c 4 control cells. northern blot analysis revealed no differences in ntpdase2 mrna levels in sham bdl, bdl, ccl, control, or cc14 pf. confocal micrographs of normal human biopsy specimens revealed ntpdase2 co-localization with vimentin in pf surrounding intrahepatic bile ducts. however, ntpdase2 fluorescence was either markedly decreased or lost in specimens from patients with primary biliary cirrhosis. no loss of ntpdase2 fluorescence was seen in biopsy specimens from patients with hepatitis c. conclusions: ntpdase2 expression is specifically decreased in biliary fibrosis, largely through a posttranslational mechanism. "portal fibroblasts" from bdl rats are in fact portal myofibroblasts, which lose expression of ntpdase2. consistent with findings in rats, ntpdase2 expression is also specifically lost in human biliary fibrosis. work is now underway to determine whether loss of ntpdase2 in the peri-biliary compartment alters nucleotide signaling in bile duct epithelia. myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. we hypothesised that marrow derived myofibroblasts contribute to liver cirrhosis. methods. we sought to analyse the origin of myofibroblasts within cirrhotic liver in two scenarios: three male recipients of female liver transplants who have subsequently developed post transplant hepatitis c cirrhosis and a female recipient of a male bone marrow transplant who later developed hepatitis c induced cirrhosis. through the use of in situ hybridisation for the y chromosome we have been able to track cells of male origin. results. we have detected significant numbers of y chromosome positive cells in fibrotic areas. by combining the in situ hybridisation with immunocytochemistry these cells were found to be positive for vimentin, alpha-smooth muscle actin and negative for cd45. these cells were therefore determined to be of a myofibroblast phenotype. in the liver transplant recipients, 15.4% (corrected count 33.6%) of the myofibroblasts were y chromosome positive. in the female recipient of the male bone marrow transplant, 12.5% (corrected count 27.3%) of the myofibroblasts were y chromosome positive. conclusions. within all the livers analysed we found frequent myofibroblasts within and adjacent to the fibrotic bands that are of bone marrow origin indicating an extrahepatic cell contribution to the pathogenesis of human liver cirrhosis. (hsc) is the major fibrogenic cell of the liver and a key target of potential antifibrotic therapies. hepatocyte growth factor (hgf) is antifibrotic in several models of experimental liver fibrosis, but its mechanism of action is uncertain. potential use of hgf as a therapeutic peptide is limited by its size and need for parenteral administration. to overcome these potential limitations, refanlin has been developed as a synthetic small molecule sflhgf mimetic, that has demonstrated antifibrotic properties in renal fibrosis. aim: to study the in vitro and in vivo antifibrotic efficacy of refanlin. methods: serum starved lx2 cells, an immortalized human hsc line, were treated with 12 mglml of refanlin for 24 hours. proliferation studies using tritiated thymidine incorporation were performed. rna was isolated, and a panel of fibrosis markers (al-smooth muscle actin (asma), collagen i, bpdgf-receptor, 9, 14 (mmp2, 9, 14) , tissue inhibitor of metalloproteinase-1,2 (timp1,2), were assessed by real time rt-pcr analysis. cirrhosis was induced in sprague-dawley rats by if thioacetamide (taa) (200 mg/kg tiw) for six weeks. in the refanlin treated group, 1 mglkg of refanlin was administered five days per week for six weeks, concurrently with the taa. control rats received ip pbs five days per week for six weeks, along with taa. liver tissue was formalin-fixed and paraffin-embedded, then stained with 1% picrosirius red and computer morphometric analysis was performed for collagen quantitation using the bio-quanttm software. results: in cultured human stellate cells, there was a 60% decrease in asma, a 70% decrease in the expression of tgfb-receptor and mmp2, and a 50% decrease in timp2. there was no effect on proliferation. in vivo, fibrosis decreased by one metavir stage in rats treated with c6 and thioacetamide for 6 weeks. furthermore, c6 led to a significant decrease in portal pressure compared with non-treated rats with fibrosis. conclusions: refanlin may have antifibrotic efficacy through its inhibition or downregulation of tgfb receptor as well as mmp2. in vivo antifibrotic activity of refanlin is associated with reduced portal pressure. these experiments better define the potential targets of hgf's antifibrotic activity. ongoing studies in mechanistically distinct models of fibrosis will broaden our understanding of this agent's effects and point towards therapeutic trials in humans. kanto, aki sato, michiyo inoue, hideki miyatake, mitsuru sakakibara, takayuki yakushijin, chika oki, tetsuo takehara, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim hepatitis c virus (hcv) infection induces a wide range of chronic liver injuries. impaired thl response against hcv may be involved in hcv persistence, however, its precise mechanism is largely unknown. dendritic cells (dc) are the most potent antigen-presenting cells that play essential roles in initiating as well as regulating anti-virus immune responses. blood dc are grouped as myeloid (mdc) and plasmacytoid dc (pdc). with respect to their functions, mdc produces il-12 and tnf-a upon stimulation and drives thl response, while pdc releases considerable amount of type-i ifn upon virus infection and mainly skews th2. we previously reported that monocytederived dc is functionally impaired (kanto t. et al. j lmrnunol 1998) , however, the roles of blood dc subsets in the direction of thllth2 in hcv infection are still elusive. to address these issues, we compared the numbers and functions of mdc and pdc between chronically hcv-infected patients and uninfected donors. subjects and methods sixty-six hcv-positive patients (43 chronic hepatitis and 23 asymptomatic carriers) and 19 agematched uninfected donors were enrolled. mdc or pdc was determined by the patterns of lineage markers, hla-dr, cdllc and cd123. each type of dc was magnetically separated or sorted under facs vantage and was subjected to the functional analyses. the ability of dc to polarize naive cd4 t-cells towards thl or th2 was estimated by the pattern of ifn-ylil.-4/il-10 production from mdc-or pdc-primed cd4. results absolute numbers of mdc, pdc and their cd34+ progenitors are lower in hcv-infected patients than in control subjects, most significantly in those with chronic active hepatitis (mdc, pdc, cd34, median, patients vs. controls; 4.2,1.0,0.511*.1 vs. 6.9,3.0,1.9/pl, p<0.05). both types of dc from patients are impaired in the stimulation of allogeneic cd4 t-cells and the production of il-12 p70 or ifn-a compared with healthy donors. on encounter with nayve cd4 t-cells, mdc from the patients are less able to drive thl response, while pdc from them prime more il-10 producing t-cells than those from donors. exogenous il-12 significantly improved the patient mdcdriven thl response, however, it simultaneously increased il-10 producers both in mdc and pdc. in contrast, ifn-y or il-15 stimulated the patient mdc to drive thl without increasing mdc-or pdc-primed il-10 producing cells. conclusion both types of blood dc in chronic hcv infection are reduced and play decisive roles in the development of an impaired thl and an il-10-rich environment, thus favoring hcv persistence. ifn-y or il-15 is feasible to restore the altered t-cell polarizing ability of blood dc in chronic hepatitis c. hepatitis c virus (hcv) replicates its genome in a membraneassociated complex composed of viral nonstructural proteins and rna as well as altered cytoplasmic membranes and other as yet unidentified host cell components. a specific membrane alteration, designated membranous web, was recently identified as the site of hcv rna replication in huh-7 cells harboring subgenomic replicons (gosert et al. j virol 2003; 77:5487-5492) . here, we describe hcv replicons that allow the direct visualization of hcv replication complexes in living cells. methods: a transposon-mediated random insertion screen followed by selection of viable replicons was performed to identify sites that tolerate insertions in nonstructural protein 5a (ns5a). the green fluorescent protein (gfp) coding sequence was subsequently inserted into selected sites. rna was in vitro transcribed from subgenomic replicon constructs harboring these insertions, followed by electroporation into huh-7 cells and selection in the presence of g418. results: two sites in the c-terminal domain of ns5a were found to tolerate the gfp insertion and allowed efficient initiation and maintenance of hcv rna replication. expression of a ns5a-gfp fusion protein of the expected molecular mass could be demonstrated by immunoblot, indicating that the gfp was stably retained and that processing of the viral protein occurred normally. more important, expression levels were robust enough to allow direct visualization of this fusion protein by fluorescence microscopy. gfp fluorescence was found in a cytoplasmic reticular pattern and in brightly fluorescing dots, the typical distribution of hcv nonstructural proteins in replicon cells. by confocal laser scanning microscopy, gfp fluorescence was found to colocalize with other hcv nonstructural proteins in the brightly fluorescing dots, suggesting that these represent hcv replication complexes, previously identified as membranous webs. metabolic labeling of the nascent viral rna with brutp and (immuno)electron microscopy studies are underway to further validate these findings. moreover, live cell imaging studies using these replicons have successfully been initiated. conclusion: we have identified sites in ns5a that allow insertion of gfp in the context of functional hcv replicons and direct visualization of hcv replication complexes in living cells. these findings have implications for the structure and function of the hcv ns5a protein as well as for the architecture of the hcv replication complex. this system will allow to gain a dynamic view of the formation and turnover of hcv replication complexes and may be useful to select for cell populations permissive for hcv rna replication. infection. masahisa jinushi, tetsuo takehara, tomohide tatsumi, takuya miyagi, takahiro suzuki, tatsuya kanto, norio hayashi, osaka university graduate school of medicine, suita, osaka, japan background and aim it has been generally accepted that the pertubation of cellular immunity plays an important role in establishment and persistence of chronicity in hcv infection as well as resistance to ifn therapy. accumulating lines of evidence have unveiled that dendritic cells (dcs), most potent antigen presenting cells, are impaired in patients with hcv infection, which may be involved in disturbance of anti-hcv immune responses. we recently reported that mhc class i-related chain a and b(mical b),ligands of nk activating receptor nkgzd, are induced on dcs upon interferonaand gain their ability to antivate nk cells; their expression is severely impaired in dcs derived from hcv-infected individuals (hcv-dc) (3. immunol. 170 1249 immunol. 170 -1256 immunol. 170 ,2003 . in the present study, we evaluated whether various types of cytokines have an effect for inducing micalb expression on hcv-dcs, thus resulting in triggering nk cell activation by dcs. methods dcs from healthy volunteers (n-dcs; n=15) or hcv-dcs (n=20) were generated from monocyte treated with gm-csf (1000 ulml) and il-4 (500 ulml) for 6 days. after stimulated with various cytokines (ifnalp, ifny, tnfa, il-12, il-15 or il-18) for 24 h, dcs were subjected to flow cytometric analysis of micalb expression. to investigate the involvement of each cytokine in mica/b expression on ifnalp-stimulated dcs, we employed elisa and rt-pcr to examine the cytokine expression (tnfa, il-lp, il-6, il-12~70, il-15 and il-18) in ifnalp-stimulated dcs, and also assessed micalb expression on dcs in the presence of neutralizing antibodies for these cytokines. to further address the stimulatory capacity of dc, allogeneic nk cells were cocultured with n-dcs or hcv-dcs at a ratio of 5:l for 24 h, and applied for flow cytometry of intracellular ifny expression, as well as 51cr release assay for the cytolysis of k562 target cells. in some experiments, masking antibody for nkg2d or micalb was added during nkldc cocultures, and then subjected to nk cell function assays described above. results micalb were induced on n-dcs, but not on hcv-dcs, upon iifnalp(% mic positive: n-dc 42.1% vs hcv-dc 3.4%; p<o.ol). in sharp contrast, micalb expression was increased on hcv-dcs at levels similar to n-dcs in the presence of of 5onglml il-15 (but not other cytokines) (%mic: n-dcs 49.4% vs hcv-dcs 47.6%). moreover, n-dcs, but not hcv-dcs, was capable of producing il-15 in response to ifnalp. little differences were detected in n-dcs and hcv-dcs for the production of tnfa, il-lp, 1l-6, il-12~70, or il-18. in addition, ifnalp-mediated micalb induction in n-dcs were completely inhibited in the presence of neutralizing antibody of anti-il15l il-15 receptorcu. even much lower doses (1nglml) of il-15 restored the ability of hcv-dcs to induce micalb and to activate nk cells in response to ifnalp. the ability of hcv-dcs for nk cell activation was largely diminished by adding the masking antibody of nkg2d or micalb. conclusion the current study demonstrated that il-15 acts as an indispensable mediator to induce micalb expression on ifnalp-stimulated dcs, and that hcv-dcs lack the ability to produce il-15 and to induce micalb expression in response to ifnalp. since defect in il-15 production is responsible for impaired nk cell activities by hcv-dcs, these findings shed light on the possibility that il-15 could improve the ifn-mediated anti-viral immune responses in chronic hcv-infected patients. the cellular immune response contributes to viral clearance as well as liver injury in acute and chronic hepatitis c virus (hcv) infection. understanding the relative immunodominance of hcvencoded peptides is important for understanding pathogenesis as well as potential vaccine candidates. we have developed a comprehensive and sensitive method to screen immune responses to all potential hcv epitopes in hcv-exposed individuals. methods: subjects with evidence of spontaneously resolved infection (eia+/hcv rna-neg, n = 15) and patients with evidence of chronic hcv infection (mean hcv rna level = 23.6 million copieslml; n = 15, all genotype la) had either leukophoresis or one-unit blood draw. we synthesized 750 peptides (15 mer-peptides overlapping by 11 amino acids) that spanned the entire hcv genotype l a polyprotein; responses to 33 pools of peptides were screened directly ex vivo by an ifn-gamma elispot assay. briefly, autologous dendritic cells (dcs) were generated from peripheral blood mononuclear cells selected by plastic adherence then cultured with gmcsf, il-4 and 10% hs for 5 d. dcs were pulsed with peptide pools to stimulate bead-purified cd8+ t cells. the remaining cd4+ t cells (5.0 x lo5 cells/well) were stimulated the same day with the peptide pools; 10 wells with no peptide (or siv peptides) were used as controls. only responses greater mean + 3 sds above controls were considered positive. the frequency and vigor of cd4+ t cell responses were statistically greater in resolved vs. chronic infection (mean 9.4 pools out of 33; median 8 in resolved vs. mean 3.86 pools; median 3.0 in chronics; p =.005). there was an inverse trend between # peptide pools with cd4+ t cell responses and hcv rna level. as shown in the figure, the hcv peptide pools differentially elicited responses; specifically, resolved infection was associated with greater cd4+ responses to pools core b, ela, ns3 protease-2, ns3 helicase-3, ns3 helicase-4, ns3 helicase-5, ns3 helicase-6, ns4b-2, ns5a-2, ns5a-3, ns5b-2. on the average, cd8+ t cells responded to 4.8 pools in resolved infection vs. 1.3 pools in chronics (p =.as). cd8+ t cell responses specifically directed against core 8, ns3 helicase-5, ns3-helicase 6, ns4b-2, ns5a-2 were more frequent in resolved as compared to chronic infection. conclusions: comprehensive mapping of hcv-specific immune responses by overlapping pools reveals significant differences in immunogenicity in resolved vs. chronic infection. in particular, regions core b (amino acids 96-203), ns3 helicase-5 (aa 1369-1479) and helicase-6 (1469-1579), ns4b-2 (1801-1899), ns5a-2 (2076-2191) induce both cd4+ and cd8+ t cell immunity and thus represent potential vaccine candidates. supported by nih r01 dk60590. sud, geoffiey c farrell, priyanka bandara, james g kench, storr liver unit, westmead hospital, westmead, nsw, australia; geoffiey w mccaughan, aw marrow gastroenterology (and liver centre, camperdown ns w, australia; jacob george, storr liver unit, westmead hospital, westmead, ns w, australia introduction: chronic hepatitis c virus (hcv) infection is associated with an increased prevalence of type 2 diabetes mellitus, which is known to accelerate fibrosis in nash. we hypothesized that viral-induced insulin resistance (ir) may be a mechanism for fibrogenesis in chronic hcv infection. methods and results: 1. to assess the influence of hcv infection on ir independent of any effect of hepatic fibrosis, 121 hcv subjects with minimal (stage 1) or no (stage 0) fibrosis were compared with 137 healthy volunteers matched by gender, body mass index (bmi) and waistlhip ratio. although the hcv subjects were younger than the healthy volunteers (p=0.004), they had significantly higher levels of markers of ir including fasting glucose (p=o.ol), insulin (p=o.o02), cpeptide (p<o.ool) and homa-ir (p=o.o02). 2. in 260 hcv patients (fibrosis stage 0 to 4), e examined the viral and diseaserelated factors (portallperiportal & lobular inflammation, viral genotype) which may be involved in the pathogenesis of ir in a multivariate model, controlling for other demographic and biochemical variables. by multiple linear regression analysis, independent predictors of homa-ir included bmi (p<o.ool), failed previous antiviral treatment (p<o.ool), portal inflammatory grade (p<o.ool) and genotype 3 status (p=o.ol). genotype 3 cases had significantly lower homa-ir than other genotypes at each stage of hepatic fibrosis. the adjusted difference in homa-ir between genotype 3 and non-genotype 3 was -0.58 (95% ci: -0.13 to -1.02; p=o.ol). 3. we then assessed if ir was associated with increased fibrotic severity. by multiple ordinal regression analysis, independent predictors for the stage of fibrosis in the 260 hcv subjects were homa-ir (or 1.3, p<o.ool), portal inflammatory grade (or: 5.3, p<o.ool), past alcohol intake (or 1.7, p<o.ool), age (or 1.1, p <0.001), alt (or 1.0, p=0.04), platelet count (or 0.93, p<o.ool), and serum cholesterol level (or 0.65, p=o.ool). as cirrhosis is known to cause ir, a separate multivariate analysis was performed for the 236 non-cirrhotic subjects. the independent predictors for fibrosis were unaltered and homa-ir remained in the model. in a subgroup of 117 patients with estimated duration of infection, multiple linear regression analysis showed that homa-ir was independently associated with an increased rate of fibrosis progression (p=0.03). conclusion: hcv infection induces insulin resistance irrespective of the severity of liver disease, and this effect appears to be genotype-specific. further, increased insulin resistance contributes to fibrotic progression. disclosures: priyanka bandara -no relationships to disclose geoffrey c farrell -no relationships to disclose jacob george -no relationships to disclose jason m hui -no relationships to disclose james g kench -no relationships to disclose geoffrey w mccaughan -no relationships to disclose background: approximately one half of hepatitis c virus (hcv) infected patients will fail to respond to current treatment regimens, pegylated interferon alfa and ribavirin. modeling of hcv rna decay in response to antiviral therapy can provide insight into viral and host determinants of viral response. aims: pharmacokinetic, pharmacodynamic, and immunologic outcomes were evaluated on 24 co-infected patients: 1) to evaluate the relationship between hcv rna decay and peg-ifn alfa2b serum concentrations in hivlhcv co-infected patients and 2) to evaluate association between hcv-specific immune responses and virologic outcome. methods: 24 co-infected, therapy-nake patients were treated with peg-ifn alfa2b 1.5pglkglwk and rbv 13 2 2 mglkglday. serum was obtained for hcv rna, hiv-1 rna and serum peg-ifn alfa2b levels at baseline, after the first dose (6,12, 24 hours) and on days 2, 3, 5, 6; after the second dose (6, 12, 24 hours) and on days 8,9; and after the third dose on days 14,15, and 16. peripheral blood mononuclear cells were obtained at baseline, weekly for the first month, and monthly thereafter. hcv and hiv-1 rna levels were determined by reverse-transcriptase polymerase chain reaction and peg-ifn alfa2b levels were measured by elisa. cd4+ cell proliferative responses to hcv antigens (core, ns3, ns4, ns5) and hiv-1 gag were performed at baseline, day 7 and 14, and weeks 4, 8, 12, and 24. among 24 co-infected patients, 21 are males, the mean age was 46 2 5.8 years, 8 are caucasian, 12 are african-american and 4 are hispanic. 20l24 patients were infected with genotype 1, 3/24 patients with genotype 2, and 1/24 with genotype 3a. results: pharmacokinetics: to date, 21 patients have been evaluated. 10/21 patients were end of treatment responders, but 2 of these were "late responders" (i.e. hcv rna declines after week 8 and is undetectable by week 24). data fitting during the first seven days revealed a free virion clearance rate c of 13.3 (day-l) and an infected cell clearance rate 6 of 0.21 (day-'). the half-lives were 2.7 hours and 4.0 days, respectively. pharmacodvnamics: viron production was blocked after the first dose of peg-ifn alfa with an average maximum effectiveness (emax ) of 89%. we estimated that the fifty percent effective concentration (ec50) of peg-ifn alfa2b, the theoretical in v i m drug concentration at which the drug efficacy is 5070, was five-fold lower in responders compared with non-responders while the maximum (cmax) and minimum (c,,,in) concentrations did not differ significantly ( table i ). the calculated minimum ( € , , , i n ) and maximum (emax) efficacy were also significantly increased in responders. immunolonic: the mean number of cd4+, cd8+ and cd56+ cells did not differ significantly at baseline or during treatment between responders and nonresponders. peripheral blood cd4+ hcv-specific proliferative responses did not differ significantly between responders and nonresponders except that the response to core antigen was increased in non-responders compared with responders at weeks 4 and 12. hiv gag cd4+ proliferative responses were stronger than hcv-specific responses at all time points evaluated. conclusions: the serum peg-ifn alfa2b concentration necessary for an end of treatment response is fivefold lower in responders compared with non-responders while there are no significant differences in the maximum and minimum peg-ifn alfa2b concentrations in responders and nonresponders. these data suggest that host factors responsible for a viral response may be different in responders compared with nonresponders, independent of serum peg-ifn alfa2b concentrations. is a significant predictor of liver disease severity in patients who are coinfected with hepatitis c virus (hcv). this accelerated liver disease is presumed to be due, in part, to hn-related immune suppression. hcv quasispecies variability is likely a marker of immune pressure on the virus. hyuotheses: 1. hcv quasispecies variability is decreased in those with hiv coinfection, and 2. treatment with highly active antiretroviral therapy (haart) is associated with increased variability, methods: hcv-infected patients with or without hiv were enrolled in a cross-sectional study. patients with genotype 1 infection and a parenteral risk factor for hcv were included. those with injection drug use (idu) were estimated to have acquired hcv 2 years after the onset of idu. alcohol use was determined using standard questionnaires. haart data were collected from the hiv clinic's database. only those with complete records were included. hcvquasispecies variability was determined using clonal frequency analysis of the second envelope gene hypervariable region. quasispecies heterogeneity was characterized according to complexity (the normalized number of unique quasispecies variants within a sample) and diversity (the average genetic distance between individual variants within the same sample). groups were compared using the student t-test for unequal variances, anova and the wilcoxon rank-sum test where appropriate. linear regression was used to assess the independent effects of hiv infection and haart on complexity and diversity. results: to date quasispecies data are available on 43 patients, 22 of whom are hivpositive. as expected, hiv+ subjects had significantly lower cd4 counts (mean 508 vs 956 cellslul, p<.oool). mean age and hcv duration were significantly lower in the hiv+ group (40.3 vs 45.5, p=.0017, and 15.7 vs 22.2 years, p=.oo18, respectively). the groups did not differ with respect to alcohol use over their lifetime or in the 6 months prior to enrollment. quasispecies complexity and diversity are detailed in the table below. while there was no significant difference among groups by anova (p=.16), the hiv+ subjects not on haart had less complexity and diversity than the other two groups, and differences between groups 1 and 2 and groups 2 and 3 were significant (table) . both hiv infection and haart were significantly and independently associated with complexity in a linear regression model; hiv infection predicted decreased complexity (p= .001), while haart predicted increased complexity (p=.007). in addition, higher alcohol use in the prior 6 months was independently associated with decreased complexity (p=.007). hiv infection and haart also were independently associated with decreased and increased diversity, respectively (p=.ool and.034). the cd4 count did not predict complexity or diversity, and the effect of haart appeared to be independent of the cd4 count. conclusion: hcv coinfection with hiv is associated with decreased hcv quasispecies complexity and diversity. while this effect is not completely reversed by haart, treatment of hiv is associated with significantly increased hcv complexity and diversity in our sample. furthermore, the effect of haart on hcv quasispecies variability appears to be independent of its effect on the cd4 count. these findings suggest that the immune pressure on hcv is enhanced in hcvlhn coinfected individuals who are on haart when compared to those who are not on haart. diversity" group (mean .t sd) (mean f sd) in the majority of cases, hepatitis c virus (hcv) becomes chronic and is associated with impaired viral-specific t cell immune responses; the mechanisms underlying viral persistence and lack of protective immunity are poorly understood. considering dendritic cells (dcs) play critical roles in initiating and modulating immune responses, we explored the effect of hcv proteins on dc genetic expression, phenotype, and function. methods: human dendritic cells (dcs) were generated following plastic adherence of monocytes from normal subjects and culture with gm-csf and il-4. autologous non-adherent cells were infected with vaccinia constructs expressing various hcv proteins (core, nssa, ns5b) or an irrelevant protein /3-galactosidase @-gal) as the control. following induction of apoptosis with uv irradiation, these vaccinia-infected cells were co-cultured with dcs. the phenotype, gene expression profiles and functions of the dcs were analyzed. dcs were evaluated after 8, 30, and 48 hrs of co-culture as well as after maturation with a cytokine cocktail (tnf-a, il-lp, pge2, il-6) for an additional 48 hrs. results: by facs analysis, we found no alteration in expression of co-stimulatory markers (cdso, cd86, cd40,) or mhc class i or i1 molecules. by elispot assay, we found that the recall response of cd4t t cells for cmv following pulsing of hcv-dc or control (p-gal)-dc was also not altered. however, between 2-10% of the genes probed in the bd clontech nylon array were differentially regulated. real time pcr has confirmed increased expression of scyc 1 (lymphotactin) in the presence of ns5a in 2 normals as well as 2 separate biological replicates. both clontech and affymetrix gene arrays revealed ns5a-induced increases in expression of il-8, a chemokine with pro-inflammatory properties. functional assays reveal that dcs which had engulfed apoptotic cells containing hcv core, ns5a, or ns5b demonstrated comparable ability to macropinocytose as measured by uptake of dextran-fitc or albumin-fitc when compared to p-gal-dcs. however, following uptake, the hcv-dcs died at a significantly greater rate than p-gal-dcs (-30%). conclusions: taken together, these results demonstrate that following engulfment of apoptotic cells containing hcv proteins, differential gene expression occurs in human dendritic cells, although the expression of mhc and costimulatory molecules and ability to stimulate memory (cmv) responses remain intact. the mechanisms underlying the surprising induction of death of dc-hcv in the macropinocytosis assay is currently under further investigation. institutes of health, bethesdu, md steatosis is a common histological feature of chronic hepatitis c virus (hcv) infection. the cause of hepatic steatosis in hepatitis c remains to be elucidated, but its association with obesity, diabetes and hyperlipidemia suggests an underlying metabolic dysfunction similar to that found in patients with nonalcoholic steatohepatitis (nash). additionally, recent data suggest an association between body mass index (bmi) and the degree of hepatic steatosis and fibrosis in hcv infection. insulin resistance (ir) is a key factor in the pathogenesis of nash and may play a similar role in the steatosis seen in chronic hepatitis c &: to compare the prevalence and severity of hepatic steatosis, visceral adiposity, j r and their relation to disease severity in african americans (aa) vs caucasians (ca) with genotype 1 hcv infection being considered for anti-viral therapy. methods: virahep-c is a prospective study of response rates to peginterferon and ribavirin in treatment-na'ive aa vs ca patients with genotype 1 hcv infection. all patients underwent liver biopsy within 18 months of enrollment. biopsies were read blindly by a central pathologist and scored for inflammation and fibrosis using the histologic activity index [i-iaii and for steatosis (0-4+) and features of steatohepatitis (zone 3 ballooning, zone 3 perisinusoidal fibrosis and mallory bodies). waist circumference and waist-hip ratio were measured as determinants of visceral adiposity. ir was determined using fasting glucose and insulin levels based upon the homa method. results: to date, 177 patients have been enrolled and 166 have complete liver biopsy information (75 aa and 91 ca). steatosis was present in 66 patients (40%) with similar rates among aa (39%) and ca (41% ca). grades of steatosis were also similar between aa and ca 1+ (38% vs 41%), 2+ (45% vs 27%), 3+ (14% vs 27%) and 4+ (3% vs 5%) (p = ns). patients with steatosis had significantly higher values for homa index (4.0 vs 2.4, p<o.ol), total hai score (10.3 vs 8.7, p<o.oool), hai inflammation score (8.5 vs 7.1, p<o.oool), waist circumference (40.2 in vs 36.5 inches, p<o.oool) and bmi (30.6 vs 26.7, p<o.oool). there were no significant differences in amount of alcohol consumption between patients with and without steatosis. spearman's non-parametric correlations were performed to assess associations between the ordinal variables (scores for fat, steatohepatitis ballooning degeneration + perisinusoidal fibrosis + mallory bodies], total hai, inflammation and fibrosis) and one continuous variable (homa index). waist circumference, waist-hip ratio, bmi, homa index, total hai, hai inflammation and hai fibrosis all correlated with both the fat score and steatohepatitis score (p<o.ol). median homa index levels were significantly greater among aa than ca (3.1 vs 2.7, p< 0.05), but step-wise logistic regression showed that race was not an independent predictor of steatosis after controlling for alt, ast, waist circumference, waist-hip ratio, bmi and homa index. conclusions: in patients with chronic hepatitis c and genotype 1, steatosis and steatohepatitis correlate strongly with the presence of insulin resistance and visceral adiposity. race is not an independent predictor of steatosis after controlling for other factors. bulk populations of antigen-presenting dendritic cells (dcs) contain myeloid dcs and plasmacytoid dcs. myeloid dcs polarize t lymphocytes to thl phenotype that produce interferon (1fn)-7. plasmacytoid dcs produce abundant amounts of interferon (1fn)-alpha. both ifn-?-producing t cells and adequate amounts of ifn-alpha are essential for viral clearance. in patients with chronic hepatitis c virus (hcv) infection, dcs are infected with hcv, which is presumed to have a role in viral persistence. however the functional implication of this has not been addressed. the aim of this study is to evaluate the functional capabilities of both myeloid and plasmacytoid dcs in patients with chronic hcv infection. myeloid dcs were defined as linage-, cdllc+, hla dr+ and cd123-cells, and plasmacytoid dcs as lineage-, hla dr+, cdllcand cd123+ cells among the peripheral blood mononuclear cells (pbmcs). the frequencies of myeloid and plasmacytoid dcs were measured in 64 patients with chronic hcv infection and 34 agematched control subjects by four-color flow cytometry. to assess the production of ifn-alpha by plasmacytoid dcs, these were cultured with graded doses of herpes simplex virus-1 (10-100 plaque-forming unitsldc). the frequencies of plasmacytoid dcs expressing intracellular ifn-alpha among total plasmacytoid dcs were counted by two-color flow cytometry. to evaluate the capacity of myeloid dcs to induce th polarization, dcs were cultured with autologous naive t cells for 7 days. polarization of t cells to thl phenotype was assumed when na'ive t cells expressed intracellular ifn-y. steatosis has been reported to occur in up to 50% of liver biopsies in patients with chronic hepatitis c virus (hcv) infection.risk factors for steatosis include alcohol abuse and those found in nonalcoholic steatohepatitis: obesity, increasing age, insulin resistance and hypertriglyceridemia. studies have shown an association between genotype 3 and hepatic steatosis in chronic hcv infection, and others have found an association between steatosis and fibrosis stage. previously published studies have been based primarily on cohorts of clinic referral patients and often have not analyzed for confounding variables. we herein report a population-based study of hepatic steatosis in hepatitis c. methods: in a study of 924 alaska nativestamerican indians with chronic hcv infection, 185 patients underwent at least one percutaneous liver biopsy as part of evaluation for treatment. all patients had a positive hcv rna by pcr and were negative for hbsag and hiv. all biopsy slides were reviewed by a pathologist blinded to patient identity, demographic, clinical and biological data. histologic activity was scored using the knodell system and fibrosis using the ishak system. steatosis was graded as 0, 1 (4 so%), 2 (30-65%), and 3 (> 65%). patients were analyzed for a) gender, age, estimated length of infection and bmi at the time of biopsy, b) genotype, current ethanol use, hcv rna within 1 year of biopsy, c) alt within 30 days of biopsy, and d) histologic activity and fibrosis found on biopsy, using univariable and multivariable analysis. results: steatosis was found in 79/185(43%) of liver biopsies; 62 (33%) had grade 1; 13 (7%) grade 2, and 4 (2%) grade 3. genotype distribution was as follows: 114 genotype 1 (62%), 44 genotype 2 (24%), and 27 genotype 3 (14%). steatosis was found in 39% (441114) of genotype 1,48% (21/44) of genotype 2, and 52% (14/27) of genotype 3 patients (p = 0.34). grade 2 or 3 was found in 6% (7/114) of genotype 1, 11% (5/44) of genotype 2, and 19% (5/27) of genotype 3 patients (p = 0.09). there was no significant association found between steatosis and gender (p = 0.93); age at biopsy date (p = 0.09); alt (p = 0.58); histologic activity (p = 0.27), viral load (p = 0.27) and estimated length of infection (p = 0.22). there was a significant association of steatosis with bmi (< 0.01), fibrosis score (p < 0.01) and current ethanol use (p = 0.03). bmi 2 30 occurred in 52% (42/81) of genotype 1, 39% (11/28) of genotype 2, and 27% (6/22) of genotype 3 patients (p = 0.10). after controlling for bmi, there was no significant relationship found between steatosis and genotype. for bmi < 30, 23% (9/39) of genotype 1, 47% (8/17) of genotype 2, and 50%(8/16) of genotype 3 patients had steatosis (p = 0.08). for bmi 2 30,60% (25142) of genotype 1,55% (6111) of genotype 2, and 67% (4/6) of genotype 3 patients had steatosis (p = 1.00). multivariable analysis was performed on 5 variables (p 0.25 in univariable analysis) and genotype. these included age (categories < 30,30-40,40-50, so+), bmi (< 30,30+), current ethanol use (yes, no), ishak fibrosis score (0-1, 2 21, and estimated length of infection (5 10, 11-15, 16-25, 25+ years) . a significant association with steatosis was found in 3 variables: ishak fibrosis score 2 2 (or 4.2, 95% ci 1.5-11.6, p = 0.005), bmi -> 30 (or 3.7, 95% ci 1.6-8.8, p = 0.003), and current ethanol use (or 2.5, 95% ci 1.0-5.8, p = 0.04) after adjustment for hcv infection length, which itself was not statistically significant (p = 0.82). conclusion: contrary to a number of previously reported studies, this population-based study did not find an association between steatosis and genotype, including genotype 3 in chronic hcv infection after multivariable analysis. we did find that ishak fi-brosis score, bmi and current ethanol use were associated with steatosis after adjustment for hcv infection length, but age and histologic activity were not associated. infection results in necroinflammatory liver disease characterized by the insidious progression of hepatic fibrosis and loss of functioning hepatocyte mass. a cell-mediated immune response with prominent lymphocytic infiltration of liver is thought to play a major role in pathogenesis, although little is known about the molecular mechanism underlying the liver injury associated with this viral infection. on the other hand, non-immune mechanisms have also been proposed as another mechanistic candidate for such sophisticated processes. the recent intensive interest is that the expression of hcv proteins seemingly alters lipid metabolism and transport in the liver in association with the reactive oxygen species (ros) mediated carcinogenesis, although the molecular basis of underlying mechanisms as well as responsible elements of hcv gene for this is yet to be established. thus, the aim of this study was to clarify whether hcv core protein expression directly alters lipid metabolism-relating enzymes to (cause steatosis, especially focusing on nuclear receptors and atp binding cassette transporter proteins expression. methods: 1. the complementary dna clone of full length hcv core (aa 1-191) was derived from serum of hcv i b patient by reverse transcription and nested polymerase chain reaction, and hcv-core expression plasmid (pcag-hcvcore) was prepared by a standard procedure. control plasmid was also prepared with p-galactosidase (pcag-lacz). 2. hepg2 cells were transfected with pcag-hcvcore or pcag-lacz, thereafter harvested for enzymatic triglycerides (tg) assay, hcv core antigen rneasurement by elisa at 24 h or 48 h. also, lipid metabolism-related enzymes mrna expression was estimated by semi-quantified rt-pcr; mitochondrial and peroxisomal fatty acids ,&oxidation, o-oxidation, abc transporters (mdr3, mrp2, bsep), microsomal tg transfer protein (mtp), ldl receptor, and nuclear receptors. resultss: 1. hcv core expression was evidenced at 24 and 48 h at the similar level. 2. hepatic tg was increased in hcv core expressing cells, along with down-regulation of carnitine palmitoyl transferase 1 a(cptla), a precious protein in liver mitochondrial fatty acid@-oxidation (50% at 24 h, 85% at 48) and up-regulation of acyl-coa oxidase 1 (acol), a precious protein in peroxisomal fatty acidso-oxidation (130% at 48 h). cyp4a11, a precious protein in microsomal o-oxidation, and mtp, a precious protein in vldl assembly, were not detected or unchanged. superfamily 2 (sf2) helicases and helicase-like proteins share 6 conserved motifs. alignments reveal that several additional conserved motifs are present in the sf2 helicase encoded by the hepatitis c virus (hcv). the roles of two such motifs are examined here using structure-based site-directed mutagenesis. the first motif (yrgxdv) forms a loop that connects sf2 helicase motifs 4 and 5, at the tip of which is arg393. when arg393 is changed to ala, the resulting protein retains a nucleic acid stimulated atpase but cannot unwind rna, unwinds dna poorly, and does not translocate on ssdna. dna and rna stimulate atp hydrolysis catalyzed by r393a like the wildtype but the mutant protein binds dna more weakly than wildtype both in the presence and absence of a non-hydrolysable nucleotide analogue. thus, this "arg-clamp" motif anchors the protein on nucleic acid enabling processive unwinding. the second motif (dfsldptf) forms a beta-loop between sf2 motifs 5 and 6 that connects domains 2 and 3. when f444 in this "beta-arm" is changed to ala, the resulting protein is devoid of all activities. when f438 is changed to ala, the protein retains nucleic acid stimulated at-pase, but unwinds dna and rna poorly. in this case, uncoupling of atp hydrolysis and unwinding is due to the fact that the f438a mutant does not release dna upon atp binding like the wildtype. the f438a mutant also has a lower melting temperature than the wildtype indicating the hydrophobic pocket formed by the beta-arm and residues in domain 3 stabilizes the protein. data support an inchworm model for helicase action and identify two new potential sites for rational hcv drug design. this work was supported by the aasld liver scholar award from the american liver foundation. tat is an early gene product of hiv-1 that is essential for replication and viral gene expression. this transactivating protein is secreted by hiv-infected cells and taken up by neighboring cells. tat modulates expression of specific cellular genes and may be a key player in the interactions between hiv and other infections. one of the most common coinfections observed among hiv patients is hepatitis c virus (hcv). if hiv tat has an effect on hcv replication, this could help explain the rapid development of liver disease and cancer among hiv patients. aim this study was designed to test the hypothesis that tat alters hcv replication. methods: soluble tat was added to the media of huh-7 cells containing a hcv replicon. replicon replication was quantitated using a ribonuclease protection assay. using an ltr-luciferase plasmid to transfect hela cells, we developed a series of controls outlining the relative oxidative state of the tat protein. results exposure to tat protein in the reduced state substantially increased hcv replicon replication. oxidized tat was found to have no significant effect on the replication of the replicon. the increase was dependant on length of exposure to tat and the concentration of tat. present work: further assays seek to define the nature of the effect by tat on hcv replication. conclusion: tat may upregulate hcv replication directly or indirectly, and thereby play a role in modulation of hcv infection and pathogenesis. elucidating the complex interactions between hiv and hcv will be critical in evaluating and developing workable treatments for the increasing number of coinfected hiv/hcv persons with liver disease. acknowledgements: this research was supported by nih grants ai34764, ai54626, ai54238, ca54576 and ca89121. we have found previously that expression of hepatitis c virus proteins in cell lines or in the liver of transgenic mice inhibits interferon alpha induced signaling through the jak-stat pathway (heim et al., j virol, 1999, 73:8469 and blindenbacher et al., gastroenterology, 2003, 1241465) . the aim of the present study was to investigate if the same inhibition takes place in livers cells of patients with chronic hepatitis c. from february 2001 to april 2002, all patients with chronic hepatitis c referred to the outpatient liver clinic of the university hospital basel were asked for their permission to use part of the liver biopsy for this study. for non-hcv controls, patients that underwent ultrasoundguided liver biopsies of focal lesions (mostly metastasis of carcinomas) were asked for their permission to obtain a biopsy from the normal liver tissue outside the focal lesion. the protocol was approved by the ethical commission of basel. written informed consent was obtained from all patients that agreed to participate in the study. after removal of a 20 to 25mm long biopsy specimen for routine histopathological workup for grading and staging of the liver disease, the remaining 5 to 20 mm long biopsy cylinders were immediately incubated in pbs or pbs with human interferon alpha (lo00 iulml) for 10 to 60 minutes at 37°c. the samples were then used for the preparation of cytoplasmic and nuclear extracts and in some cases for immunofluorescence studies. in a first part, the ex vivo stimulation of biopsy tissue was validated. biopsy samples of patient with different degrees of fibrosis were incubated for 0, 10, 20,30 and 60 minutes, and the nuclear translocation (a surrogate marker for activation) of statl was visualized by immunofluorescence. we found a transient activation of stat1, with a peak after 20 minutes of stimulation with interferon alpha. the signal was shut down in all biopsies after 60 minutes. interferon alpha diffused readily in the biopsy cylinders regardless of the degree of fibrosis. in the second part, 44 consecutive biopsies of patients with chronic hepatitis c and 12 consecutive control biopsies of patients who underwent ultrasound-guided biopsy of focal lesions in otherwise healthy livers (mainly liver metastasis of carcinomas) were used for semiquantitative assessment of interferon alpha induced statl activation using gel shift assays. we found a signhcant inhibition of statl dna binding in patients with chronic hepatitis c compared to controls. as in our previous work with hcv protein expression systems, statl phosphorylation was not impaired in human liver biopsies from patients with hepatitis c. we conclude that interferon induced intracellular signaling can be analyzed semi-quantitatively in human liver biopsies ex vivo by gel shift assays. using this newly validated method, we found that signaling is impaired in liver biopsies from patients with chronic hepatitis c. the block in stat signaling is at the level of dna binding in the nucleus, whereas stat activation at the interferon receptor functions normally. methods we studied the intrahepatic and peripheral hcv-specific cds+ t cell response in a cohort of 15 hla-a2 positive chronically hcv infected patients by tetramer staining, intracel-m a r ifngamma staining and cfse labeling. in addition, viral amino acid sequences corresponding to the four ctl epitopes used in the study were deduced by nucleotide sequence analysis to assess the potential role of viral escape variants. results (1) substantial higher numbers of hcv specific cd8+ t cell responses were detectable in the liver compared to the peripheral blood, suggesting compartmentalization at the site of infection. in addition, intrahepatic cds+ t cells were multispecific whereas peripheral hcv specific cds+ 'i' cell responses were primarily monospecific. these results suggest, e.g., the persistence of hcv-specific t cells at the site of disease or the direct priming of t cells in the liver. (2) epitope-specific cd8+ t cells that were detectable in peripheral blood as well as liver were characterized by a good peripheral proliferative capacity after peptide specific stimulation suggesting that proliferation is a prerequisite of peripheral virus-specific cd8+ t cells to accumulate in the liver. this is further supported by the observation in one patient that the lack of proliferation of virus-specific cd8+ t cells in the peripheral blood was associated with the absence of the same cds+ t cell response in the liver. (3) despite the strong accumulation of hcv specific cd8+ t cells in the liver, the majority of those cells was unable to secrete cytokines after peptide specific stimulation indicating that dysfunction of intrahepatic hcv specific cd8+ ' i cells might contribute to viral persistence. importantly, ifn gamma producing tetramer positive cd8+ t cells were only detectable in patients with low viral titers or in patients with high viral titers but with sequence variations in the corresponding viral epitope, suggesting viral escape. the relative contribution of t cell dysfunction versus viral escape to viral persistence is not known. it is important to note, however, that both mechanisms can operate within the same patient. insulin resistance (ir) is a frequent feature in chronic hepatitis c while risk factors of steatosis are body mass index in patients infected with genotype 1 and viral load in those infected with genotype 3. in patients with chronic hepatitis c, we adressed the following issues: 1) is ir the cause or consequence of steatosis and fibrosis ? 2) what are the risk factors of ir ; 3) does ir play a role (and to what extent) in the occurrence of steatosis ? 4) is ir involved in the progression of fibrosis ? therefore, this study was designed to assess relationships between ir, steatosis and fibrosis according to hcv genotypes in non diabetic patients. methods: 152 non-diabetic patients with biopsy proven non-cirrhotic chronic hepatitis c had fasting serum glycemia and insulinemia measurements. ir was evaluated by using homa model assessement. 4 groups of patients were defined according to hcv genotypes (1 or 3) and degree of steatosis (absent or mild vs moderate to severe). results: the 4 groups were similar in terms of age, sex ratio, bmi and disease duration. prevalence of ir (homa higher than1.64) was significantly higher in genotype 1 patients with steatosis than that of other patients (77% vs 27,23 and 21% respectively, p=o.oool). ir was significantly associated with extensive fibrosis in genotype 1 patients (p=0.008) but not in genotype 3 patients. among genotype 1 patients, independent parameters associated with ir were age (p=0.002) and steatosis (p=o.o3) but not fibrosis. independent risk factors for steatosis in genotype 1 and genotype 3 patients were dr (p=o.o4) and viral load (p=0.02) respectively. steatosis was associated with significantly higher fibrosis score whatever the genotype (p=o.o1). among genotype 1 patients, the median progression rate of fibrosis was significantly higher in those having steatosis and ir together than in other patients (0.1 vs 0.05, p =0.02). conclusions : in non-diabetic patients with non-cirrhotic chronic hepatitis c 1) steatosis and fibrosis are associated with ir in genotype 1 patients but not in genotype 3 patients, 2) among genotype 1 patients, ir mainly depends on age but not fibrosis 3) ir is a major risk factor of steatosis in genotype 1 patients, 4) the combination of steatosis and insulin resistance is a risk factor of fibrosis progression. these results suggest that ir is not the consequence but rather the cause of steatosis and fibrosis progression. . however, in the first month of life, in 4 of these 13 newborns, hcv rna was only detected in pbmcs and not plasma. in 2 neonates, the sscp band patterns of pbmc-derived viral sequences were different from maternal sequences in serum or pbmc; however, they were identical to hcv rna negative strand amplified from mothers' pbmc. the latter strongly suggests that the infection was transmitted through maternal infected pbmcs. another infant harbored different hcv strains in serum and pbmc; both strains were present in the mother's serum. only 7 of 13 hcv-rna positive children developed hcv antibody at one year. hiv infection was found in 2 out of 13 hcv rna-positive and in 6 out the 35 hcv rna-negative infants (ns). conclusions: we have found that 1) hcv strains infecting neonates may be derived from strains residing in maternal pb-mcs. 2) hiv+ pregnant women who were hcv rna positive in pbmcs were more likely to transmit hcv to their infants. these data suggest that one mechanism for perinatal transmission of hcv is maternal-fetal transfusion of hcv-infected pbmcs late in pregnancy or during labor and delivery. additionally, hcv infected neonates may have a limited ability to develop hcv antibodies in the first year of life and thus there may be an underestimation of the prevalence of hcv infection by anti-hcv determination among children born to hcv rna positive mothers. (hcc) in this clinical setting is very high. however, the molecular mechanisms of how hcv related proteins may contribute to hcc is not well defined. hcv core protein, a viral structural protein, has been implicated in tumor development. the goal of this study was to characterize the transcriptional regulation induced by core protein expression with respect to generations of a malignant phenotype. meth0ds:a cdna fragment encoding hcv core protein of l b genotype was subcloned into the ptre2hyg vector and co-transfected with ptet-on vector (clontech) into a human huh 7 hepatoma derived cell line using polyamine (mirus) as a carrier. stably transformed clones were selected by growth in culture medium containing g418 and hygromycin. clones positive for hcv core protein expression were screened by immunoblotting from cells grown in the presence and absence of tetracycline. one clone with core expression tightly regulated by tetracycline was chosen for further analysis. cell proliferation was evaluated by a modified mtt assay, at 6,12,24, 36 and 48 hours after induction of core protein expression. twenty-four hrs after hcv core expression, the cellular transcriptional changes were analyzed by microarray analysis using human genome u133 array sets (affymetrix). the microarray data were validated by real-time pcr on selected genes. results hcv core protein expression was tightly regulated by the presence of tetracycline and the protein levels were dependent on the amount of the tetracycline present in the culture medium. core protein expressing cells showed significant increase in growth compared to non-expressing cells at 6,12, 24,36 and 48 hours after addition of tetracycline indicating a proliferation stimulus provided by core protein. microarray analysis using human gene chip u133 revealed that 1072 of 39,000 genes were significantly changed (> 3 fold), with 626 up-and 446 down-regulated. this screen was quite informative since 52 genes involved in regulation of cell proliferation (38 genes) and apoptosis (14 genes) were changed respectively. conclusions: enhanced growth of liver-derived cells by hcv core protein is due to cellular transcriptional changes induced upon core expression; two major molecular pathway(s) are involved 1) those involved in celi growth control and 2) those involved in cell survival. such gene regulation by hcv core protein is important to the molecular pathogenesis of hcc daudi-cd4 cells were infected with pnl4-3, a t-tropic hiv molecular clone. 24 hours later, uninfected and hiv-infected cells were incubated with either 10% high titer hcv-positive patient serum, 10% hcv-negative patient serum, or 10% fbs (no human serum). infections were continued for 3,4,5, and 6 days; cells were harvested, stained with annexin v and propidium iodide, fixed, and apoptosis was measured by flow cytometry. annexin v is a marker of early apoptosis, and propidium iodide indicates cell death either by apoptosis or necrosis. results: six high titer hcv-positive sera and six hcv-negative sera were tested in five separate experiments, and in each case, the percentage of viable cells was higher in the hivihcv-coinfected cells compared to those infected with hiv only. to further elucidate dynamics of the course of coinfection, we did a time course consisting of harvests every eight hours beginning at day 3 (the earliest point at which apoptosis had been observed in prior experiments) and continuing until day 6 plus 16 hours. the results of the five harvests from day 5 plus 8 hours to day 6 plus 16 hours are shown in the figure below. the percentages of viable cells and those undergoing apoptosis are shown as measured by flow cytometry. during this 32-hour interval the percentage of viable (negative for apoptosis) cells fluctuated very little in untreated cells, or in cells incubated with hcv-positive serum, or even in hn-infected cells incubated with hcv-positive serum. in striking contrast, hiv-infected cells that were incubated with hcv-negative patient serum dropped to only 16% viability by the end of the 32 hours, compared to 64% viability in the hiv-infected cells incubated with hcv-positive patient serum. this effect was not observed when apoptosis was induced by either of two chemical inducers of apoptosis in daudi cells: peroxisome proliferator-activated receptor gamma ligand and calpain inhibitor 11. next, we sought to determine if the effect of apoptosis inhibition was dependent on hcv particles. when h ninfected cells were kept separate in culture from hcv-infected cells by a membrane that prevented virus passage, apoptosis in the hn-infected cells was still inhibited. conclusions: collectively, these results suggest that in our experimental conditions: 1) hcv-positive sera, but not hcv-negative sera provide a protective effect to hiv-induced apoptosis in daudi-cd4 cells, and 2) a cellular factor induced by the presence of hcv, rather than hcv particles themselves, is responsible for the inhibition. methods: serum and pbmc samples from an individual with acute hcv-infection (genotype l a ) were collected every two to three months over a time period of 20 months beginning 2 months after infection. pcr amplification of vrna using a set of overlapping primers for the entire hcv genome was performed and pcr-products population sequenced using an abi automated sequencer. cd8+ t cell responses were defined using an ifngamma elispot assay using an overlapping synthetic peptide set spanning the whole hcv-genome (genotype la). based on the hla-type the complete viral genome was also screened for putative cds+ epitopes using a web based epitope prediction program (syfpeithi). results: elispot screening with the whole hcv-genome peptide set detected only two ex-vivo ifn-gamma responses (ns3 1073-1081 and ns5b 2594-2602), although neither epitope was associated with the development of mutations. however, over the 20 month period of observation we detected a total of 25 mutations throughout the genome (2 in el, 4 in e2,3 in ns2,5 in ns3, 2 in 353a ns4 and 9 in ns5), in addition to multiple changes in the hypervariable regions. interestingly, only five of these mutations resided within known hla-restricted optimal epitopes. however, 17/25 mutations (68%) were located within predicted epitopes based on known hla-binding motifs of the subject. in order to begin to define the rate at which these mutations develop an intermediate time point of 12 months was also sequenced. interestingly, 19/25 (76%) of the mutations had already occurred by this time. it is likely that at least some of these mutations are the direct result of immune pressure exerted through cd8+ t lymphocytes. we are currently generating peptide-specific cds+ t cellines against these regions exhibiting viral evolution, providing an opportunity to determine which of these regions are associated with previously undescribed cd8+ responses. conclusions: hypothesizing that evolving mutations in hcv might be the result of immune pressure by ctl, longitudinal full length sequencing of hcv may represent a powerful tool to define additional hcv-specific cd8+ t cell responses, especially those exerting substantial selective pressure. using this approach we were able to identify a number of candidate regions where cd8+ t cell responses may be present and driving viral evolution. determining the extent to which viral escape from cd8+ responses occurs during hcv infection will elucidate its overall impact in preventing proper control of hcv. indicate that the main open reading frame of hcv contains rna structures in the corelarfp (alternate reading frame protein) and nssb (polymerase) genes. we hypothesized that one or more of these structures are cis-acting replication elements (cre)s. methods. we used custom software, thermodynamic rna folding programs, and classical comparative phylogenetic analysis to build secondary structural models. we then used directed mutagenesis in the subgenomic replicon system to seek stem-loop elements in ns5b that are required for viability. the mutations we introduced maintained the sequence of the polymerase protein, i.e., they were silent, synonymous codon substitutions. structural probing was carried out on replicon rna. rnase t1 and lead (ii), and nuclease v1, were used to identify loops and helices, respectively. results. mutations in ns5bsl3.2 blocked replication, indicating that this novel structure is an essential cis-acting replication element (cre the aims of this study were: 1) to correlate mean alt level with hcv rna by pcr positivity or negativity, 2) to compare clinical, demographic and risk factor data as well as viral load and genotype between persons with pnalt, persistently elevated alanine transaminase (pealt) and fluxuating alanine transaminase (fluxalt) who had at least 3 years of data available. methods: for each person in the cohort from whom 2 2 pcr results were available (n=278), we calculated the mean level of all alts measured over a 3-year period after enrollment. for a subset of the cohort, we selected persons who met the following criteria: 2 positive pcr tests 2 1 year apart with the first positive pcr occurring prior to date of the first alt and a minimum of 6 alt levels measured over the subsequent 3 years with a minimum interval of 1 month between alt measurements (n=129). we defined a person as having pnalt or pealt when 2 5 of 6 alt levels were normal or elevated, respectively, during the 3-year follow-up period. persons who did not fit into either of the above 2 categories were defined as fluxalt. we reviewed clinical data via chart review, demographic and risk factor data via interviews, as well as data on viral load (performed by branched dna assay version 2.0) and genotype (restriction polymorphism analysis background: hcv infection rarely presents acutely, but when it does it offers a potential early "window" for effective therapy. it has been suggested that such therapy is efficacious due to enhanced activity of t cell responses, which are most active during acute disease. methods: 8 subjects with acute hcv infection were comprehensively mapped for cd8+ t cell responses with an interferongamma elispot assay using 301 overlapping peptides, spanning the entire hcv polyprotein. responses were confirmed by establishing peptide-specific t-cell lines and intracellular cytokine staining. the screening for hcv-specific cd8+ t cell responses was repeated during and after therapy. responses detected in the screening assay were longitudinally studied before, during and after therapy using single peptide elispot ,as well as tetramer assays. we also studied cd4 proliferative responses over time in some subjects using recombinant hcv proteins. results: cd8+ t cell responses were detected in 6/8 subjects before therapy was initiated and were typically multispecific, with up to 5 epitopes targeted. after the start of therapy, frequencies of hcv-specific cd8+ t-cells declined following suppression of hcv viremia. therapy also did not increase the breadth of the hcv-specific response as no additional specificities were detected at later timepoints, when elispot screening was repeated. in 5/8 subjects viral relapse occurred after therapy was stopped, with 2 subjects being retreated successfully. relapse was not predicted by the presence or absence of hcv-specific cd8+ t-cell responses. during viral relapse, a vigorous expansion of pre-existing hcv-specific cd8+ t-cells was observed for most specificities, with single responses reaching almost 3% of cd8+ t cells as measured by tetramer staining. these responses were unsuccessful in containing hcv. in the two subjects who were retreated after viral relapse, we observed the identical pattern of declining cd8+ t cell responses as in the first treatment course. in contrast to cd8+ t cell responses, cd4 proliferative responses usually became more vigorous after virus was suppressed on therapy. however, such responses also did not protect from viral relapse. conclusions: these data suggest that although multispecific functional cd8+ t cell responses may be present during acute disease, this does not predict a successful outcome of antiviral therapy. rather than boosting cd8+ t cell responses, therapy and viral suppression appear to lead to their attenuation, even though cd4+ t cell responses may be restored. figure) in the fibrous septum of portal tract in cirrhotic liver. significantly less fltl immunopositivity occurred in nd liver. hepatocytes were negative for fltl. conclusion: the greater de of genes involved in immune activation, fibrosis, cellular proliferation and cell signalling indicated that these processes are more active in the cirrhotic liver that has developed hcc than the cirrhotic liver without hcc development. plgflfltl signalling has an important role in neo-vascular development within organs. the decreased expression of plgf and its receptor flt 1 in cirrhosis with hcc implies that vascular proliferative signals although normally active in cirrhosis itself may be decreased in cirrhotic livers with hcc. prospective analysis of cirrhosis prior to hcc development is needed to indicate whether these findings represent a premalignant phenotype. background: hepatitis c virus (hcv) infection often leads to chronic liver diseases including liver cirrhosis and hepatocellular carcinoma (hcc). at least 10 hcv proteins have been identified, which serve as viral structural and non-structural proteins required for viral replication and virion formation. several viral proteins have been implicated in hcv persistence and the development of hcc. we have previously reported that the ns2 protein inhibits gene expression f+om various cellular and viral promoters in liver and non-liver derived cells. thus, expression of endogenous cellular proteins was significantly reduced in ns2 expressing cells. in this regard, the ns2 protein was recently found to be an inhibitor of the pro-apoptotic cide-b protein and therefore may contribute to hepatic oncogenesis. to understand the molecular basis of repressed gene expression, we constructed successive deletion mutants of the ns2 protein and tested their effect on luciferase expression driven by cmv promoter. meth-ods: the cdnas encoding the full-length (1-217), n-terminal part (1-91) and internal parts of ns2 were generated by pcr and cloned into the pcr3.1 vector for expression under control of the cmv promoter. a liver-derived huh-7 cell line was co-transfected with a plasmid encoding the luciferase reporter gene and plasmid encoding various deletion mutants of the ns2 gene. inhibition of gene expression was measured by luciferase activity assay at day two after transfection. cell viability was also evaluated as well. results: deletion constructs 61-121, 61-111, 71-121 and 71-111 exhibited a significant inhibitory effect on luciferase activity comparable to the fulllength ns2 protein, whereas the deletion constructs 81-111 and 81-121 lost the inhibitory effect. therefore, the minimal amino acid residues required for the inhibition of gene expression by this viral non-structural protein was mapped to residues 71-111. interestingly, this region partially overlaps with the binding site of the ns2 to a newly identified cide-b pro-apoptotic protein. il12 plays an essential role in the antagonism of t helper 2 differentiation and in the induction of antiviral host defence. we postulated that genetically determined attenuation of il12 production could provide a plausible immunological mechanism able to determine outcome of hcv infection and have investigated a recently described polymorphism in the il12b gene. methods: we have extracted genomic dna from whole blood taken from 196 hcv antibody positive patients. 124 patients had chronic hcv with detectable hcv rna and 72 had spontaneously resolved infection, testing hcv rna negative on several occasions. genotyping for a single nucleotide polymorphism (snp) at position (a16974c) on the ill2b gene was performed using polymerase chain reaction and restriction digest. maximal in-vitro il-12 production by cultured mononuclear cells in response to sac (staph aureus cowan) stimulation was determined in 24 cases by elisa. results: of the hcv rna positive cases 81 (65%) were homozygous for the 'a' allele, 39 (31%) heterozygous 'ac'; and 4 (4%) were homozygous 'cc' whereas of the hcv rna negative cases 36 (50%) were 'aa', 33 (46%) were 'ac'; and 3 (4%) were 'cc'. hcv rna negative cases were significantly more likely to be heterozygous for lac' than hcv rna positive cases (p=0.04). of the 24 patients studied for il-12 production, 13 were genotype 'aa' and 11 'ac'. 13 were hcv rna positive (8 genotype lac' and 3 'aa') and 11 hcv rna negative (3 genotype 'aa' and 3 'ac'). maximal il-12 production with sac stimulation was lower in the 11 genotype 'aa' cases (mean 104 units) than in the 13 genotype 'ac' cases (mean 228 units) (p= 0.08). hcv rna positive cases produced significantly less il-12 than did hcv rna negative cases (mean 84 units compared to 266 units, p= 0.016). comparison of hcv rna positive or negative cases only, revealed a trend for higher maximal il-12 production with genotype 'ac' regardless of hcv outcome. conclusion: cases heterozygous for the variant 'c' allele at position 16974 of the il-12 p40 gene are significantly more likely to be hcv rna negative than those homozygous for the 'a' allele. recent data found carriage of the variant 'c' allele to be associated with greater il-12 production capacity and our data from a small number of cases supports this. genetically influenced enhancement of il-12 production appears to be a factor influencing the outcome of hcv infection. we have previously demonstrated that hepatitis c virus (hcv) core protein expression in huh-7 hepatoma cells increased reactive oxygen species (ros) derived from mitochondria without inducing apoptosis. the aim of this study was to investigate whether hcv core protein inhibits the rosassociated apoptosis induced by deoxycholic acid (dca). methods:: we measured ros level and 8-hydroxy-2'-deoxyguanosine (8-ohdg) content, and evaluated apoptosis in the presence or absence of dca (500 pm), using a human hepatoma-derived cell line with tightly regulated hcv core protein expression under the control of a tet-offrm promoter. cells were incubated with 5pm of chloromethyl2',7'-dichlorodihydrofluorescein diacetate for 30 min for measurement of ros. cellular 8-ohdg content was quantified with enzyme-linked immunosorbent assay. fragmented nuclei were assessed with 4',6-diamidino-2-phenylindiole staining and dna fragmentation was evaluated by genomic dna laddering. the degree of apoptosis was quantified with enzyme-linked immunosorbent assay. we also examined whether the general caspase inhibitor (zvad-fmk) inhibited the ros-associated apoptosis induced by dca. in some experiments, cells were incubated with 500wm of ursodeoxycholic acid (udca) in addition to dca. the experiments were repeated 3 or 4 times. results:: strong core expression was detected 72 hours after withdrawal of tetracycline from the culture medium. the expression of core protein increased the basal ros level (1.6 2 0.17-fold, p<0.05) and 8-ohdg content (1.13 i 0.005-f0ld, p<0.05) of huh-71191-20 cells without inducing apoptosis. dca stimulation produced a 4.0-fold increase in ros content in the presence of core expression and a 2.5-fold increase in the absence of core expression. similarly, the 8-ohdg content was significantly increased by dca regardless of hcv core expression (1.13-fold, p=0.02 for core expression, 1.15-fold, p=o.o1 for core non-expression). nevertheless, hcv core protein significantly suppressed the ros-associated apoptosis induced by dca (w0.05). also apoptosis induced by dca was almost completely inhibited by zvad-fmk. udca significantly decreased the ros (p<0.05) and the 8-ohdg content (p=o.oos) in the core-expressing cells and attenuated dca-induced apoptosis. on-treatment virological response (otr) was defined as complete loss or greater than 100-fold drop in hcv viremia within 3-6 months of therapy by quantitative or qualitative rt-pcr (roche cobas amplicor v2.0), based on recommended early virological testing for continued therapy. forty-six patients with at least 3 months of continued antiviral therapy were examined thus far, including 30 with genotype 1 (cl) and 16 with either genotypes 2 or 3 infection (c2). on-treatment response (otr) was achieved in 29/46 (63%) patients overall, including 47% among c1 and 94% in c2 groups. positive and negative control groups included 19 healthy hcv seropositive but nonviremic subjects without history of antiviral therapy ("recovered") and 23 healthy hcv seronegative volunteers, respectively. hcv-specific cd4 t cell response was examined using recombinant hcv core, ns3l4, ns5 and control proteins in standard proliferation and ifn-gamma (ifng) elispot assay at baseline (pre-treatment) and at 1, 3 andlor 6 months during antiviral therapy. results: hcv-specific cd4 t cell response was significantly greater in the recovered compared to the chronic patients, consistent with its expected role in natural hcv clearance. among the chronics, c2 patients (genotype non-1) displayed a baseline t cell response to hcv ns3/4 that was modestly but significantly greater than c1 patients ( the progression of fibrosis in chronic hepatitis c virus (hcv) infection differs among individuals and determines the ultimate prognosis and thus the need for therapy. the molecular mechanisms associated with the progression of fibrosis are poorly understood. gene expression profiling technologies allow the analysis of gene networks whose expression is associated with specific pathological conditions. we used real-time quantitative rt-pcr assays to compare the mrna expression of 148 selected genes in liver biopsies of controls (n=10 normal liver samples) and of 57 untreated patients with chronic hcv infection and different stages of fibrosis according to metavir, i.e. stage flal (n=11), f1a2 (n=9), f2a1 (n=10), f2a2 (n=lo), f3a2 ( n = l l ) and f4a2 (n=6). in order to limit the number of pcr experiments, we first studied total mrna pools which were prepared by mixing amounts of individual liver biopsies mrna of each group. this pooled sample analysis allowed the selection of genes displaying significant different expression (> 2-fold variation) in comparison to "normal livers". the selected genes were then studied at the individual level and their diagnostic performance was assessed using roc curves. the most informative genes were used to construct specific gene expression signatures. we identified several genes specifically involved in various stages of fibrosis. the dysregulated genes mainly encoded extracellular matrix proteases, growth factors, cytokineslchemokines, and ifncu-induced genes. we also observed a statistically significant association between hcv rna amount (as determined with the same real-time quantitative rt-pcr technology) and expression of several genes, mainly ifn-a-induced genes including stat1, ifi27, mix1, oas2 and gip2. understanding the correlates of protective immunity in this setting is an important first step in the development of potential vaccine candidates. methods: two recipients of frozen patellar allograft tissue procured from the same donor developed evidence of acute hcv within 6 months of surgery. in retrospect, the donor was confirmed to be hcv antibody negative but hcv rna positive (genotype la). both patients were enrolled in a prospective study of t cell immunity requiring whole unit blood draw at baseline, 2, 4, 6 and 12 months. comprehensive hcv-genomewide analyses were determined by ifn-y elispot responses to 33 overlapping peptide pools that spanned the entire hcv polypeptide (genotype la, 3010 aa, total 750 peptides). peptide responses were defined as greater than mean plus 3 sd compared to 10 control wells. results: patient 1 (51 yo wf) cleared the virus spontaneously within 6 months; when first evaluated, the pt. demonstrated cd4+ t cell responses to 8 of 33 (24%) peptide pools, with effector frequencies as high as 1 in 5,000 (to ns3helicase-7, amino acids 1569-1667). ifn-y cd8+ t cells were detected following stimulation with 6 of 33 (18%) pools (highest frequency to pool ns5b-5, aa 2829-2927). remarkably, 6 months later, the repertoire of the hcv-specific cd4 t t cell response had expanded further, and patient 1 demonstrated responses to 13 of 33 (39%) pools. in contrast, patient 2 (49 yo wm) demonstrated persistent viremia (4.4 million copieslml, bayer assay) and lacked cd4+ t cell responses to any peptide pools when first evaluated; only one peptide pool (ns3-helicase-5, aa 1369 -1478) elicited responses in cd8+ t cells. despite virologic clearance with antiviral treatment (pegylated interferon and ribavirin), elispot screening failed to reveal emergence of new cd4+ or cd8+ t cell responses (6 months later). conclusions: comprehensive analyses of hcv-genome-wide cd4+ and cd8+ t cell responses reveal significant differences in patients exposed to the same hcv innoculum and correlate with spontaneous clearance versus chronicity. in hcv infection that resolves spontaneously, the repertoire and strength of the hcv-specific immune response may continue to expand in the first year after infection (and may target more than one-third of the hcv polypeptide). in contrast, the profile of t cell responses remains narrow, weak, and constant in the acute infection that becomes chronic, even after successful antiviral therapy. alcohol consumption exacerbates liver injury in chronic hepatitis c and enhanced oxidative stress is one possible pathophysiological mechanism. we have previously developed a hepatoma cell line with conditional, stable expression of hcv core protein and constitutive expression of cyp2e1. these cells demonstrate dosedependent ethanol toxicity when core protein is expressed. the aims of this study were to determine whether reactive oxygen species (ros) production and mitochondrial dysfunction are responsible for ethanol-induced cytoxicity. methods: huh-7 cells with conditional expression of core protein and constitutive expression of cyp2e1 (l14 subclone) were exposed to 0.1 micromolar tbooh and/or ethanol (25 mm) for up to 24 hrs. cytotoxicity was measured by mtt assay or trypan blue exclusion. ros production was assayed with the oxidation sensitive fluorescent dye dcfda. mitochondria1 membrane potential was analyzed by flow cytometry using the dye jc-1 as a probe. apoptosis was detected by cellular dna content analysis with flow cytometry. results: in the presence of 0.1 pm tbooh, there was a progressive increase in cell death in huh-7 cells expressing no heterologous proteins (2 2 2%), cypzel only (12 2 l%), core only(l5 2 5%), or core plus cypzel (3927 x). addition of 25 mm ethanol to core/ cyp2e1 expressing cells further increased cell death to 5326%. dna content analysis and nuclear morphology showed that this type of cell death represented necrosis and not apoptosis. effects on mitochondrial membrane potential were also examined. under control conditions, only 250.5% of cells had depolarized mitochondria. expression of corelcyp2el and exposure to tbooh depolarized mitochondria in 47211% of cells. similar to cell death, ethanol (25 mm) addition increased depolarization to 7027% of cells. compared to control cells, core protein increased ros by a factor of 1.220.3, and the combination corelcyp2el by 2.150.3. furthermore, cells expressing corelcyp2el ampiified the effect of exogenous tbooh. incubation with tbooh (0.1 pm) had no effect on ros content of control cells. it approximately doubled the ros content of cells expressing either core or cyp2e1 and it increased ros content of cells expressing both corelcyp2el by 4.120.5 fold. in all cases the increase in ros was completely blocked by the antioxidant n-acetyl cysteine (20 mm). the antioxidant also completely blocked both mitochondrial depolarization and cytotoxicity. conclusions: hcv core protein and cyp2el synergistically enhance ros production in hepatoma cells, amplify the oxidative stress produced by extracellular ros, and sensitize cells to mitochondrial depolarization and necrotic cell death caused by alcohol. since all these effects are blocked by antioxidants, the formation of ros is likely to be the primary event which subsequently produces mitochondrial dysfunction and cell death. sponse associated with different outcomes of hcv infection may provide important insights into our understanding of hcv pathogenesis. for this purpose, we compared the functional features of hcv-specific cd8 cells in patients with chronic hepatitis c (23 patients, ch), chronic asymptomatic carriers of hcv with persistently normal alt and positive serum hcv-rna (12 patients, as) and in subjects with resolved hcv infection, either spontaneously (8 subjects, sp) or following anti-viral treatment (12 subjects, rt). functional analysis was carried out with 5 highly immunogenic peptides corresponding to ns31073-1081 , ns31406-1415 , ns41812-1821 , ns41992-2000 , ns52627-2635 , containing previously identified hla-a2 restricted epitopes. since the different hcv genotypes were represented in the different groups of patients in different proportions, different sets of peptides designed on genotype 1, genotype 2 and genotype 3 sequences were synthesized and used to reproduce more closely the sequence of the viruses infecting each individual patient and responsible for in vivo priming of the cds response. the immunological parameters tested were: a) frequency of hcv specific, cd8+ t cells by tetramer staining, both ex-vivo and after in vitro stimulation with synthetic peptides; b) ifn-7 production by intracellular cytokine staining; c) cytolytic activity by a standard %hromium release assay. the results of our study indicate that subjects recovered from hcv infection following treatment show lower ex-vivo frequencies of circulating hcv-specific cd8 cells compared to ch patients but their cells are able to expand and to produce ifn-7 more efficiently after in-vitro stimulation. indeed, hcv-specific t cell lines were induced in 83% of rt subjects and in 74% of ch patients; moreover, ifn-7 production was induced in 100% of rt subjects compared to 67% of ch patients (p<0.05). in the group of subjects spontaneously recovered from infection, ex vivo cd8 frequencies were below the threshold of tetramer detection; however, peptide stimulation in vitro was able to expand tetramer+ cd8 cells and to induce ifn-7 production in 37% and 63% of the subjects, respectively. these lower levels of reactivity of sr subjects compared to tr patients are likely due to the different time elapsed from recovery, which was approximately 3 years in tr subjects but much longer in sp subjects, who had recovered even decades before the time of immunological analysis. finally, the group of as patients showed the lowest levels of response to hcv, in terms of both frequencies of hcv-specific circulating cd8 cells and cytokine secretion. in conclusion, the hcv-specific, cd8-mediated response has different features in patients with different outcomes of infection, showing a hierarchy of efficiency declining from subjects recovered after treatment who displayed the best responses, to chronic hepatitis patients, subjects recovered spontaneously and asymptomatic hcv-carriers, who appear to be the least responsive as a likely result of a deeper condition of tolerance to hcv. hepatitis c virus (hcv) infection is the most frequent cause of chronic liver disease in western countries and it has been involved in the development of cirrhosis with the risk of hepatocellular carcinoma. cyclooxygenases (cox) are crucial enzymes in the biosynthesis of prostaglandins and cox-2, the inducible isoform, has been implicated in inflammation, fibrogenesis and carcinogenesis. to address whether cox-2 may play a role in hcvrelated hepatic inflammation and fibrosis, we determined the cox-2 expression pattern in the liver tissue from 27 patients with hcv-induced chronic hepatitis (low histological activity=22, high histological activity=5) and 5 with end-stage cirrhosis, searching for correlations between the expression level of this enzyme and the histological activity of liver disease as well as with the intrahepatic expression and activity of metalloproteinases (mmps). we also investigated whether structural and non-structural hcv proteins may promote cox-2 expression in the human hepatocytederived cell line ccl13. western-blot and rt-pcr analysis for cox-2 demonstrated that cox-2 expression levels were higher in mild chronic hepatitis (mch, 2.6 fold), severe chronic hepatitis (sch, 3 fold) and cirrhosis (2.4 fold) than in normal liver. moreover, pge2 levels were also higher in liver samples from patients with sch (1.9 fold) and in those with cirrhosis (3.3 fold) than in normal liver. besides other cell types, hepatocellular cox-2 immunoreactivity was markedly observed in hcv cirrhosis, and although some cox-2 positive hepatocytes were observed at the edge of hepatic lobules, the majority of them were mainly restricted to the regenerative nodules. in contrast, none or few cox-2 expressing hepatocytes located in periportal areas were observed in patients with mch and sch, respectively. interestingly, we found a significant correlation between the intrahepatic expression and activity of cox-2 and mmp-2 and -9 in all the histological groups of patients analyzed. cox-2 mrna, protein and activity was de novo induced in resting ccl13 cells stably transfected with hcv core and ns5a, and this effect was higher (1 fold and 2.5 fold) when activated with cytokines and phorbol esters, respectively. in conclusion, our results provide evidence that a virus-induced hepatocellular cox-2 upregulation could mediate important pathogenic events in the course of chronic hcv infection, suggesting that specific cox-2 inhibitors might be useful for chemoprevention and therapy of hcv-related liver disease. were nahe to hbv. thl responses to hcv proteins ns3, ns4 and core were sought by elispot. as a control thl responses were also sought to hbsag, where results were analysed according to recovery from hbv infection or nayve to hbv. thl responses to each of the 5 proteins was performed before and after depletion of t-regs using anti-cd25 (>80% depletion). results: following treg depletion thl responses were revealed de novo in 3/12 and enhanced in 8112 patients with ns3,2 and 9 with ns4 respectively and 4 and 7 with hcv core respectively. the overall increase in the thl responses with t-reg depletion was significant for both hcv core (median increase 13-fold p = 0.004) and ns3 (5backaround: in both chronic viral hepatitis b and c viral persistence is thought to be due to an inadequate antiviral t cell response, which in turn may be caused by inappropriate priming of t cells by dendritic cells. an important subset of peripheral blood dendritic cells, the plasmacytoid dendritic cell, has been identified to be the major interferon-alpha producing cell in humans. since both hepatitis b and hepatitis c infection can be successfully treated by exogenous interferon-alpha, an impairment of endogenous interferon-alpha producing cells is an attractive hypothesis for the pathogenesis of chronic viral persistence. methods: patients with acute symptomatic ( n = l l ) and chronic hepatitis c (n=25), sustained responders to interferon-alpha therapy (n=22) and patients with acute hepatitis b (n=lo) as well as healthy controls (n=20) were included in this study. plasmacytoid dendritic cells were stained for facs-analysis in peripheral blood mononuclear cells with antibodies against bdca-2 and cd4 and by exclusion of lineage marker positive cells (cd3, cd14, cd16, cd19). in addition pdc were stained with various activation and maturation markers (e.g. cdso, cd83, cd86). production of interferon-alpha was measured by elisa after stimulation with cpg. results: in acute hepatitis c, ifn-alpha production was about 45-fold reduced as compared to healthy controls (15 pg/50000 pbmc vs. 701 pg150000 pbmc). this reduction was caused by both a significant reduction in absolute numbers of pdc (3.8/~1 vs. 8.4/pl, p=0.0032) as well as by a reduction of ifn-alpha production per cell (0.19 pg/pdc vs. 3.45 pg/pdc; p=0.0004). in chronic hepatitis c, ifn-alpha production was still reduced by over 50%, although absolute numbers of pdc were similar to healthy controls. following spontaneous or treatment induced viral clearance both number and ifn-alpha secretion of pdc were not different from healthy controls. importantly, in acute hepatitis b, which runs a self-limited course in the majority of cases, also the ifn-alpha production was reduced (21 pg150000pbmc). this reduction was also caused by both the reduction of absolute pdc count (5.291~1, p=o.l) as well as by the ifn-alpha secretion per pdc (0.32 pg1pdc; p=0.0007). using a panel of dc activation and maturation markers we did not find any evidence of a more immature or more activated phenotype of pdc in acute hepatitis c. exogenous ifn-alpha administration during acute hepatitis c led to a further reduction of ifn-production by pdc. conclusions: acute viral hepatitis has a dramatic impact on frequency and function of plasmacytoid dendritic cells in the peripheral blood, indicating that pdc play an important role during this phase of viral hepatitis. however, no differences were found between patients with self-limited vs. chronically evolving acute hepatitis c or patients with acute self-limited hepatitis b. future studies have to clarify whether the reduced ifn-alpha secretion by pdc contributes to viral persistence or whether this is rather part of a physiological response to acute viral disease. it has been suggested that hepatocellular steatosis, a frequent histological feature of chronic hepatitis c, is more frequent in hcv genotype 3 infection, and disappearance of steatosis in patients who clear hcv genotype 3 infection after antiviral therapy suggests a possible direct role of this hcv genotype. in order to assess the direct causal role of hcv in steatosis, we studied the relationship between hcv rna load and steatosis according to the hcv genotype. methods: we studied 176 patients with chronic hepatitis c (genotype 1, n = 83 ; genotype 3, n = 93). the serum hcv rna level was measured at the time of liver biopsy by means of a third-generation "branched dna"-based assay (versant hcv rna 3.0 quantitative assay, bayer diagnostics). steatosis was graded as absent, mild (< 10% of hepatocytes), moderate (10 to 30% of hepatocytes) or marked (> 30% of hepatocytes). daily alcohol intake during the 6 months prior to liver biopsy, and the body mass index (bmi), were recorded. results: steatosis was more frequent in patients with genotype 3 infection than in those with genotype 1 infection (84.9% vs 74.7%, ns). steatosis was significantly more severe in hcy genotype 3 than in genotype 1 infection (moderate or marked in 54.8% vs 25.3%, p=o.oool). in patients infected by genotype 3, the severity of steatosis was significantly related to hcv rna load but not to alcohol intake or bmi. in contrast, in patients infected by hcv genotype 1, the severity of steatosis was not related to the hcv rna level but was significantly influenced by alcohol intake and bmi. bivariate analysis demonstrated the effect of the hcv genotype on the association between the severity of steatosis and viral load in genotype 3-infected patients, and between the severity of steatosis and exogenous metabolic factors in genotype 1-infected patients. multivariate analysis showed that : (i, in patients infected by hcv genotype 3, only hcv rna load was independently related to the severity of steatosis (odds ratio (or) = 2.5, 95% confidence interval (ci): 1.4-4.4; p = 0.001), (ii) in patients infected by hcv genotype 1, bmi higher than 28.0 kglm2 (or = 5.8, 95% ci: 1.3-26.9; p = 0.016), alcohol intake exceeding 30 glday (or = 29.6, 95% ci: 3.2-272.0; p = 0.009), and histological grade (or = 20.0, 95% ci: 2.6-152.0; p = 0.001) were independently related to the severity of steatosis. conclusion: our results suggest : (i) steatosis is a cytopathic lesion induced by hcv genotype 3 ; (ii) hcv genotype 1 is not steatogenic per se or at the usual in vivo expression levels, and liver steatosis is a feature of associated steatohepatitis in these patients. the effect of hcv genotype 3 sequences and the role of their expression levc4s must be tested in vitro, in order to better reflect the in vivo situation. in animal models, steatosis is associated with oxidative stress and lipid peroxidation which is known to promote fibrogenesis. this study was aimed to assess in patients with chronic hepatitis c whether steatosis contributes to fibrosis through oxidative stress. methods: markers of lipid peroxidation and antioxidant status were measured in blood from 192 chronic hepatitis c patients and age and sex matched healthy controls. intrahepatic levels of these markers were also assessed in a subgroup of 23 patients and controls. results: the lipid peroxidation marker malondialdehyde was significantly higher in the blood and in the liver of patients compared to controls (p=0.05 and p=o.o01 respectively) while the antioxidant glutathione peroxidase was significantly lower (p=0.05 and p =0.0001 respectively). the antioxidant superoxide dismutase was significantly higher in the blood and lower in the liver compared to controls (p=o.o01 and p=0.002 respectively). autoantibodies to soluble liver antigen (sla) are specific for autoimmune hepatitis. the molecular characterization of the sla antigen has allowed the establishment of highly sensitive and specific radioligand assays. to determine serum anti-sla a specific radioligand assay was used. total rna was isolated and reverse transcribed from hepg2 cells. the cdna encoding sla was amplified by pcr and used as a template to express sla protein eukaryotically in a tnt coupled reticulocyte lysate system (promega corporation, southampton, uk). anti-sla was measured retrospectively in 16 consecutive patients (10 girls, median age at transplant 52 months; range 8-160) with dn-aih, in whom sera were available before transplant, 1,2,6,12 and 24 months post-transplant and at the time of the diagnosis of dn-aih. eight patients had biliary atresia, 2 alagille syndrome, 2 cryptogenic cirrhosis, 2 alpha-1-antitrypsin deficiency, 1 druginduced acute liver failure and 1 bsep deficiency. the patient with acute liver failure did not have pre-transplant serum specimen stored. twelve patients had developed smooth muscle andlor antinuclear antibodies, two anti-liver kidney microsomal (one atypical) and two anti-mitochondria1 antibody. before transplantation, anti-sla was negative in 13/15 cases, the two positive patients having cryptogenic cirrhosis and biliary atresia. post transplant, anti-sla remained positive in these two and became positive in further 9 patients. of these, in 7 patients anti-sla became positive at a median of 17 months (range: 1-115) before the clinical diagnosis of dn-aih. in 5 children anti-sla became detectable as early as 2 months post-surgery. in 6 patients anti-sla levels were highest at the time of the clinical diagnosis of dn-aih. the presence of anti-sla in dn-aih supports the autoimmune nature of this condition; the frequent appearance of anti-sla before the clinical manifestation of the disease makes it a potential predictive marker for development of dn-aih. all 38 patients showed positive response to the therapy, with respect to remarkable release of severe meteorism, active diet, and significant improvement of liver and kidney functions. however, no difference was presented in the markers of electrolytes, blood routine and blood gas analysis before and after the mars, while the effects on serum levels of alanine aminotransferase, aspartate aminotransferase and y-gt were remained uncertain since the alt decreased from 941 ull to 690 ullduring a in conjunction with observations from other centres, our data show that mars treatments resulted in a ramarkable removal of bilirubin, uric acid, bun, cr and ammonia, which could therefore decrease the toxic effects that higher concentrations of these compounds exert on liver and kidney function and could thus contribute to improvements in multiple organ dysfunctions. we found additionally that the higher concentration of serum toxin before detoxication therapy, the more efticacy removal could be achieved which presents mars could be {of therapeutic results even in the very serious cases. it remains questionable whether some of useful substances characted in low molecular weight such as trhlthyrotrophin-releasing factor), gnrh), adh(anti-diuretic hormone and calcitonin will be also removed by albumin dialysis, whether the added synthesis function is necessary for an artificial liver support, and whether this encouraging survival rate applies to long term outcome remains open for further investigation. scoring system has recently been introduced to determine priority for organ allocation in liver transplantation (lt). there is limited available data on resource utilization in the post-meld era.aims: 1. evaluate the effect of the meld system on lt-associated resource utilization and post-lt survival. 2. compare the costs of lt in the periods before and after the implementation of meld. methods : patients undergoing lt at our center in the 6 months following meld implementation(cases) were compared to those undergoing lt in the immediately preceding 9-month period (controzs). the outcome parameters studied were: 1) resource utilization: defined as ( there were no significant differences between the two groups in terms of age, sex distribution or presence of hepatocellular carcinoma (hcc). the average meld among cases was 25.9, compared to 24.1 in controls (p =0.19, ns). overall los was similar in the two groups. however, pre-lt los, especially in the intensive care unit (icu), was significantly higher in the pre-meld or control group. the pre-lt cost was also higher in this group(p=0.05), translating into significantly higher total costs of lt in the pre-meld period (p=o.ol). the need for post-lt hd, pmv and pressors was no different between the two groups. this data is summarized in table 1. the meld score was found to be significantly correlated with bile d u d damage is a maior feature in liver graft rejection. ductopenia (dp), defined as loss of more than 50%-of bile ducts, is a major hallmark of chronic liver graft rejection (cr), which usually leads to graft failure within the first post transplant year. in a previous study we have shown that in patients who developed dp and cr a deficient proliferative response of canals of hering (coh) was present in the preceding biopsies showing acute rejection (ar) when compared with a control group who experienced ar but did not progress to cr. these findings support the postulation that coh represent a regenerative compartment of the biliary unit in the liver. aim of the present studv: to analyze whether preservation of coh might contribute to the reversibility of dp. patients and methods we studied 2 groups of patients. the index group (ig, n=5) developed loss of bile ducts without progression to graft failure due to cr during a follow up of 5 years after transplantation. the second group (crg, n=12) were all cr patients, retransplanted at a median time of 7 months (range 2-19 months). reperfusion (rb), 1 week (1-w), 1 month (1-m) biopsies were studied in both groups. in the crg the last biopsies (lb) before retransplantation taken at a median time of 160 days (range 42-329 days) were also studied. additionally, in the ig the 1& (1-y), 2nd (2-y) and 5* (5-y) annual biopsies were also included. bile ducts and coh were identified by immunohistology using cytokeratin 7. ki67 (mib) was applied to investigate the proliferative activity. the number of bile ducts and coh were counted per portal tract and the number of k67+ cells was counted as ratio of the total number of cells in these structures. clinical follow-up of the ig group was studied based on liver function tests at similar time points as the biopsies. the ig group showed damaged bile ducts and decreasing numbers of bile ducts in the course of time, leading to dp at a median of 3 years. contrastingly, the crg developed dp at a median time of 160 days, observed in the last biopsies taken before retransplantation. when compared with the crg, the ig showed significantly less proliferative activity in bile ducts at 1-w (p=o.o43) but not in rb, 1-m and 1-y, the latter was compared with the lb of the crg. coh showed an initial increase at 1-w in the ig, but a progressive decrease in the subsequent biopsies, reaching significant loss at 5-y (p=0.043). in the crg, progressive loss of coh started at 1-w, leading to a significant loss within 1 year. numbers of coh were consistently lower in the crg at all time points except rb, although not statistically significant. there were also no significant differences in proliferative activity both within the ig in the course of time and when compared with crg. all ig patients continuously showed abnormal liver function tests apart from the bilirubin levels, which were elevated during the first month after transplantation but were normal after 1-y. at 5-y, median serum level of alkaline phosphatase was 228 u/l, gamma glutamyl transpherase was 312 ull, bilirubin was 26 micromoll1 and asatlalat were 96193 ull. conclusion: the ig showed a much slower course of loss of bile ducts when compared with crg, leading to graft survival of more than 5 years. a higher proliferative activity in bile ducts at 1 week in the crg did not prevent the progressive development of dplcr in crg. the initial increase of coh at 1 week in the ig might be responsible for the prolonged preservation of coh in the ig. as coh are believed to represent a regenerative compartment of the biliary unit, our findings indicated that prolonged preservation of coh might contribute to the delayed occurrence of dp but apparently not to reversibility of dp. absence of reversibility of dp is supported by the abnormal liver function tests. (ltx) . recurrent hcv poses a significant and possibly increasing threat to graft and patient survival. diagnosis of acute rejection and alterations in immunosuppression (is) may significantly impact the tempo of post-tx hcv. these associations motivated us to re-examine risk factors for early acute rejection (ear) in a large and contemporary cohort of ltx recipients. methods: the study cohort comprised of 282 consecutive adults undergoing primary ltx between 1/1/99 and 10/31/2002 for chronic liver disease with a minimum of 7 day graft and patient survival. recipients received triple is, typically with steroids, tacrolimus, and mycophenolate mofetil. ear was defined as biopsyproven rejection or treatment for presumed rejection within 6 months of ltx. risk factors for ear were determined by cox proportional hazard methods. spearman rank and phi correlations were used to determine associations between factors. results: 177 men (63%) and 105 women (37%) underwent deceased (n = 236; 84%) or living donor (n = 46; 16%) ltx. the etiologies of chronic liver disease were hcv (n=138; 49%), aih i pbc / psc (n=40; 14%), hbv (n= 34; 12%), cryptogenic (n=27; lo%), alcohol (n=24; 9%), and miscellaneous (n=19; 7%). our overall incidence of ear was 45% (126 i282 recipients); 118 recipients (94%) had biopsy-proven rejection while 8 recipients (6%) were treated for rejection without histologic confirmation. cox univariate models showed that hcv, female gender, meld 2 28, year 2000 compared to year 1999, and lower day 5-7 tacrolimus (tac d5-7) level were positively associated with ear while asian compared to caucasian ethnicity was negatively associated with ear (table la) . factors such as recipient and donor age, recipient african american or hispanic ethnicities, donor type (deceased or living), other years (2001 and 2002) , child's score and class, pre-ltx icu location, and cold and warm ischemia times were not significant risk factors. cox multivariate models showed that hcv diagnosis and female gender remained independent and significant risk factors for ear (table lb) . hbv etiology and asian ethnicity were significantly correlated (phi coefficient 0.53; p = <0.0001) and thus, did not remain independent risk factors in multivariate analysis. while lower tacrolimus level tended to predispose to ear, meld 2 28 and year 2000 were no longer associated with ear. lower tacrolimus level was significantly correlated with both higher meld score (spearman rank correlation -0.34; p = 0.0000) and later transplant year (spearman rank correlation -0.17; p = 0.019). conclusions: hcv etiology of liver disease is strongly associated with ear after ltx. risk of ear is higher for hcv than etoh, cryptogenic, and hbv etiologies and comparable to aih / psc i pbc etiologies; this effect is independent of gender, ethnicity, year, meld, and post-ltx is. recipients with high meld scores are also at increased risk for ear, at least partly because of lower post-ltx is. it is unclear whether the strong association of hcv etiology with ear is because hcv infection results in an immunologic environment predisposing to ear or whether we are unable to accurately diagnose rejection in the setting of hcv recurrence. aim: although the donor pool has expanded in response to increasing waiting lists the quality of donor livers has suffered as a result. it remains unclear whether such marginal organs can be safely used in high-risk recipients or whether they should be implanted solely into good recipients. we aimed to define recipient selection criteria for implantation of these grafts. methods: a prospectively collected database containing 397 patients who underwent orthotopic liver transplantation between 1994 and 2002 was analysed. donors were scored using a formula derived by logistic regression that identified 3 covariates (graft steatosis, donor age and cold ischaemia time) that independently correlated with primary graft dysfunction. this enabled them to be stratified into either marginal or non-marginal groups. using logistic regression analysis, recipient factors independently correlated with 1-year post transplant survival in the marginal group (recipient age, plasma albumin and urea) were built into a mathematical model and each patient was given a score accordingly. patients with scores higher than the defined optimum cut-off value were considered as high-risk and the others with lower scores as low-risk recipients. outcomes were then evaluated in these subpopulations results: marginal and non-marginal donor groups consisted of 65 and 332 patients respectively. the recipient score derived from the multivariate analysis was as follows : score = 1.989 x plasma albumin+ 1.22 x age group + 2.076 x urea, with the recipient albumin level coded as 1 for < 3.1 gldl and 0 for 2 3.1 gldl, the recipient age group coded as 2 for > 55 years, as 1 for 43-55 years, and as 0 for 22.4 mgldl, and as 0 for 5 22.4 mgldl. the roc curve analysis showed that the score had a good discriminating power (area under the curve 0.78, p =.001) and the ideal cut-off value demarcating high-risk from low-risk recipients was 3.25. therefore the recipients were classed as high-risk if they had a plasma albumin level below 3.1 gldl with either an age of over 55 or a urea level of above 22.4 mgldl or when they had a urea level over 22.4 mgldl with an age of over 42. of the 65 recipients in marginal group, 28 (43%) were classified as high-risk and 37 (57%) were classified as low-risk. there was a huge 1-year survival difference between high-risk and low-risk recipients (60.7% and 91.9%, respectively, p <.0001 background single-center experience with pretransplant use of rabbit anti-human-thymocyte globulin suggests that despite early depletion of lymphocytes, a third of all pediatric liver recipients develop early rejection, while the remainder demonstrate clinical graft adaptation. purposelmethods: to identify potential mechanisms, 31 pediatric liver recipients, median age 7.8 years, median followup 8 months, received pre-and post-transplant measurements of 1. whole blood concentrations of tacrolimus (tac), 2. interdose changes in mitogen-stimulated t-and b-cell responses (slr), 3. dendritic cell (dc),and peripheral blood mononuclear cell subsets, and 4. cd8+28-suppressor effect on donor antigenpresenting cell types (cd34fmonocytes and cd19+ b-cells). all patients received ratg preconditioning with a total dose of 5 mglkg in two divided doses. the first dose was given before liver transplantation (ltx). maintenance agent was tac without steroids. in 9 patients this was replaced with sirolimus (srl). mitogen-stimulated t and b-cell responses were compared with those seen in a historical population, who had not received ratg pretreatment. results: all measurements were performed at a median interval of 47 days (22-57 days) after ltx. 1. in slr, the frequency of t-cells expressing the cytokines ifn-g, tnf-a, and il-2 decreased with increasing total exposure (auc) to tac in historical controls (n=6). this relationship was markedly dampened in ratg patients (n=15), as suggested by slopes of 0.0023, 0.032, and 0.0006 relating ifn-g, tnf-a and il-2 on the y-axis to tac auc. 2. significant decrease in cd4 absolute counts at 1 week and partial reconstitution at 2 months after ratg pretreatment (mean pretx-3120 vs 1 week-1920 vs month 2-2117 celllmm3). during this period, monocytes and b-cells remained stable. 3. dc2 remained unchanged, while dc1 frequency increased significantly, from 0.15 to 0.23, p=0.05. 3. in coculture experiments, purified cd8+28-subpopulations from two recipients receiving minimal tac doses induced decreased cd86 expression in donor apc, but increased its expression in hla-mismatched apc. eleven of 31 subjects experienced rejection, while 18 subjects are being maintained on daily (n=13) or every other day (n=5) doses of tac or srl. conclusions: despite lymphocyte depletion, rejection in the setting of t-cell anergy can be explained by relative sparing of antigen-presenting cells, which can recruit alternative effector mediators. the appearance of donor-specific cd8 + suppressor cells in recipients on low immunosuppression suggests that these conditions may also foster a favorable immunomodulatory response toward the liver allografts. (dropout) . the objective of this study was to evaluate the impact of the hcc-adjusted model for end-stage liver disease (meld) organ allocation scheme on the intention-to-treat outcome. under the meld scheme, patients with hcc meeting the united network for organ sharing (unos) t1 (single lesion under 2 cm) and t2 (single lesion between 2 to 5 cm, or 2 to 3 lesions none exceeding 3 cm) criteria were eligible for an initial meld priority score of 24 and 29, respectively. they were also entitled to an increase in meld score, corresponding to a 10% increase in mortality, for every 3 months on the waiting list. methods: excluding patients undergoing living-donor liver transplantation, we prospectively evaluated 102 consecutive patients with hcc listed for olt since january 1998. the kaplan-meier probabilities of olt and dropout among 44 patients with hcc listed under the meld system between february 2002 and january 2003 were compared with 58 patients listed between january 1998 and january 2002 under the previous system of organ allocation. follow-up in the pre-meld group was censored on february 27,2002, when the meld system for organ allocation was implemented by unos. for patients under meld, follow-up was censored on february 27,2003, when further refinements of the meld policy for hcc were made. results: the baseline characteristics were not significantly different between the two groups. eighteen of 44 patients (41%) in the meld group and 26 of 58 patients (45%) in the pre-meld group received chemoembolization or various ablation treatments before olt (p=0.89). all patients in the meld group met t1 (5 patients) or t2 criteria (39 patients). in the pre-meld group, 6 patients had hcc stage exceeding t2 but meeting our proposed expanded criteria (single lesion not exceeding 6.5 cm or no more than 3 lesions none greater than 4.5 cm with total tumor diameter not exceeding 8 cm) by the time of olt. the kaplan-meier cumulative probabilities for olt at 3, 6, and 8.5 months of longest followup were 22.5%, 64.0% and 88.0%, respectively, in the meld group, versus 17.2%, 24.7%, 35.8%, and 47.2% at 3, 6, 9, and 12 months, respectively, in the pre-meld group (p=0.0006). under meld, none of the 5 patients with t1 lesion had received olt. the cumulative probabilities for olt for the 39 patients with t2 hcc in the meld group were 25.5%, 69.2%, and 89.2%, respectively at 3, 6, and 8.5 months (p=o.oool versus the pre-meld group). the cumulative probability of dropout was 5.6% at 8.5 months of longest followup without olt under meld, whereas the cumulative probability of dropout increased from 7.2% at 6 months to 37.8% at 12 months in the pre-meld era. the difference did not reach statistical significance (p=0.74) largely due to low dropout rates in the first 6 months for both groups. among the patients who received olt, 3 of 22 patients in the meld group versus 9 of 25 patients in the pre-meld group had pathologic hcc stage in the explant exceeding t2 criteria (p=0.78). unfavorable histologic tumor features in the explant, including either poorly differentiated grade or microvascular invasion or both, were observed in 4 of 22 patients in the meld group versus 5 of 25 patients in the pre-meld group ( i " 1.0). the short duration of follow-up under meld precluded comparison of intention-totreat survival or hcc recurrence between the two groups. con-clusion: the hcc-adjusted meld system significantly improved the probability of timely olt, and was not associated with selection of a greater proportion of hcc with unfavorable explant tumor histology. given the low probabilities for dropout in the first 6 months following listing for olt even in the pre-meld era, patients with hcc might have indeed received too high a priority score in the first year under meld. disclosures: nancy l ascher -no relationships to disclose nathan m bass -no relationships to disclose randomized at d7 to receive maintenance is regimen with ciclosporine microemulsion + prednisone (group 1) or without steroids (ciclosporine microemulsion + placebo, group 2) after a 7 days blinded oral steroid tapering period. results : 193 patients were recruited and a total of 174 were randomized at d7 (group 1 = 90, group = m). there was no difference between the 2 groups for baseline characteristics, proportion of patients with hepatitis c (18.9% and 20.2%), or cyclosporine blood levels. the incidence of treated biopsy confirmed acute rejection at 6 months was 24.4% in group 1 and 38.1% in group 2 (p= 0.03), with a trend for higher incidence of grade 213 rejection ( 18.9% vs 28.6% ; p=0.12). this difference was maintained in hcv pos and hcv neg patients. no difference was observed between the 2 groups in terms of adverse events, infections, incidence of hypertension and renal dysfunction. changes from baseline were similar with regards to metabolic parameters. a trend towards a better glucose tolerability was observed, less patients receiving an antidiabetic treatment in the placebo group ( 2 vs 10). conclusion : peritransplant immunosuppression with basiliximab, cyclosporine microemulsion, and steroids resulted in excellent low rejection rates in lt patients. early steroid withdrawl strategy at day 14 is not supported by the results of this study, which showed an higher incidence of acute rejection and a trend to more severe acute rejection, only balanced by a trend to a lower need of antidiabetic treatment. to determine if an association exists between meld score and post transplant graft loss within 3 months. methods from 2/28/02 through 10/31/02,2,745 patients underwent liver transplantation, 92% of whom were followed for a minimum of three months. cox regression analysis was utilized to assess the relationship between meld score and post transplant outcome. transplantation. seventy one percent of patients were positive for autoantibodies: 52.9 % were positive for anti-nuclear antibody, 23.5% for anti-smooth muscle antibody and 0% for anti-liver kidney microsomal antibody. the average alt elevation was 224 (range 61-486) and ast elevation 166 (range 52-346). an elevated ggtp was seen in 70.6% of patients. all patients were hepatitis c pcr negative. at the time of diagnosis, 12 patients were being treated with cyclosporine, 5 with f'k506, and 7 with steroids in addition to calcineurin inhibitors. after diagnosis, all patients received standard therapy with l-zmg/kg of steroids. in addition, 89% (15117) began hthioprine and 24% had their calcineurin inhibitor dose decreased. after a mean of 2.6 years of follow-up (range 0.53-5.93), 59% remain steroid dependent and 12% are off steroids. one patient was re-transplanted for biliary complications and 1 patient required conversion to sirohus. conclusion: diagnosis of de novo am requires a high index of suspicion as auto-antibodies are often negative. steroid therapy, often with the addition of azathioprine, is effective in controlling disease. many patients, however, become steroid dependent in order to remain in remission. background. liver allografts have an improved outcome compared with other solid organ grafts, and rodent studies have suggested that activation-associated lymphocyte death may play a key role. studies from our laboratory have shown increased levels of apoptotic lymphocytes in human liver grafts compared with renal grafts and native liver. although the levels of apoptosis did not differ significantly between those who developed acute rejection (rej) and those who did not (nr), the earliest timepoint studied was 3 days post-olt. we hypothesized that the very early postoperative period (within 48 hours) would reveal a relationship between leukocyte apoptosis and rejection. aims. the aim of this study was to determine the amount of early leukocyte apoptosis and its relationship with the degree of lymphocyte activation, subsequent rejection status and degree of donor cell chimerism. methods. peripheral blood mononuclear cells were isolated from 76 patients undergoing olt and were collected on the day prior, 5 hrs after reperfusion (day 0) and 24 hrs post-olt (day 1). dna and rna were prepared and real-time pcr used to quantify apoptosis (ligation-mediated pcr), lymphocyte activation (il-2, ifn-gamma, il-10, cd40 ligand, using gapdh as an internal standard) and donor cell chimerism (y chromosome dyz3 or donor-specific drb1). results. the mean level of circulating apoptotic cells in day 1 recipient pbmc was higher than healthy controls (0.9% t-0.2 vs 0.2% ? 0.1, ~~0 . 0 1 3 ) . apoptosis was greater in nr (1.1% 2 0.3) compared with rej (0.3% t-0.1, p=o.o21). on day 1 the pbmc from nr had increased expression of ifn-gamma (p=0.006), il-10 (p=0.016), cd40 ligand (p=0.02) and il-2 (trend) compared with rej, despite no difference in lymphocyte counts. donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased leukocyte apoptosis in the nr group. interestingly, the level of chimerism 5 hrs postreperfusion (day 0) was significantly higher in nr (3.8% f 0.6) compared with rej (1.2% t-0.4, p=0.004) and there was a close correlation between chimerism on day 0 and pbmc cytokine expression on day 1 (r=0.58, p=0.006). conclusions. patients who did not experience rejection had a paradoxical increase in markers of lymphocyte activation at day 1, in association with higher levels of leukocyte apoptosis. this suggests that recipient cell death may have a role in graft acceptance and reduced acute rejection in human olt. the higher donor cell chimerism seen in non-rejectors implicates the passenger leukocytes in the process of heightened cell death. disclosures: andrew clouston -no relationships to disclose wenyi gu -no relationships to disclose julie r jonsson -no relationships to disclose elizabeth e powell -no relationships to disclose daina m vanags -no relationships to disclose amadeo marcos, bridget flynn, paulo fontes, thomas cacciarelli, wallis marsh, michael devera, obaid shakil, noriko murase, anthony demetris, john fun% thomas e stanl, university of pittsburgh, pittsburgh, pa the seminal mechanism of organ engraftment is thought to be immune activation-dependent clonal exhaustion-deletion. the conventional use of heavy immunosuppression may depress the initial step of donor-specific activation to the extent that the treatment is anti-tolerogenic. to avoid this pitfall, we have applied 2 therapeutic principles in management of 78 adults cadaveric liver recipients transplanted between 9/2002-5/2003. the treatment principles were, first, host conditioning prior to transplantation, and second, minimum post-transplant immunosuppression. the host conditioning was done with one gram of methylprednisolon and a single infusion of 30 mg of alemtuzumab, completed before liver revascularization. there were 49 males and 29 females in the group with mean age 52.3110.3 years (32-73). main causes of liver disease were hepatitis c (40%), cholestatic liver disease (18%), alcoholic liver disease (22%). daily post-transplant monotherapy with tacrolimus (trough target 10 ng/ml) was started 12-24 hours postoperatively (starting tacrolimus dose: 10.5+5 mg, median 10). because of suspected neurotoxicity, 5 patients were switched to cyclosporin. in addition sirolimus monotherapy was substituted for tacrolimus in one patient for management of nephrotoxicity. immune activation diagnosed by liver function tests or by mild or equivocal rejection in liver biopsies frequently was not treated, and tended to resolve spontaneously. clinically and pathologically significant rejection was treated with a bolus of methylprednisolone and/or a 30 mg dose of alemtuzumab. nine patients (11.5%) died 5 sepsis; 2 pnf; one coagulation disorder (hypercoagulable state), and one congestive heart failure. of the 69 surviving recipients four experienced rejection during the first two months which was readily reversed with a bolus of methylprednisolon. after demonstrating the absence of immune activation in liver biopsies at 120 days post-transplantation, weaning from monotherapy was initiated in 49 patients (with the aim of completely stopping treatment in selected cases). seven patients experienced one episode of rejection that was reversed with single dose of methylprednisolone and/or infusion of 30 mg of alemtuzumab. presently, 14 patients are on once a day monotherapy, 14 patients on every-other-day, 10 patients on three timedweek, 10 patients on twicelweek and one patient on once-a-week immunosuppressive regimen (latest tacrolimus dose 5.7t3.5 mg, median 5.5). the patients on cyclosporin and sirolimus are also following the same stepwise weaning pattern. no cases of new onset diabetes, renal failure or hypertension was found in these patients. mean serum creatinine level in the study group at the time of transplantation was 1.020.4 mgldl and at the latest follow up 1.220.3 mgldl. cmv infection was seen in 22% of the patient population but was easily treatable with antivirals. no ptld was seen during the follow-up period. hepatitis c recurrence was seen in over 70% of the patients. response to anti-hcv therapy in this group, after reduction of immunosuppression and especially during the weaning process was promissing. conclusion: by applying the principles of immunosuppression outlined above, it has been possible to drastically reduce the amount of total immunosuppression relative to any (of our) pre-vious experience with liver transplantation. to see less side effects of chronic and high dose immunosuppressive therapy, and probably to have a chance to get a better response to treatment in our hepatitis c population. the results suggest the possibility of systematically achieving drug-free tolerance after liver transplantation. background:end stage liver disease as a consequence of hepatic sarcoidosis is an uncommon indication for liver transplantation (lt). consequently, there is a paucity of information on the pre-lt findings and postoperative course of individuals transplanted for hepatic sarcoidosis. the purpose of this study was to evaluate our experience with lt for sarcoidosis. methods: cases were identified by review of the mount sinai hospital lt database. patient records were reviewed; including the pathologic analysis of the hepatic explant, to confirm that sarcoidosis was responsible for liver failure. for each case, two control patients with other causes of liver failure matched for age, gender and date of transplant were selected. data collected encompassed a mean follow up period of 5 years (range 1-8 years) post-lt. two-tailed student's t-test was used for comparison of continuous data, two-tailed fisher's exact test for comparison of categorical data and log rank test for comparison of survival d,ita. results: hepatic sarcoidosis was the indication for lt in 7 of 2016 adult-lt (0.3%) performed from september 1988 -june 200.3. the mean age at transplant was 56 years and 4/7 (57%) were males. the diagnosis of sarcoidosis was established by findings of extensive, non-caseating granulomas in pre-lt biopsy specimens or in the native liver explant. in 2/7 cases, sarcoid had been previously diagnosed by biopsies of lung parenchyma or mediastinal lymph node. 6 of 7 patients were ama negative. the sole exception was a patient with an ama titer of 1:320 in whom sarcoid was still considered the most likely diagnosis based on liver biopsy findings of granulomas present in both portal and lobular areas and granulomas in a lung biopsy performed 10-years prior to lt. extrahepatic disease was limited to pulmonary involvement in 4 patients with radiographic findings of either interstitial infiltrates (2) or interstitial infiltrates and hilar adenopathy (2). the mean age at transplant and gender distribution were identical in cases and controls. the indications for lt in the control group were: hepatitis c (7), pbc (2) and one patient each with hereditary hemochromatosis, cryptogenic cirrhosis, hepatitis b and laennec cirrhosis. no statistically significant differences between the groups were present with respect to pre-lt levels of ast, alt, alkaline phosphatase, total bilirubin, serum creatinine or prothrombin time. cases and controls had a similar prevalence of ascites and anti-hepatitis b core antibodies. in contrast, sarcoid cases were much more likely to have a diagnosis of diabetes mellitus (100% vs. 21%, p=.002) and less likely to have antibodies to hepatitis c (0% vs. 50%, p=.05). standard orthotopic lt was performed (two control patients received right trisegment grafts). rates of acute cellular rejection were 71% in cases and 43% in controls (p=o.44) with cases experiencing a greater number of acute rejection episodes per patient (1.9 vs. 0.6 episodeslpatient). de novo autoimmune hepatitis developed in one case and one control patient. both de novo autoimmune hepatitis and chronic rejection developed in one case patient. recurrence of hepatic sarcoidosis was diagnosed in two patients at 1.5 and 5.6 years of follow-up. among cases, the one-year graft and patient survival rates were 100% and five-year graft and patient survival rates were 86%. there was no statistically significant difference in five-year graft or patient survival between cases and controls. conclusions: end-stage liver disease as a consequence of sarcoidosis is a rare indication for lt. these patients share many features in common with those transplanted for other indications. despite the small number of patients, a relatively high rate of acute rejection and de novo autoimmune hepatitis was observed in patients with sarcoidosis. recurrence of hepatic sarcoidosis was observed. five-year graft and patient survival rates were comparable to those of patients transplanted for other indications. disclosures: sander florman -no relationships to disclose leona kim schluger -no relationships to disclose kevin m korenblat -no relationships to disclose evan j lipson -no relationships to disclose 440 transplantation. james d eason, ari j cohen, safheesh nair, george loss, ochsner clinic foundation, new orleans, la sirolimus has been used successfully in improving renal function in olt recipients previously treated with tacrolimus and steroids. we report our experience with early sirolimus conversion in steroid-free olt recipients to determine safety and efficacy in patients never treated with steroids. methods: we performed 220 olt over a threeyear period. steroid-free immunosuppression with rabbit atg induction and tacrolimus and mmf was used in 140 of these patients. twenty-five of these steroid-free recipients were converted from tacrolimus to sirolimus within one week to ll months of transplant. results of these patients receiving sirolimus conversion were reviewed. results: renal insufficiency was the reason for conversion in18 patients, while seven patients were converted because of neurotoxicity. six patients were dialysis-dependent at the time of conversion. one-year patient survival in this high-risk group was 80% (20/25) compared to 88% in patients who remained on tacrolimus. the four deaths were in patients converted because of renal failure, three of whom were on dialysis. the incidence of rejection was 31% in sirolimus patients compared to 24% in patients who remained on tacrolimus. rejection was treated by the addition of mmf or reintroduction of low-dose tacrolimus. only one patient required steroids to reverse rejection. conclusion: early sirolimus conversion is safe and effective lawal, sandy florman, isabel fiel, ronald gordon, myron schwartz, charles miller, thomas d schiano, the mount sinai medical center, new york, ny introduction: primary non-function (pnf) fter liver transplantation occurs in approximately 5% of cases and is fatal without timely retransplantation. pnf has been associated with many risk factors, however the etiology remains unknown. some evidence suggests that ultrastructural changes in the liver may be causative. aim: we sought to examine the hepatic ultrastructure of donor allografts in patients with and without pnf using electron microscopy. medical center, over 2000 adult orthotopic liver transplants were performed. patients with the classic clinical presentation of pnf requiring retransplant were identified. archived pre-and postreperfusion donor liver biopsies were examined by electron microscopy in 9 patients with pnf and in 9 matched controls. each pnf case was matched by donor age t5years, gender, cold ischemic time klhour and the donor's cause of death. all biopsies were blindly reviewed by the same pathologist. particular attention was paid to abnormalities of the mitochondria, endoplasmic reticulum and sinusoidal endothelial cells. in addition, the glycogen content of the cells was also assessed. the recipient age and creatinine, as well as the donor serum peak transaminases and bilirubin were compared.non parametric tests with p values <0.05 were regarded as significant using the spss 11.5 program. results: overall, patients with pnf were older (55.7 t 10 vs. 48.99 years, p=0.03) and had higher peak alt levels (141 ? 185 vs. 40 2 32 u/l, p=0.04). there was no significant difference in recipient peak serum creatinine, donor peak serum ast, sodium or donor peak serum bilirubin. in all cases, the endoplasmic reticulum and sinusoidal endothelial cells were ultrastructurally normal. the hepatocytes had variable degrees of glycogen pooling and moderate to severe fatty infiltration. in 7/9 (78%) pnf cases vs. 2ly (22%) control had intramitochondrial crystalline inclusions on pre-perfusion biopsy. conclusion: liver allografts from patients with primary non-function have significant mitochondria1 ultrastructural changes on pre-perfusion biopsies, and may thus have some intrinsic mitochondria abnormalities. introduction: the ideal liver allocation system would allocate donated livers to patients who are most likeiy to die without a transplant, but who have the lowest predicted mortality once they have been transplanted. this mode of allocation assesses the condition of both the recipient and the donor at the time of transplantation. in this study we have constructed a self-organising map (som: this is a neural network or non-linear mode of decision analysis suitable for modelling complex multidimensional relationships) and then validated it by using the som to classify survival in an unrelated population. patients and methods: we have previously described a som consisting of 72 (50 recipient and 22 donor) factors (inputs) constructed from 827 (449 male; median age 52 years; median meld score 16) consecutive primary liver transplants undertaken between 01/01/1993 and 31/07/2002 in the queen elizabeth hospital, birmingham, uk. using a 3 neuron version of this som with three output functions (patient survival at 3 months; 1 year and 5 years) we used the som to classify the post-transplant survival of all primary graft recipients (n=200 patients) over a 5 year period from a north american centre (125 male; median age 48 years; median meld score 16). the birmingham (uk) patients were first classified by the som and then the probabilities of survival (p,) at 3 months, 1 year and 5 years calculated by dividing the number of surviving patients by the total number of patients in neuron i. using this som, each of the wisconsin (us) patients was classified to each of the neurons and the distribution of survival probes for each neuron compared between the two populations using the chi squared test. results: there was no significant difference in the survival probabilities of patients in each neuron when the wisconsin population was compared with the birmingham population. thus, the birmingham som was able to classify successfully the survival of the wisconsin patient population at 3 months, 1 year and 5 years following transplantation by using the same donor and recipient factors ( table 1) . analysis of the inter-neuronal survival probabilities demonstrated that for 3 month and 1 year survival, neuron 3 was associated with a significantly lower survival probability than neurons 1 and 2 (p=0.006 for 3 months; p= 0.006 for 12 months). further, if recipients from neuron 1 received livers from patients classified to neuron 3 by "computer simulated transplantation", a proportion of patients then had a lower predicted survival probability (by som re-distribution to neuron 3). this analysis indicates that patient survival post-transplantation is significantly infuenced by both the condition of the recipient and donor factors at the time of transplantation. conclusions: (1)som analysis provides an efficient and automated method for assessing the probability of survival of individual patients in an unrelated population at 3 months, 1 year and 5 years following liver transplantation. (2)the model not only assesses the pre-transplant condition of the patient, but also considers a wide range of donor factors when predicting the probability of survival post-transplant. (3)this unique resource can match a single liver to a population of recipients most likely to die without a transplant whilst ensuring the best possible post-transplant survival for the most suitable recipient (4)som analysis can also assess the probability of survival of individual recipents matched to a range of possible donated livers when they are being considered for transplant programmes. purpose of study: to assess the efficacy of targeted prophylaxis in patients identified to be at highest risk of if1 after orthotopic liver transplantation. introduction: historically, invasive fungal infection (in) has complicated up to 20% of cases of orthotopic liver transplantation (olt). retrospective analysis has identified a variety of risk factors associated with a high risk for the subsequent development of ifi. these include patients undergoing retransplantation, transplantation for fulminant hepatic failure (fhf), requiring haemodialysis and prolonged intensive care unit (icu) stay. prophylactic anti-fungal therapy is safe and can reduce the incidence of ifl in olt recipientri. in recent years our unit has moved towards targeting prophylaxis to higher risk transplants. we assessed the efficacy of our policy in reducing the incidence of invasive fungal infection. methods: a retrospective audit was conducted comparing two groups of adult olt recipients over two 5 year time periods, 1990-1994 when targeted prophylaxis was not used (group 1) and 1997-2001 when targeted prophylaxis was used (group 2). data were collected with respect to risk factors for ifi, anti-fungal prophylaxis use and development of ifi. results: there was no difference in the overall number of risk factors for if1 associated with olt between the two groups. however, a higher proportion of patients in group 2 had high risk factors for if1 compared to group 1 (p=0.07). there was a significant difference in the use of targeted anti-fungal prophylaxis given to high risk patients in group 2 compared to group 1 (52.3% cf 23.8%, p=0.03), and there was a significant reduction in the number of ifis in group2 compared with group 1 (4.5% cf 28.6%, conclusion: a policy of targeting anti-fungal prophylaxis to highest risk liver transplant recipients leads to a significant reduction in if1 this group. there remains a background incidence of infection in low risk or long term recipients in whom prophylaxis is not given. background: only few studies have described risk factors associated with cellular rejection. the diagnosis of cellular rejection requires histology. there are no data evaluating the absence of rejection in a protocol biopsy population. aim: to assess predictive factors associated for the absence of cellular rejection in a protocol liver biopsy population and to evaluate the usefulness of protocol liver biopsies. method 449 consecutive patients transplanted on a data-base at our centre. protocol liver biopsies were performed between 5 and 10 days post transplantation and then when clinically indicated, during the first 3 months. rejection was scored prospectively. the following variables were examined with respect to absence of rejection over 3 months and in the first protocol liver biopsy: a) donor factors: age, gender, race, donorlrecipient gender match, abo match. b) pre-transplantation recipient factors: age, sex, aetiology, ascites, oesophageal varices, tips, encephalopathy grade, renal support, ventilation, total bilirubin, albumin, inr, ast, alt, creatinine, urea, c) graft and surgical factors: surgeon's visual assessment of graft, cold ischaemic time, use of venovenous by-pass, intraoperative blood transfusion and initial maintenance immunosuppression of either cyclosporin or tacrolimus in triple-dual or monotherapy. standard treatment of rejection was 1 g iv daily of methylprednisolone x 3 days. results: absence of rejection over 3 months was in 69 (15%), mild in 103 (21%) and moderatelsevere in 277 (62%). in the first biopsy: absence of rejection in 92 (21%), mild in 178 (39%), moderate in 159 (35%) and severe in 20 (4.5%). univariate analysis showed for both first protocol biopsies and 3 months, that 4 variables were significantly correlated with non rejection: pre-transplantation use of renal support (haemodialysis or hemofiltration), (p=0.042), cold ischaemic time >15 hours (p=0.030), suboptimal graft visually (p=0.042) and blood transfusion <= 7 units cp=0.008). no correlation was found between aetiology of liver diseases, initial maintenance immunosuppression. conclusion: a suboptimal graft with prolonged cold ischaemic time in recipients with previous need of renal support seems to be associated with absence of rejection. these data are in contrast with previous reports. these factors may be helpful in identifying patients who do not need to be submitted to protocol liver biopsy. tables 1 and 2 1 child developed abnormal transaminases and was found to have chronic hepatitis on liver biopsy and was treated with steroids. 2 required adjusting cyclosporine dosage to maintain trough levels in the identified range (range 65-90 microgramll as per protocol). summary: there was good correlation between the trough levels taken on 2 occasions in the stable paediatric post liver transplant group of patients. the range of c2 peak levels were also similar suggesting good bioavailablity conclusion: this preliminary study on long term post transplant recipients suggests that a peak c2 level is within the range of 280-460 microgramll. background hepatocytes and cholangiocytes release substantial amounts of adenosine triphosphate into bile, where it is rapidly degraded by membrane-bound ecto-atpase and 5'-nucleotidase. this degradation process leads to the generation of adenosine and inorganic phosphate (i?#. whereas adenosine is reabsorbed in a sodium-dependent manner back into hepatocytes, little is known about the fate of biliary pi. in rat, biliary pi concentration is 0.01 mm, which is about 100 fold lower than in hepatocytes (1 mm) and 200 fold lower than in plasma (2.3 mm)9 indicating active reabsorption of pi from bile canaliculi andlor from the biliary tree. aim: the purpose of the present study was to functionally characterize canalicular p, reabsorption in rat liver and to identify the involved p, transport system(s). methods: p, transport was determined in isolated canalicular liver plasma membrane (clpm) vesicles using a rapid filtration technique. identification of putative p, transporters was performed with reverse transcriptase-polymerase chain reaction (rt-pcr) from rat liver total mrna using sodiumlphosphate cotransporter specific primers for napi-iib (gggattgggaaattcatccti ttccaacacaaggttggtca), napi-111 subtype pit-1 (catctcggtgggatgtgcitgttgctctctcctccttca) and napi-i11 subtype pit-2 (gctctaccattggcttctcglaca-gaggaagtgcctggaga)3. on the protein level, phosphate transporter expression was confirmed by western blot analysis in isolated basolateral (blpm) and canalicular (clpm) rat liver plasma membrane vesicles. specific polyclonal antibodies were raised in rabbits against antigenic peptides from mouse napi-iib and human napi-iiilpit-2 showing >88% identity with rat homologues. results: transport studies in isolated clpm vesicles demonstrated sodium-dependent p, uptake (najut > nag 3877 pmollmin; k&t > kg 107 pmollmin). initial na+-dependent p, uptake was linear for at least 6 sec and exhibited a clear overshoot indicating transient intravesicular concentration of p, (secondary active p, transport). initial p, uptake rates (4 sec) were saturable with increasing p, concentrations and exhibited an apparent k,,, value of -11 km. furthermore, sodium-dependent p, transport was higher at an acidic (phout = 6.5; ph,, = 7.4) as compared to an alkaline extravesicular ph (8.0). in addition, an intravesicular negative membrane potential stimulated sodium-dependent p, transport indicating a na:p, stoichiometry of > 1. these data are comparable with the transport characteristics of sodiumlphosphate cotransporters napi-iib, napi-iiilpit-1 and napi-iiilpit-23. mrnas of all these three napi's were found to be expressed in rat liver by rt-pcr. however, on the protein level only napi-iib was found to be expressed selectively in clpm, whereas napi-iiilpit-2 was detected in blpm. no clearcut localization of napi-iiilpit-1 protein could be obtained. conclusions: the canalicular membrane of rat hepatocytes localizes the sodiumlphosphate cotransporter napi-iib, which can reabsorb p, from primary hepatic bile back into hepatocytes. in contrast, napi-iiilpit-2 was found to be expressed at the basolat-era1 hepatocyte plasma membrane. the results indicate that napi-iib regulates p, concentration in bile and may play an important role in the overall p, homeostasis in rat liver backgroundlaims. organic anion transporting polypeptides (oatps) are a family of transport proteins of the basolateral hepatocyte membrane. human oatp8 (slc21a8) is predominantly expressed in hepatocytes and is an uptake system for xenobiotics such as digoxin. the oatp8 gene promoter possesses an inverted repeat (ir1) element at nt -82l-70 relative to the transcription start site, that binds and is activated by the farnesoid x receptorlretinoid x receptor (fxrlrxr). ligands of fxr include bile salts such as chenodeoxycholic acid (cdca), previously shown to activate the oatp8 gene. however, because oatp8 is only a poor bile salt but an efficient xenobiotic transporter, we investigated whether xenobiotics such as rifampicin regulate the oatp8 gene. rifampicin is a prototypic ligand of the xenobiotic receptor pxr (pregnane x receptor). methods. endogenous oatp8 mrna levels in caco2 cells were quantified by real-time pcr. an oatp8 gene promoter construct containing nucleotides -120l +38 relative to the transcription start site (luc-120) was assayed for reporter activity in transiently transfected cells. the fxr binding site in the oatp8 gene (ir1 element) was characterized using the luciferase construct ir1-tk-luc that contained a thymidine kinase promoter under the control of the irl element. results. endogenous oatp8 mrna levels in caco2 cells were induced 7fold by cdca (100 pmolll). interestingly, incubation of cells with rifampicin (10 pmolll) resulted in a 4.5fold increase in oatp8 mrna levels. to study the mechanism of rifampicinmediated induction of oatp8, the promoter sequence was searched for potential pxr binding sites. because the ir1 element, previously shown to bind the fxrlrxr heterodimer, was the only nuclear receptor binding site found, we hypothesized that the induction by rifampicin could be mediated through the fxr element. we, therefore, studied the effect of cdca and rifampicin on the ir1 element. in cells cotransfected with the ir1-tk-luc construct and expression plasmids coding for fxrlrxr, pxrlrxr or car (constitutive androstane receptor)lrxr, fxrlrxr induced the irl element llfold in the presence of cdca and 2.5fold in the presence of rifampicin, indicating that rifampicin is capable of activating fxr. in contrast, pxrlrxr or carlrxr did not activate the ir1 element in the presence of cdca or rifampicin. to confirm that an oatp8 promoter construct is also activated by rifampicin, huh7 cells cotransfected with the luc-120 construct and fxrlrxr expression plasmids were incubated with cdca or rifampicin. cdca induced oatp8 promoter activity -zfold, rifampicin -15fold, confirming that rifampicin transactivates the oatp8 promoter. to investigate whether other xenobiotics also activate the fxr element, huh7 cells cotransfected with the ir1-tk-luc construct and fxrlrxr expression plasmids were incubated with rifampicin, rifamycin, ru486, clotrimazol, phenobarbital or pcn. a 1.4fold (rifamycin) to 2.5fold (clotrima-201) induction of the ir1 element was seen in the presence of these xenobiotics. conclusions. the human oatp8 gene is induced transcriptionally by xenobiotics that represent prototypic ligands of the xenobiotic receptor pxr. however, activation does not occur through pxr but through activation of fxr bound to the ir1 element in the oatp8 gene promoter. the data thus indicate that pxr ligands such as rifampicin and clotrimazol can also activate fxr and thereby induce the fxr-regulated transporter gene oatp8. disclosures: may-britt becker -no relationships to disclose michael fried -no relationships to disclose diana jung -no relationships to disclose gerd a kullak-ublick -no relationships to disclose peter j meier -no relationships to disclose epidemiologie de population epi 106 seema sonnad -no relationships to disclose 428 preservation of canals of hering mitigates bile duct loss in liver graft rejection. ivlarius c van den michael angelis -no relationships to disclose jeffery cooper -no relationships to disclose erick edwards -no relationships to disclose richard b freeman jr -no relationships to disclose ann harper -no relationships to disclose abigail mithoefer -no relationships to disclose no relationships to disclose the data was not available. the ca 19-9, cea, and afp levels were obtained using standard commercially available assays results: out of a total of 108 patients with esld fifty-eight patients fulfilled the inclusion and exclusion criteria. of these, thirty-three patients had evidence of ascites and twenty-five did not. the etiology of liver disease in the two groups was similar. the mean levels of ca 19-9, cea and afp levels in patients with ascites were not significantly different from those without ascites (ca 19-9 48.92 % 65 furthermore, 15 out of these 58 patients with esld had levels -> 37 ulml, the upper limit of normal in this assay unitslml] vs 6.53 5 6.13 conclusions: ascites does not seem to make an impact on the serum levels of ca 19-9 chhaya hasyagar -no relationships to disclose savant mehta -no relationships to disclose 445 severe mitochondrial toxicity after liver transplantation in hiv-hcv coinfected patients liver transplantation (lt) may be the only potentially curative treatment available to these patients at this stage. however its feasibility and benefit has still to be established. severe recurrence of hepatitis c on the liver graft may complicate the post operative course as recently suggested by us and others. mitochondrial toxicity of highly active antiretroviral therapy (haart) may also play an active role on the liver graft of htv-hcv co-infected patients. we aimed to study this complication on the liver graft of hiv-hcv coinfected patients. patients and methods: between he had an history of episode of pancreatitis and haart therapy was azt-3tc-nelfinavir. at m10 post lt, microvesicular steatosis was noted. at m18 a low content of liver mtdna was found (mtdnalnuclear dna = 0.28). 3 other patients had microvesicular steatosis respectively at m2, m4 and m6 post lt. one of these 3 patients had low content of liver mtdna (mtdnalnuclear dna = 0.20). severe defect of complex iv of the respiratory chain was noted in 1 of these 3 patients. no deletion of mtdna was observed by southern blot or long range pcr conclusion: mitochondrial toxicity on the liver graft may become a major problem during post lt c o m e and could worsen graft lesions related to hcv recurrence on the liver graft in hiv patients daniel azoulay -no relationships to disclose henri bismuth -no relationships to disclose denis castaing -no relationships to disclose duclos-vallee -no relationships to disclose cyrille feray -no relationships to disclose michelle gigou -no relationships to disclose catherine guettier -no relationships to disclose philippe ichai -no relationships to disclose claude jardel -no relationships to disclose anne lombes -no relationships to disclose bruno roche -no relationships to disclose faouzi saliba -no relationships to disclose didier samuel -no relationships to disclose elina teicher -no relationships to disclose daniel vittecoq -no relationships to disclose 381a 457 identification of sodiumlphosphate cotransporter type iib marek nowicki, usc, los angeks, ca; jorge rakela, tomasz laskus, there is growing evidence that patients with chronic hepatitis c are more likely to have significant changes in their physical and mental well being, commonly manifested as fatigue and depression, than patients with liver disease of other etiology. recently published studies demonstrated also that hcv infection is associated with cognitive dysfunction. hepatitis c virus (hcv) was reported to replicate in monocyteslmacrophages and lymphoid cells. we have recently demonstrated that leukocytes carry hcv across the blood-brain barrier and we found hcv rna in the central nervous system (cns) in some infected patients (j virol 2002, 76, 10064-10068; 76, 600-608) . however, biological basis for neurocognitive abnormalities observed in hcv-infected patients remains unclear. aim: to determine the pattern of gene expression in cns in hcv-positive patients as compared to hcv-negative controls. material and methods we analyzed samples of brain tissue obtained at autopsy from 3 hcv-positive patients and 3 hcv-negative control patients. all were men of similar age, none was infected with hiv. all 3 hcv+ patients and 2 out of 3 controls had liver cirrhosis. only 2 deaths (one in each group) were liverrelated. the analysis of gene expression was conducted using three different techniques: differential display (genhunter inc), reverse northern, and microarray analysis (bd atlas plastic miroarray). reverse northern analysis was used for confirmation of differential display findings. analysis of microarray data was done using atlas image 2.01 (bd) and cluster 2.20 and treeview 1.60 (m.eisen; ucb). only those genes that were up or downregulated 1.8 times in reverse northern and/or microarray analysis were considered differentially expressed. results: the most striking finding was downregulation of mitochondria] oxidative phosphorylation genes in all hcv-infected patients as compared to controls; impairment of brain oxidative/ energy metabolism has been previously suggested to be the proximate cause of many disorders that impair mentation. another consistent finding in differential display and microarray analysis was downregulation of multiple ribosomal proteins genes and several genes involved in transcription regulation. these could indicate reduced metabolic activities perhaps secondary to deficiencies in oxidative phosphorylation. we also observed upregulation of several genes involved in immune response. there was upregulation of mhc class i and class i1 and several lymphokine receptors like interferon (alpha, beta), il-6, il-9, il-17 as well as lfa-1 ligand intracellular adhesion molecule icam-2 and ceacam1. furthermore, upregulation of cd8o and allograft inflammatory factor-1 gene (aif-1) suggested the presence of activated microglia cells and/or activated macrophages. conclusions: we found downregulation of oxidative phosphorylation genes and upregulation of genes involved in immune response in brains from hcv-positive patients when compared to hcv negative patients. our findings provide the likely substrate for neuropsychiatric symptoms and cognitive impairment associated with hcv infection. medicine, ehime, japan; emmett v schmidt, raymond t chung, massachusetts general hospital, boston , m a background/aims: we previously reported cell-based hcv replication using a novel binary expression system in which cells were transfected with a t7 polymerase-driven full length hcv cdna plasmid (pt7-flhcv-rz) and infected with vaccinia-t7 (pnas 989847). to circumvent vaccinia-induced cytotoxicity, we sought to determine whether replication-defective adenoviral vectors expressing t7 or cell lines stably transfected with t7 could support hcv replication. because vaccinia interferes with pkr function, we further sought to define the antiviral activity of interferon-ar (ifn) in these models. methods: 24 hours after transient transfection of cv-1 and huh7 cells with pt7-flhcv-rz, cells were treated with recombinant replication-defective adenovirus vectors expressing t7 polymerase (ad-t7pol). medium with or without 1000 iulml of ifn was changed at day 1 post-infection and every 2 days thereafter. cells were harvested on day 1, 2, 3, 5, 7 and 9 post-infection. for t7-stably transfected cell lines (huh-t7), we transiently transfected with pt7-flhcv-rz, then added ifn to selected cells in analogous manner. hcv positive and negative strand were measured by strand specific real-time rt-pcr. hcv core protein expression was assessed by western blotting. results: in the hcv replication system with recombinant adenovirus vectors and with t7 stable cell lines, no cytotoxicity was observed at 9 days. with pt7-flhcv-rz and ad-t'lpol, hcv positive and negative strand rna expression was strongest in the first 3 days post-infection and diminished thereafter, but were expressed throughout the 9 days. in the 2 days after adding ifn, hcv positive strand was significantly decreased (0.48520.213 vs. 0.162+0.017 in cv1, 0.123t0.051 vs. 0.035?0.012 in huh7, hcv/ gapdh copy ratio, pc0.05) than the samples without ifn. sustained expression of hcv rna and ifn inhibitory effect was also observed in t7-stable huh-t7 cell lines. pkr expression was strongly induced by ifn, suggesting that pkr is appropriately induced by ifn. conclusions: we have successfully substituted recombinant adenovirus vectors or t7 stable cell lines in our cell-based binary hcv replication system. in these systems, ifn inhibits hcv replication, and upregulates the pkr pathway. these improved binary systems are a durable and more authentic model for identification of host cellular processes critical to hcv replication. disclosures:jason blackard -no relationships to disclose raymond t chung -no relationships to disclose yoichi hiasa -no relationships to disclose norio horiike -no relationships to disclose yoshitaka kamegaya -no relationships to disclose morikazu onji -no relationships to disclose emmett v schmidt -no relationships to disclose the meld scoring system for the allocation of cadaveric donor liver for transplantation gives significant priority to cirrhotic patients with a small, t1 or t2, hepatocellular carcinoma. patients on the wait list were given adjusted meld scores equivalent to 3-month mortality rates of 15% and 40% for t1 and t2 lesions, respectively based on theoretical tumor doubling times. frequently these patients have compensated cirrhosis and hence their intrinsic meld score is lower, yet they are not candidates for surgical resection secondary to the underlying cirrhosis. to assess whether the additional meld points for hcc is valid, we sought to examine the impact of the meld scoring system on a single center's waiting list one year after implementation.methods: records of all patients undergoing liver transplantation (oltx) at uthsc-san antonio from feb 27,2002 through feb 27,2003 were retrospectively reviewed. pathology and radiology reports as well as data from unet were collated for analysis and review. results over a 1-year time period following implementation of the meld system, 126 patients underwent oltx at our center. of these 24 were excluded from analysis because of the following reasons: status 1 (lo), living-related recipient (6), pediatric non-tumor (8). the table below summarizes the pertinent findings. of the 102 remaining patients, 36 (35%) underwent oltx with suspected hcc (4 tl, 32 t2).none of the hcc patients had their meld score downgraded because of tumor progression (t2-t3). at transplant, 2 patients were found to have extra hepatic disease(one with known hcc and the other unknown) and the cases were aborted. sensitivity of radiological evaluation for the presence of hcc was 92.5% and specificity 94%. the calculated meld score at the time of listing for oltx and at the time of oltx was significantly lower for those patients thought to have an hcc. exception points for hcc gave those patients a significantly higher average meld score at the time of oltx than patients without hcc. patients wlo hcc had a aupper; true meld of +3.1 at the time of oltx compared to listing as opposed to hcc patients whose a-upper; true meld was -0.3. during the study period 34 patients (none with suspected hcc) died on the waiting list or were too sick to transplant while 215 new patients were listed compared to 24 dying the prior year with 163 new registrants (15.8% vs 14.7%). mean meld score of those dying during the study period was 22.6 8 (median 22.5) and mean time on the list was 284 421 days (median 99 days). of those patients dying. 16 had a meld score of 1 2 4 . conclusion: meld has effectively prioritized cadaveric donor livers to the sicker patients. however, patients with hcc are given too much priority as evidenced by all of the wait list deaths occurring in patients without hcc and lack of tumor progression (to t3) sufficient to loose meld exception points in those with hcc. likewise, the mean meld score of those dying was less than that given to patients with t1 lesions(22.6 vs. 24). the recent downward adjustments of meld exceptions to 20 and 24 for ti and t2 lesions, respectively, are reasonable. ongoing analysis will be needed to accurately place hcc patients in the current allocation system. there was no significant difference (p>0.05) in weight gain between the sexes, those with a bm1>30 pre-transplant and those with a bmi<30 pre-transplant or those on prolonged courses (>3 months) of steroids. weight gain was significantly greater (p<0.05) in patients aged over 50 compared to those under 50 and those transplanted for chronic liver disease compared with fulminant liver failure. a pre-transplant bmi>30 was a strong indicator that the patient would still have a bmi>30 at 3 years. there was no effect of immunosuppression on weight gain: corticosteroids were discontinued in 59.3% by 3 months and long term use of steroids was not associated with weight gain. weight gain was similar in those on cyclosporine and on tacrolimus.conclusions: a bm1>3o is common in the liver transplant population, but seems to unrelated to specific immunosuppression and may be more closely linked to lifestyle. most weight gain occurs after the first 6 months, and intervention with dietary and lifestyle advice at this point could be implemented to minimise the long-term morbidity and mortality risks associated with obesity. serum tumor marker estimations are often performed as a part of evaluation for liver transplantation. there is a paucity of information on the effect of esld on the levels of these tumor markers making interpretation difficult one tumor marker, ca-125, has been consistently reported to be elevated in patients with ascites from liver disease. purpose:-to compare the levels of serum ca 19-9, cea and afp in patients with esld with or without ascites.-to compare the serum ca 19-9 levels in patients with esld vs normal controls methods: all patients referred for liver transplantation between jan 2000 and dec 2002 at our center were included in the study. men with end stage liver disease (esld) develop a plethora of debilitating symptoms, including severe muscle wasting, that render many of these patients non-suitable for liver transplantation. esld-associated hypogonadism, i.e. testosterone deficiency, may, in part, be responsible for the development and progression of many of these incapacitating symptoms. in general, hypogonadism can be effectively treated with topical testosterone replacement (ttr), which avoids the first pass effect via the hepatic system in contrast to oral anabolic steroids. ttr has been shown to effectively improve muscle mass and overall well being in patients with hiv. however, little is known about the effects of ttr in men with esld. the aim of this study was to determine the potential benefits and safety of ttr in patients with esld and muscle wasting. method:the medical records of liver transplant patients treated for at least 3 months with testosterone gel 1% (5 grams per day) therapy for muscle wasting (mw) from january 2002 to march 2003 were reviewed. information collected included; demographics, albumin, pre-albumin, transferring, testosterone, estrogen, lh and fsh. drug safety results were also collected and included, acute cellular rejection, cholestasis, malignancy, and patient or graft loss. tumor markers for afp, ca19-9, cea, psa, prolactin levels and radiographic imaging was reviewed to rule out and potential malignancy. patients not treated with testosterone but with a similar clinical setting were compared (group 2). results: thirteen patients were identified with esld and mw, group 1 (n=8), group 2 (n=5). demographics were as follows: (group 1) 6 males, 2 females, and mean age 56 years; (group 2) 4 males, 1 female, mean age 53 years. disease etiology for group 1: (hcv (n=5), hcv/hiv (n=l), aih (n=l), cryptogenic (n=l)) group 2: hcv (n=4), hcvilaennec's (n=l). most patients in group 1 stated a subjective improvement in muscle strength, and overall wellbeing. in group 1, serum albumin levels increased from day 1, 30, 90 (2.18/2.7875/3.45 g/dl, respectively) while those in group 2 did not (2.30/2.16/2.0 g/dl) (p= 0.0002). similar results were seen for prealbumin (days 1, 30, 90) group 1 (10/18/22 mgldl) versus group 2 (10/8/6 mg/dl). in group 1, 7/8 patients received a liver transplant, while 1 remains listed for olt. in contrast, 3/5 patients in group 2 were transplanted while 2/5 died. two patients in each group were not listed at the time of evaluation due to extreme deconditioning. of those in group 1, one patient was transplanted and another patient remains listed for olt with an albumin greater than 3.5 gldl. this patient presently has no radiographical evidence of ascites. the two patients of group 2, who were not listed for lt due to extreme deconditioning, died on day 28 and 35, respectively. there was no rise in the tumor marker elevations in any of the patients. after transplantation, no patients in either group suffered from rejection or graft failure. ascites accumulation, as assessed by abdominal ultrasound imaging, appeared to be less in patients receiving ttr (group 1) compared to all 3 living patients in group 2, who had persistent ascites. one patient of the treatment group developed marked cholestasis 10 days after starting ttr and received a liver transplant 14 days later. prior to starting ttr, this patient underwent weekly large volume paracentesis and suffered from marked deconditioning and recurrent bouts of hepatic encephalopathy while maintaining a meld score of 8-9 points over a 6-month period. conclusion: our preliminary data suggest that ttr increases muscle strength (subjective measures), stimulates albumin synthesis and improves the outcome of olt. thus, ttr (testosterone gel 1%) appears to be of great benefit in patients with esld and muscle wasting. further studies are needed to determine the efficacy and safety of ttr in patients with esld. percutaneous ethanol injection (pei), radiofrequency tumor ablation (rf), and laser thermal ablation (lta) are percutaneous techniques used in the treatment of unresectable hcc. percutaneous seeding of neoplastic cells along the needle track is a rare complication of these tecniques, and it has been recently suggested to consider with caution these techniques as a "bridge" treatment in cirrhotic patients with hcc candidates to lt. the aim of this study was to verify whether percutaneous ablation treatments represent a risk factor for hcc recurrence in these patients. during the period 1990 -2002,32 patients (mean age 53 yrs) with hcc complicating liver cirrhosis previously treated with percutaneous ablation treatments underwent lt in 3 italian centers. among the 32 patients, 13 had monofocal hcc, 19 multifocal hcc. in the latter group, 5 had monolobar disease, 14 had bilobar disease. considering the 13 patients with monofocal hcc, 7 were treated with rf (associated with transarterial chemoembolization [tace] in one case), 4 with pei (associated with tace in one case), 2 with rf and pei (associated with tace in one case). among the 19 patients with multifocal hcc, 9 were treated with rf (associated with tace in four cases), 9 with pei (associated with tace in four cases), 1 with lta associated with tace. on the whole, 38 nodules were treated in the 32 patients. furthermore, 31 additional untreated hcc nodules were found at pathological examination of the explanted liver. among the 19 rf-treated nodules, 6 showed total necrosis, 12 partial necrosis, 1 absence of necrosis. among the 15 pei-treated nodules 4 showed total necrosis, 5 partial necrosis, 6 absence of necrosis. the 2 nodules treated with rf and pei showed total necrosis in one case and partial necrosis in the other one while the 2 nodules treated with lta, showed partial necrosis. the mean follow-up period was 35 months (range 3-130 months). five patients died during the follow up and the actuarial survival rate was 94% at one year, 85% at 3 and 5 years. three patients (9.4%) had post-transplant recurrence of hcc which was the cause of death in all cases; the diagnosis of tumor recurrence was made at 2 months from lt (peritoneal carcinomatosis), at 8 months from lt (bone), and at 9 months from lt (abdominal liymphnodes and bone), respectively. the 3 patients had all been treated with pei before lt and no one of them showed recurrence of hcc at the abdominal wall level. according to our results, percutaneous ablation techniques performed before lt do not seem significantly affect both survival and tumor recurrence rate in patients transplanted because of hcc complicating liver cirrhosis. in particular, no cases of recurrence at the abdominal wall level were observed in the overall series and no one case of hcc recurrence was observed in patients treated with rf alone or in combination. background: transjugular intrahepatic portosystemic shunt (tips) is valuable in the management of portal hypertension (phtn) in patients awaiting liver transplantation (olt). recurrent phtn after olt can be refractory to medical management and portosystemic shunting may be considered in rare situations, either as a bridge to retransplantation or as definitive therapy. in this report we review our experience and outcomes with tips after olt. methods: records of 309 primary adult olt recipients, between january 1995 and december 2003, were retrospectively reviewed. evidence of refratory post-olt phtn was noted. those who required tips were the subject of this review. demoraphics, indications for olt and tips, evidence of allograft dysfunction and outcomes after tips were described. results: during the study period 6 tips were placed in 6 patients at a mean of 13.5 months after olt (range 2-36 months). there were 3 males and 3 females, age 53.0l7.8 years. hepatitis c was the primary indication for olt in 5 and primary biliary cirrhosis in 1. indications for tips included refractory ascites (6), variceal bleeding (2) and various degree of associated hepatic vein outflow stenosis (3). five patients had resolution of phtn and 1 patient with refractory ascites had severe hepatic vein outflow stenosis and associated hepatitis c in the allograft. two patients required re-olt for recurrent hepatitis c. there were 3 deaths: liver failure 1 month after tips done in the setting of allograft dysfunction, 3 months after tips with subsequent re-olt and organ failure, and lung cancer 5 months after tips. bridging fibrosis was present in 3 patients, 2 needed re-olt and 1 died from liver failure while waiting for olt. currently, 3 patients are alive without evidence of phtn 3, 9 and 58 months after tips. conclusions: 1-tips effectively and safely controls phtn after olt. 2-tips in setting of a moderate to severe allograft dysfunction does not abrogate the need for re-olt and can be associated with a high mortality. 3-timely re-olt should be considered in the presence of fibrosis from recurrent hcv and phtn. 4-tips has been successful in the setting of mild-moderate hepatic vein outflow stenosis and phtn. introduction: biliary tract complications after liver transplantation (lt) are reported to occur in 13% to 35% of patients. the frequency of biliary infections is not well documented. therefore, we prospectivly obtained bile samples during diagnostic and or therapeutic endoscopic retrograde cholangiography (ercp) in all liver transplant patients from 0112001 to 1112002. methods: in 67 patients (mean age 53.6 years, range 24-73 years) a total of 172 ercp's were performed (2.57 ercflpatient). all patients had a lumen adapted, end-to-end biliary anastomosis. lt was performed between 3.2 months and 2.8 years before the intervention. cholangitis was defined as the presence of cholestasis with clinical and biochemical signs of infection not explained otherwise. only in 19 cases of 172 interventions, positive culture results were combined with clinical signs of cholangitis. the follwoing risk factors were identified stenosis of any type, plastic endoprothesis, choledocholithiasis and previous papillotomy. patients with these risk faktors had significantly higher incidence of positive bile cultures (77.6% vs 40%, p<o,ool; chi-square). in addition, they were more often infected with multiresistant bacteria (p<0.05). 49.3% were resistant to gentamycin, 44.0% to ceftriaxone. vancomycin (1.7% resistance) was the most effective antibiotic. imipenem (18.2%), ciprofloxacin (27.5%) and amoxicillin (28.8%) had intermediate incidence of resistant bacteria. conclusion: in patients with biliary tract complications after lt, biliary infection is common. clinical cholangitis, however, occurs predominantly in those with risk factors. gram-positive bacteria were more commonly demonstrated than gram-negative. cocolonisation with anaerobes andlor candida can occur. ciprofloxacin and amoxicillin seem to be appropriate antibiotics for prophylaxis. selection of an empiric antibiotic therapy for patient after liver transplantation with sings of cholangitis should be directed toward agents with a low incidence of resistance or a combination therapy. disclosures: thomas buratti -no relationships to disclose adolescent health related quality of life after liver transplantation. shikha s sundaram, katie neighbors, lisa amaruso, james sinacore, estella m alonso, northwestern university, chicago, il background: adolescence is an important developmental stage, during which children with chronic diseases begin to assume some of the responsibility for their own medical care. many children who received liver transplants as infants are now reaching adolescence and their unique health perceptions should be considered in providing appropriate healthcare. the aims of this study were to 1) quantify the health related quality of life (hrqol) in adolescent liver transplant recipients and compare this to a published healthy population, 2) compare parental ratings of adolescent hrqol to adolescent self-reports of hrqol, and 3) assess adolescent hrqol and it's relationship to disease specific disability. methods: a cross sectional mailing survey of liver transplant recipients between ages 11-18 years, surviving transplant by at least 1 year and receiving care at our center was conducted. primary caregivers of these patients were also recruited. adolescents completed the child health questionnaire, child form 87 (chq-cf87) and primary caregivers the child health questionnaire, parent form 50 (chq-pf50). demographic and medical data was collected using an institutional transplant database and standard demographic survey. a disease specific disability score was calculated using the liver transplant disability scale (ltds). mean scores for 12 chq-cf87 subscales, including general and mental health, physical and social functioning and self-esteem were calculated. these were compared to published normative values from a healthy population and mean chq-pf50 subscale scores. results: thirty-seven of 53 surveys mailed were received for a response rate of 69.8%. adolescent respondents had a mean age of 14.122.13 years, were 65% female and 81% caucasian. cadaveric donors accounted for 67.6% of transplants and 38% of patients were transplanted for biliary atresia. a majority (86.5%) of respondents had private insurance and 86.5% indicated mothers were their primary caregivers. after transplantation, adolescents had a mean global health score of 59.28518.47 as compared to mean normative score of 66.38214.62, p=0.031. they had higher scores than the normative population for role behavioral, 94.89211.29 vs. 86.542 21.45, p=o.ool and mental health, 77.53z10.81 vs. 72.65215.95, p=0.021. all other scores were similar between the 2 groups. significant positive correlations between adolescent and parental scores were observed in relation to physical functioning, (r =0.64, p<0.0005), self-esteem (r = 0.72, p < 0.0005) and mental health (r = 0.59, p<0.0005). the remaining sub-scales lacked statistically significant correlation. an inverse relationship between inpatient hospitalization during the previous 6 months and mean global health score was observed 0 inpatient days had a mean of 76214.77,l-4 inpatient nights had a mean of 65235.5 and greater than 4 inpatient nights 38.3243.1. there were no other significant correlations between subscale scores and ltds scores. conclusion: adolescent liver transplant recipients report their hrqol as equal or better than the normative population. parents may be adequate proxies for some, but not all domains of adolescent hrqol in this population. were quantitated from wild type and fxrnull mice. results:transfection of the moatp4-pgl3 into hepg2 cells led to a 5.5fold ( 2 1.0 ) increase in reporter gene activity over vector controls. co-transfection with the bile acid receptor fxr and addition of its ligand, chenodeoxyacholic acid (cdca), resulted in a further 6.25 ( t 0.35 ) -fold increase suggesting fxr-mediated transactivation.fxrlrxr cotransfection and addition of ligands, cdca and 9-cis retinoic acid decreased transactivation suggesting rxr-mediated antagonism ( jbc, 278: 10028,'03 ). hnf-1 a also transactivated moatp4 promoter as shown previously. mutation of ir-1 sequence in moatp4 promoter abolished fxr-transactivation. emsa analysis using rat liver nuclear extracts showed specific binding of nuclear proteins to moatp4 fxre which was supershifted by fxr and rxr antibodies. mrna levels for moatp4 in fxrnull mice were reduced by 48.3 % compared to wild-type mice ( 0.612 in wild-type vs 0.296 infxr-null mice). conclusion: based on the above data, we suggest that moatp4 is regulated by bile acids by binding to fxr via ir-1 in its promoter. , and g1249a encoded for amino acid changes i1173f, a1450t, and v4171, in the third membrane spanning domain, the second nucleotide binding domain, and the second membrane spanning domain, respectively. the full-length coding regions of the reference mrp2 and three mutants sequences were inserted into the baculovirus genome. sf9 insect cells were infected with the baculovirus stocks, and membranes were isolated from the infected cells expressing mrp2 or the mutants. atpase activity was measured by determining release of inorganic phosphate by a colorimetric assay. atp-dependent transport of 3h-e23g (estradiol-p-(3p-d-glucuronide)) or 3h-e217g (estradiol-p-(17p-d-glucuronide)) was determined by uptake into inside out membrane vesicles collected on filters and counted by liquid scintillation. results: the total expression levels of mrp2 and the three mutants in the infected sf9 cells were comparable by densitometry of western blots of three levels of each protein, indicating total expression in sf9 cells was not influenced by the mutations. mrp2 atpase activity was significantly stimulated by the uricosuric probenecid lmm, the cholestatic 250pm, and the mild choleretic e23g 1mm. although the control and stimulated at-pase activities of the v417i mutant were not different from mrpz, the i1173f mutant had significantly less baseline and less stimulated atpase activities, indicating impaired function. a1450t had less probenecid-stimulated atpase activity, but activity stimulated by e217g and e23g was not different from mrp2. ursodiol saturably stimulated atpase activity of mrpz by 520% with km =72138pm. although the three mutants transported 3h-e23g with affinities not significantly different from mrp2 (km=122223 pm), the vmax of i1173f was only 5.1% (15414 pmollmglmin) and the vmax of v417i was 157% (4701251 pmollmglmin) compared to the vmax of mrpz (29912186 pmollmglmin); the vmax of a1450t was not different (2967287 pmollmglmin). ursodiol activated transport of 3h-e23g (60nm) with a peak effect of 950% at 100pm; higher ursodiol concentrations (up to 1mm) returned transport to near control values. mrpz-mediated atp-dependent transport of 3h-e217g (44nm) in the presence of ursodiol (1oopm) for the mutants differed from mrp2 (lo%, 67%, and 140% for i1173f, a1450t, and v4171, respectively). conclusions: ursodiol markedly activated mrp2 transport of e23g. although ursodiol has several anticholestatic effects, these data show its direct effect on transport of mrpz substrates, and may suggest activation of mrp2 as a target in the treatment of cholestatic liver disease. the mrp2 mutants showed several important differences in activity, including changes in transport vmax, and transport in the presence of ursodiol, and activation of atpase activity by ursodiol, probenecid, and the mrp2 substrates e217g and e23g. further characterization of these mrp2 mutants and others will lead to a better understanding of mrp2 structure and function and may yield new therapeutic approaches. also, although e217g is an established mrp2 substrate, these data show that its noncholestatic isomer e23g is also an mrpz substrate, implicating mrp2 in the disposition of other estrogen metabolites, which can be interconverted to cholestatic compounds like e217g. argentina; nicholas f larusso, mayo medical school, clinic and foundation, rochester, m n previous work from our laboratory is consistent with an important role for aquaporins (aqps), a family of water channel proteins, in canalicular bile secretion principally via agonist-induced exocytic insertion of aqps into the hepatocyte canalicular membrane. to further define the pathways and molecular mechanisms for water movement across hepatocytes, our aim here was to directly assess osmotic water permeability (pf) and activation energy (ea) in basolateral (blmv) and canalicular membrane (cmv) vesicles using stopped-flow spectrophotometry. scattered light intensity was used to follow changes in vesicle volume in response to gradients induced by sucrose solutions of different osmolarities. results: the time course of scattered light for blmv and cmv fit a single-exponential function. in blmv, pf was 10824 pm.sec-' (25 oc) with an ea of 7.7 kcallmol; in cmv, pf was 8615 pm.sec-' (25 oc) with an ea of 8.0 kcallmol. the aqp blocker, dimethylsulfoxide, significantly inhibited the pf of both basolateral (8124 pm.sec-l; -25%) and canalicular (59?4 pmsec-'; -30 %) vesicles consistent with the presence of aqps in both vesicle populations. experimental data for cmv from dibutyryl camp-treated hepatocytes fit best to a double-exponential curve implying the presence of two functionally distinct cmv populations; one population had pf and ea values similar to those of cmv from untreated hepatocytes and the other population had a very high pf (6552135 pm.sec-l, 25 oc) and very low ea (2.8 kcallmol). dimethylsulfoxide completely inhibited the high pf value in this second vesicle population. in contrast, the pf of blmv was unaltered by camp treatment of hepatocytes. conclusions: our data are consistent with the notion that the canalicular but not the basolateral membrane becomes more permeable to water after a secretory stimulus due to the insertion of aqp molecules. the data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation. disclosures: ariel j caride -no relationships to disclose sergio a gradilone -no relationships to disclose bing q huang -no relationships to disclose key: cord-022754-ehq9qnoo authors: nan title: liver date: 2012-07-25 journal: canine and feline gastroenterology doi: 10.1016/b978-1-4160-3661-6.00061-4 sha: doc_id: 22754 cord_uid: ehq9qnoo nan the basic elements of the biliary tract are the hepatic canaliculi, bile ductules, intralobular ducts, interlobular ducts, hepatic ducts, cystic duct, gallbladder, common bile duct, and the pancreaticobiliary sphincter (of oddi). 2 there are many variations on this central theme, the most important of which are (a) the pancreaticobiliary sphincter is a common physiologic and anatomical channel at the duodenal papilla in the cat 3 and (b) there are many anatomic variations in the feline gallbladder, from single gallbladder to bilateral gallbladders, body duplication, fundic duplication, complete duplication, septate, and y-shaped gallbladder. 4 hepatocytes account for 60% to 80% of the liver cell mass (see table 61 -1) and contribute to a wide range of metabolic activity, including carbohydrate, protein, lipid, nucleic acid, porphyrin, metal, vitamin, glutathione, hormone, and xenobiotic metabolism; coagulation factor synthesis; biliary secretion; and immune surveillance. 1, 5 hepatocytes have an eosinophilic cytoplasm reflecting numerous mitochondria, and basophilic stippling caused by large amounts of rough endoplasmic reticulum and free ribosomes. hepatocyte nuclei are round with dispersed chromatin and prominent nucleoli. anisokaryosis is common and often reflects various degrees of polyploidy, a normal feature of more than 50% of hepatocytes. the average life span of the hepatocyte is 5 to 6 months reflecting their ability to regenerate. hepatocytes are organized into plates separated by vascular channels (sinusoids), an arrangement supported by a reticulin (collagen type iii) network. the sinusoids have a discontinuous, fenestrated endothelial cell lining. the endothelial cells have no basement membrane and are separated from the hepatocytes by the space of disse, which drains lymph into the portal lymphatics. hepatocytes are supported by a number of other cell types, which account for 40% of the liver cell mass. representing 3% to 10% of liver cell mass, cholangiocytes are also known as biliary epithelial cells. 6 they secrete water, bicarbonate, and cations into the bile in the physiologic state, but they may also participate in the immune response as antigen-presenting cells in disease states. the biliary tract is a convergent system of canals that begins in the canaliculi, followed by the bile ducts, and ending with the common bile duct. bile secretion depends on the function of membrane transport systems in hepatocytes and cholangiocytes and on the structural and functional integrity of the biliary tract. the hepatocytes, constituting the most abundant liver cell population, generate the so-called primary bile in their canaliculi. biliary canaliculi are blind tubular structures, with a very high surface-to-volume ratio that by means of osmotic gradients favors the formation of bile flow. cholangiocytes, which constitute 3% to 10% of the liver cells, modify the canalicular bile by secretory and reabsorptive processes as bile passes through the bile ducts, and they are responsible for approximately 30% of bile volume. in contrast to hepatocytes, where secretion is constant and poorly controlled, cholangiocyte secretion is broadly regulated. 5, 6 the immunoglobulin family, without which the liver cannot clear complement system-coated pathogens. in disease states, kupffer cells contribute to the pathology of ethanol and other toxic principles through production of inflammatory mediators, activation of toll-like receptors, and elaboration of tumor necrosis factor (tnf-α). 7 kupffer cell activation is responsible for early ethanol-induced liver injury, common in chronic alcoholics. ethanol activates the toll-like receptor 4 and cd14, receptors on the kupffer cell that internalize the endotoxin lipopolysaccharide. internalization activates the transcription of tnf-α and production of superoxide (a prooxidant). tnf-α then enters the stellate cell in the liver, leading to collagen synthesis and fibrosis. fibrosis eventually causes cirrhosis or loss of function of the liver (see the role of the stellate cell, which is discussed in "stellate cells" section that follows). hepatic stellate cells (hscs) (also referred to as vitamin a-storing cells, lipocytes, interstitial cells, fat-storing cells, and ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50% to 80% of vitamin a in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. [7] [8] [9] [10] in physiologic conditions, these cells play pivotal roles in the regulation of vitamin a homeostasis. in pathologic conditions, such as hepatic fibrosis or liver cirrhosis, hscs lose vitamin a and synthesize a large amount of ecm components, including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. the morphology of these cells also changes from that of the star-shaped stellate cell to that of the fibroblast or myofibroblast. hscs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. 11 activation of hscs, a key event in liver fibrosis, is caused by diminished adipogenic transcription. 12 wnt signaling inhibits antiadipogenic activation of hscs and liver fibrogenesis; wnt antagonism inhibits hsc activation and liver fibrosis. 9 also known as natural killer (nk) cells or large granular lymphocytes, pit cells represent 1% of liver cell mass, and serve as part of the immune surveillance mechanism in the hepatic sinusoids. pit also known as browicz-kupffer cells or stellate macrophages, these cells represent 2% to 5% of the liver cell mass, and are specialized macrophages localized to the sinusoids as part of the mononuclear phagocyte system. kupffer cells begin their development in the bone marrow with the genesis of promonocytes and monoblasts into monocytes, and then on to peripheral blood monocytes, completing differentiation into kupffer cells within the liver. in health, kupffer cells are involved in the metabolism of erythrocyte hemoglobin. during perfusion of the liver, senescent red blood cells are phagocytized by the kupffer cells, and the hemoglobin molecule is further metabolized into its component parts. globin chains and amino acids are reutilized; the iron-containing portion of heme is removed, transported, and stored; and heme is further oxidized into bilirubin, conjugated with glucuronic acid within hepatocytes, and secreted into the bile. kupffer cells also express a complement receptor of progenitor cells have multilineage potential and similar characteristics to stem cells. the late progenitor cells have differentiated further and produce progeny in only a single lineage. although they divide rapidly, they are capable of only a short-term tissue reconstitution and they do not self-renew. 15 early studies in hepatocyte turnover and liver regeneration showed that the parenchymal cell, the hepatocyte, was the primary and only cell involved in tissue renewal. however, new studies of liver regeneration, hepatocarcinogenesis, liver transplantation, and various cell lines show that a variety of cell types participate in maintaining hepatocyte number and mass. recent studies indicate the presence of both intrahepatic and extrahepatic stem/progenitor cell populations that serve to maintain the normal organ and to regenerate damaged parenchyma in response to a variety of insults. the intrahepatic compartment most likely derives primarily from the biliary tract, particularly the most proximal branches, that is, the canals of hering and smallest ductules. the extrahepatic compartment is at least in part derived from diverse populations of cells from the bone marrow. embryonic stem cells are considered as a part of the extrahepatic compartment. 16 the precise role(s) of each of these individual cells remains to be determined, but it is clear that in the aggregate they confer the vast regenerative capacity of the liver . the liver is involved in many aspects of intermediary metabolism. 1 the liver is at the center of carbohydrate metabolism through its role in maintaining normoglycemia. glucose is the primary energy source for most mammalian cells, and its metabolism is tightly regulated to guarantee that a sufficient supply is available to glucosedependent organs, particularly the brain. glucose can be made available from two sources: absorption of dietary glucose from the intestine, and release of glucose from organs such as the liver and kidney. early in fasting, the majority of endogenous glucose is generated by glycogenolysis where glycogen in the liver is converted to glucose-6-phosphate under the regulation of debranching enzyme, hepatic glycogen phosphorylase, and phosphorylase kinase. with more prolonged fasting, endogenous glucose is generated by gluconeogenesis from certain substrates such as amino acids, lactate, and glycerol. both processes generate glucose-6-phosphate, which must then be dephosphorylated in order to transport glucose out of the cell. • early fasting: glycogen → glycogenolysis → glucose → normoglycemia • prolonged fasting: amino acids → gluconeogenesis → glucose → normoglycemia the enzyme responsible for the dephosphorylation of glucose-6phosphate is glucose-6-phosphatase-α. alterations in quantity, location, or activity of glucose-6-phosphatase, such as those seen in type 1 glycogen storage diseases, effectively result in a lack of all endogenous glucose production and severe hypoglycemia develops during periods of fasting. 17 the liver is an important site of protein metabolism. amino acids and proteins absorbed from the intestine or produced in the body are delivered to the liver. the liver deaminates amino acids and cells belong to the group of sinusoidal cells, together with kupffer, endothelial, and fat-storing (stellate) cells. pit cells use the fasl fas ligand (fasl) and perforin/granzyme pathway to kill target cells. fasl on effector cells binds the fas that is present on the target cell membrane, which results in oligomerization of fas and activation of caspase 8. perforin and granzymes, of which granzyme b is the most potent, reside in granules of the cytotoxic lymphocytes and are released by exocytosis. intracellular delivery of granzyme b results in the initiation of the caspase cascade by proteolytic activation of caspase 3, either directly or through a mitochondrial-dependent pathway. caspases play a central role in the execution of apoptosis. lymphocyte recruitment from the circulation into tissue is dependent on the ability of the lymphocyte to recognize and bind molecules on the endothelial cell surface that promote transendothelial migration. a multistep model of leukocyte adhesion to vascular endothelium has been described and is broadly applicable, although the details of the signals involved differ between tissues. 13, 14 in a generally accepted model, tethering or rolling receptors expressed on endothelial cells capture free-flowing leukocytes. these receptors may be either selectins or members of the immunoglobulin superfamily. once captured, the leukocyte can receive activating messages presented by endothelial cells in the form of chemokines that activate specific g-protein-coupled receptors on the leukocyte surface. occupancy of these receptors triggers a cascade of intracellular signals that results in the presentation of high-affinity integrin receptors on the leukocyte surface that bind to immunoglobulin family of counterreceptors on the endothelium to promote leukocyte arrest on the vessel wall. in the presence of the appropriate migratory signals the leukocyte will migrate across the endothelium into tissue, where it follows a hierarchy of chemotactic signals toward the focus of inflammation. representing 2% to 5% of the liver cell mass, smooth muscle cells are located primarily in the hepatic artery and portal vein and their tributaries, and serve primarily to regulate the hepatic microcirculation. hepatic fibrosis is a common outcome of hepatic injury in the dog. activated fibroblasts that develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: (a) portal or septal myofibroblasts, (b) interface myofibroblasts, and (c) the perisinusoidally located hscs. it is difficult to arrive at a universally applicable definition of a stem cell because some of the defined properties of a stem cell can be exhibited by the stem cells in some tissues or organisms but not in others. in spite of that, a generally acceptable consensus defines a stem cell as an undifferentiated cell that has capacity to self-renew, for production of progeny in at least two lineages, for long-term tissue repopulation after transplantation, and for serial transplantability. in addition, stem cells exist in a mitotically quiescent form and clonally regenerate all of the different cell types that constitute the tissue in which they exist. they can undergo asymmetrical cell division, with production of one differentiated (progenitor) daughter and another daughter that is still a stem cell. the offspring of stem cells are referred to as progenitor cells, also named as transit amplifying cells and therefore cannot be serially transplanted, and are classified as early and late. the early progenitor or stem/ either removed from the circulation by the liver or undergo further transformation by lipoprotein lipase and/or hepatic lipase to form intermediate-density lipoproteins and ldls. ldls, which are relatively depleted of triglyceride and enriched in cholesteryl esters and phospholipid, circulate in the blood and bind to specific ldl receptors that are widely distributed throughout tissues in order to deliver cholesterol. hdls produced by the liver play an important role as donors and acceptors of apo c, apo e, and various lipids from other lipoproteins in the circulation. reverse transport. hdls play an important role in the reverse transport of cholesterol from the periphey to the liver. lecithin cholesterol acyl transferase esterifies hdl cholesterol and cholesteryl esters move to the core of the hdl molecule to allow more free cholesterol to be absorbed into the particle. continued absorption of free cholesterol and subsequent esterification by lecithin cholesterol acyl transferase leads to the formation of the larger, cholesteryl ester-rich hdl2s. hdl2 molecules continuously acquire cholesteryl esters, resulting in the formation of the hdl1 molecules. on hdl1, cholesteryl esters are transferred from tissues to the liver for disposal or reuse, and not to ldl or vldl molecules (as in humans), which transfer cholesterol to peripheral tissues. this function of hdl1s may account for the lower incidence of atherosclerotic disorders in dogs compared with humans. 23, 24 nucleic acids pyrimidine biosynthesis is one of the classic roles of the liver in nucleic acid metabolism. more recently, micrornas have been impugned in the normal development and regeneration of the liver, as well as in hepatic pathology. micrornas are small noncoding rnas that regulate both messenger rna and protein expression of target genes, which results in alterations in messenger rna stability or translation inhibition. micrornas influence at least one-third of all human transcripts and are known regulators of various important cellular growth and differentiation factors. micrornas recently emerged as key regulatory molecules in chronic liver disease. 25 porphyrins porphyrins are intermediates of the heme biosynthetic pathway. porphyrins are found in hemoglobin, myoglobin, cytochromes, catalase, and peroxidase enzyme. the liver and biliary tract serve as an excretory route for the porphyrins. the liver stores iron, which can be toxic in excessive amounts (hemochromatosis). the amount of iron in the body is largely determined by regulation of its absorption in the upper small intestine. iron is stored intracellularly as ferritin in a number of tissues, with the liver having a large storage capacity. when the capacity of the liver is exceeded, iron accumulates as hemosiderin. the liver incorporates copper into specific copper proteins such as cytochrome c oxidase, mitochondrial monoamine oxidase, and ceruloplasmin. mobilization of copper from hepatocytes takes place by at least two mechanisms: ceruloplasmin and bile secretion. cholestatic liver disease is associated with secondary copper retention, which may then induce hepatocyte injury. 26, 27 vitamins the liver is importantly involved in vitamin metabolism. the liver produces bile for absorption of fat-soluble vitamins (a, d, e, k), and the liver is an important site for vitamin storage. converts them to carbohydrates and lipids. [18] [19] [20] [21] deamination produces α-keto acids, which can be metabolized for energy or used for synthesis of monosaccharides and fatty acids. 20 the liver synthesizes amino acids from intermediates of carbohydrate and lipid metabolism by amination and transamination. 21 examples of amino acid transaminations include: • alanine + α-ketoglutarate ↔ pyruvate + glutamate • aspartate + α-ketoglutarate ↔ oxaloacetate + glutamate the liver synthesizes many proteins, including albumin and fibrinogen, most α globulins, and some of the β globulins. prothrombin and clotting factors v, vii, viii, ix, and x are produced in the liver, as well as ceruloplasmin, ferritin, and many serum enzymes. lipid metabolism and transport is organized into three basic transport systems: (a) exogenous transport, which is associated with the metabolism of exogenous (dietary) lipids, (b) endogenous transport, which is associated with the metabolism of endogenously produced lipids, and (c) reverse transport, which is associated with the transport of lipids from the periphery (e.g., skeletal muscle, adipose, connective tissue) to the liver. exogenous transport. triglyceride is the major dietary lipid, along with cholesterol, phospholipids, and fat-soluble vitamins. 22 the digestion of dietary lipids begins in the proximal gi tract with the action of lingual and gastric lipases, and is completed in the small intestine with the actions of pancreatic lipase, cholesterol ester hydrolase, and phospholipase a 2 . lipid digestion and absorption is more complicated than carbohydrate and protein digestion and absorption because of lipid solubility characteristics, and involves emulsification of lipids by bile salts, hydrolysis by pancreatic lipase and colipase, solubilization of fatty acids and monoglycerides into mixed micelles, absorption, reesterification, chylomicron formation, and transport into the intestinal lymphatics or portal capillaries. chylomicrons containing short-and long-chain triglycerides, and the newly incorporated b-100 apoprotein, are preferentially absorbed into the intestinal lymphatics where they are transported into the cisterna chyli, thoracic duct, and systemic circulation where they acquire apolipoproteins c and e from circulating high-density lipoproteins (hdls). apolipoprotein (apo) c-ii activates lipoprotein lipase in the capillary beds of adipose and skeletal muscle, where they are stored as is or hydrolyzed into free fatty acids, β-monoglyceride, and glycerol. the cholesterol-rich remaining particles (now referred to as chylomicron remnants), return apo c-ii molecule to hdl and are recognized by specific hepatic apo e and apo b-100 receptors that rapidly remove them from the circulation by endocytosis. the cholesterol found in chylomicron remnants can be used in very-low-density lipoprotein (vldl), lipoprotein synthesis, bile acid formation, or cholesteryl storage. endogenous transport. while chylomicrons are the apoprotein responsible for transport of dietary lipids, vldls, intermediatedensity lipoproteins, low-density lipoproteins (ldls), and hdls are instead involved in the metabolism of endogenously produced lipids. triglycerides and cholesterol produced by the liver combine with phospholipids, apo b-100, and apo b-48 to form vldls. when secreted from the liver, vldls acquire the apo c and apo e from hdl. vldl apo c-ii activates lipoprotein lipase located in the capillary beds, where once again triglyceride hydrolysis takes place with the production of free fatty acids and glycerol. the vldl molecules remaining after hydrolysis of vldl triglycerides are intestinal bacteria to produce the secondary bile acids, deoxycholic acid, and lithocholic acid. 2, 5, 28 prior to secretion, bile acids are conjugated with taurine and/or glycine to form tauro-and glycoconjugated bile salts (see figure 61 -3, c). conjugation lowers the pk a to well below the physiologic range of biliary and intestinal ph, and conjugated bile acids become ionized anions (referred to as bile salts) rather than undissociated bile acids. in the ionized form, they are less likely to be absorbed by the small intestine and so maintain a higher intraluminal concentration appropriate for emulsification, digestion, and absorption of lipids. dogs and cats conjugate primarily with taurine. dogs can convert to glycine conjugation if taurine is deficient, but cats cannot. cats are obligate taurine conjugators, and have an essential dietary taurine requirement. 2, 5, 28 bile salts are amphipathic molecules with polar and nonpolar domains imparting two important functions. bile salts have an initial detergent effect on fat particles in food permitting the breakup of fat globules into smaller sizes. this is the initial emulsification phase of bile salts that facilitates intraluminal lipid hydrolytic digestion. bile salts further assist in the absorption of fatty acids, monoglycerides, cholesterol, and other lipids through the formation mixed micelles. these micelles serve to transfer digested lipids across the unstirred layer of the mucosa. following emulsification and micellarization of fat, most of the secreted bile salts are transported along the gi tract to the ileum where they are absorbed into ileal enterocytes and portal blood flow via na + -bile salt cotransporters. 2, 28 the liver plays an important role in maintaining hemostasis. the liver produces procoagulant, anticoagulant, and fibrinolytic proteins, and also removes normal and abnormal clotting factors from the circulation. 29 hepatocytes synthesize most of the clotting factors including clotting factor i (fibrinogen), ii (prothrombin), v, vii, ix, x, xi, and xiii. the site of biosynthesis of factor viii remains controversial, but it is probable that the liver plays an important role in this factor, too. the liver is also responsible for the activation of the vitamin k-dependent factors ii, vii, ix, and x and protein c. in addition to the production and activation of coagulation factors, the liver is also essential for the clearance of activated coagulation water bilirubin -vitamin a is stored in both stellate cells and hepatocytes. approximately 95% of total body vitamin a is stored in the liver, representing a 1-to 2-year supply. the liver continues to release vitamin a to maintain normal blood concentrations despite reductions in its content. liver and plasma vitamin a concentrations are reduced by malnutrition, liver disease, and intestinal malabsorption, but signs of deficiency do not appear until abnormalities become severe. fat-soluble vitamins a, d, e, and k require normal bile secretion for absorption. vitamin k is particularly essential for synthesis of the prothrombin-complex clotting factors. water-soluble vitamins, with the exception of vitamin b 12 (cobalamin), are readily absorbed from the small intestine. these vitamins are used primarily as coenzymes in metabolic processes. vitamin phosphorylation, occurring primarily in hepatocytes, is required to produce some coenzymes. thiamine is phosphorylated to thiamine pyrophosphate, for example, primarily in the liver and kidney. nicotinic acid is a precursor in pyridine nucleotide synthesis, and an initial step in its conversion is nicotinamide synthesis in the liver. pyridoxine is phosphorylated to its active form in the liver, as is the transformation of pantothenic acid to coenzyme a. folic acid is converted to its active form in the liver. large amounts of all water-soluble vitamins except vitamin c are stored in the liver. glutathione is synthesized in most if not all mammalian cells. the liver is particularly active and has relatively high levels of glutathione. glutathione performs a variety of physiologic and metabolic functions, including thiol transfer reactions that protect cell membranes and proteins; thiol-disulfide reactions involved in protein synthesis, protein degradation, and catalysis; reduction of capacity; detoxification of hydrogen peroxide, organic peroxides, free radicals, and foreign compounds; and metabolism of various endogenous compounds. seen in dogs with chronic hepatitis plus cirrhosis, which may be a result of reduced synthesis rather than increased consumption of coagulation factors. numerous foreign compounds, including drugs, are so hydrophobic that they would remain in the body indefinitely were it not for hepatic biotransformation. cytochrome p450 (p450 or cyp) comprises a superfamily of enzymes that catalyze oxidation of a variety of xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens, as well as endobiotic chemicals including steroids, fatty acids, prostaglandins, and vitamins. the cytochrome p450 enzymes in families one to three mediate 70% to 80% of all phase i-dependent metabolism of clinically used drugs and participate in the metabolism of a huge number of xenobiotic chemicals. there are 57 known active p450 genes and 58 pseudogenes in the human genome. with 54 active genes, dogs are phylogenetically closest to the human. although there are many similarities between dogs and humans, there also are many important differences. [30] [31] [32] [33] dogs present an products and the production of clotting factor inhibitors, such as antithrombin and α 1 -antitrypsin, as well as fibrinolytic proteins like plasminogen. in liver disease, factor and inhibitor synthesis and clearance of activated factors in both the coagulation factors and fibrinolytic system may be impaired. the extent of coagulation abnormalities depends upon the degree of disturbed liver function. 29 patients with hepatic failure may present with the entire spectrum of factor deficiencies and may even develop disseminated intravascular coagulation. in a study of 42 dogs with histologically confirmed liver disease, one or more coagulation abnormalities were found in 57% of dogs with liver disease. 29 activated partial thromboplastin time was significantly prolonged in dogs with chronic hepatitis with or without cirrhosis. mean platelet numbers, antithrombin, and factor ix activity were significantly lower in dogs with chronic hepatitis with cirrhosis, compared to dogs with other hepatopathies. d-dimers were not significantly increased in any group. only three dogs, all with different histologic diagnoses, satisfied the criteria for disseminated intravascular coagulation. hemostatic abnormalities were primarily where nh3 = ammonia; adenosine triphosphate = adenosine triphosphate; adp = adenosine diphosphate; p i = inorganic phosphate; and amp = adenosine monophosphate. mineralocorticoids (aldosterone), glucocorticoids (cortisol, corticosterone), and sex steroids (androgens, estrogens, progesterone) are metabolized by the liver. changes in the concentrations of total and free cortisol and of the binding capacity of corticosteroid-binding globulin have been reported in canine liver disease. as a consequence of hypercortisolemia, dogs with liver disease and hepatoencephalopathy have clinical and biochemical characteristics of pdh, including polyuria, high basal cortisol levels, and α-melanotropin. 37, 38 chronic hypercortisolism is associated with impaired osmoregulation of the release of vasopressin and inadequate urinary concentration. 39 the multiple physiologic functions of the liver require an immune response that is locally regulated. pathogenic microorganisms must be efficiently eliminated, while the large number of antigens derived interesting challenge in the assessment of p450-mediated drug-drug interactions because most of the enzymes have not been completely characterized, diet and aging induce significant changes in gene expression, and dogs are often treated off-label with a number of human drugs with little idea of risk for drug-drug interaction. drug metabolism takes two general forms: phase i metabolism (modification reactions) and phase ii metabolism (conjugation reactions). phase i metabolism typically subjects a drug to oxidation or hydrolysis. it involves the cytochrome p450 (cyp) enzymes, which facilitate reactions that include n-, o-, and s-dealkylation; aromatic, aliphatic, or n-hydroxylation; n-oxidation; sulfoxidation; deamination; and dehalogenation. phase ii metabolism conjugates the drug to hydrophilic substances, such as glucuronic acid, sulfate, glycine, or glutathione. phase i metabolism usually precedes phase ii metabolism, but this is not always the case. 34 the liver is an important site of drug toxicity and oxidative stress because of its proximity and relationship to the gi tract. seventyfive percent to 80% of hepatic blood flow comes directly from the gi tract and spleen via the main portal vein. portal blood flow transports nutrients, bacteria and bacterial antigens, drugs, and xenobiotic agents absorbed from the gut to the liver in a more concentrated form. drug-metabolizing enzymes detoxify many xenobiotics but might activate the toxicity of others. hepatic parenchymal and nonparenchymal cells may all contribute to the pathogenesis of hepatic toxicity. the toxicity of drugs can be considered in five contexts: on-target toxicity, hypersensitivity and immunologic reactions, off-target pharmacology, bioactivation to reactive intermediates, and idiosyncratic drug reactions. 35, 36 ammonia ammonia is an important by-product of amino acid metabolism. organisms that cannot easily and quickly remove ammonia usually have to convert it to some other substance, like urea or uric acid, which are much less toxic. insufficiency of the urea cycle occurs in from the gi tract must be tolerated. the liver favors the induction of tolerance rather than the induction of immunity. although hepatocytes constitute the major cell population of the liver, direct interaction of hepatocytes with leukocytes in the blood is unlikely. sinusoidal endothelial cells, which line the hepatic sinusoids and separate hepatocytes from leukocytes in the sinusoidal lumen, and kupffer cells, the resident macrophage population of the liver, can directly interact with passenger leukocytes. in the liver, clearance of antigen from the blood occurs mainly by sinusoidal endothelial cells through very efficient receptor-mediated endocytosis. liver sinusoidal endothelial cells constitutively express all molecules necessary for antigen presentation (cd54, cd80, cd86, major histocompatibility complex [mhc] classes i and ii, and cd40) and can function as antigen-presenting cells for cd4 + and cd8 + t cells. 40, 41 thus, these cells probably contribute to hepatic immune surveillance by activation of effector t cells. antigen-specific t-cell activation is influenced by the local microenvironment. this microenvironment is characterized by the physiologic presence of bacterial constituents such as endotoxin and by the local release of immunosuppressive mediators such as interleukin-10, prostaglandin e 2 , and transforming growth factor-β. 42 regeneration liver regeneration after partial hepatectomy is a very complex and well-orchestrated phenomenon. it appears to be carried out by the participation of all mature liver cell types. 43, 44 the process is associated with signaling cascades involving growth factors, cytokines, matrix remodeling, and several feedbacks of stimulation and inhibition of growth related signals. 45, 46 the liver manages to restore any lost mass and adjust its size to that of the organism, while at the same time providing full support for body homeostasis during the entire regenerative process. in situations when hepatocytes or biliary cells are blocked from regeneration, these cell types can function as facultative stem cells for each other. gene expression in the regenerating liver is a multistep process with at least two critical steps: the transition of quiescent hepatocytes into the cell cycle ("priming"), and the progression beyond the restriction point in the g 1 phase of the cell cycle. hepatocytes must first be primed before they can fully respond to growth factors. as many as 70 different genes participate in the early response to hepatectomy, but tumor necrosis factor (tnf), interleukin (il)-6, and interleukin-22 appear to be the major cytokines involved in the priming of hepatocytes. 47 the proliferative effect of tnf on hepatocytes is further influenced by reactive oxygen species, nitric oxide, and glutathione content, and multiple transcription factors (e.g., nuclear factor kappa b, stat3, ap-1, and c/ebpβ) play major roles in the initiation of early liver regeneration. 47 progression through the cell cycle beyond the initiation phase requires growth factors, primarily hepatocyte growth factor and transforming growth factor-α (tgf-α). the subsequent expression of cell-cycle genes establishes the stage at which replication becomes growth factorindependent and autonomous. at this point, the hepatocyte is irreversibly committed to replicate and the cell cycle replication machinery takes over. the proliferation of hepatocytes advances from periportal to pericentral ares of the lobules, as a wave of mitoses. hepatocytes surrounding the central veins are the last ones to undergo cell replication. proliferation of biliary epithelial cells occurs a little later than hepatocytes. proliferation of endothelial cells starts at 2 to 3 days and ends around 4 to 5 days after partial hepatectomy. the kinetics of proliferation of stellate cells is incompletely understood. the regenerative capacity of the residual hepatocytes may restore liver mass and function after as much as 65% to 70% hepatectomy and it takes place over 7 to 14 days in most animal species. 48 a small wave of apoptosis in hepatocytes occurs at the end of regeneration. the liver is the second largest organ in the body and performs an estimated 1500 essential biochemical functions. 1 these diverse functions include drug metabolism; removal of exogenous and endogenous toxins (e.g., ammonia, food antigens); synthesis of vital substances such as albumin and blood clotting factors; protein, fat, and carbohydrate metabolism; vitamin storage and activation; glycogen, triglyceride, and mineral (e.g., copper, iron) storage; activation, conversion, secretion, deactivation, and excretion of various hormones; bile salt synthesis; conjugation and excretion of bilirubin in bile; among others. symptoms (defined here as abnormalities noted by the owner), clinical signs (defined here as abnormalities found during the physical examination), and diagnostic results reflect impairments in these functions. hepatitis represented approximately 1% of the clinical population of the companion animal teaching hospital of utrecht university. box 61-1 summarizes the most common liver diseases with their possible etiologies in dogs and cats. a properly taken history is pivotal to defining the most clinically relevant problems that need to be resolved. a structured interview process and understanding the basics of communication are important success factors to retrieve this crucial information. fortunately, the knowledge about communication in the medical profession and the focus on the veterinary curriculum, has increased considerably during the last few years. 2, 3 some basic principles should be kept in mind to understand the symptoms in dogs and cats with diseases affecting the hepatic parenchyma, portal vasculature, and the biliary system. first, for most of its functions, the liver has a tremendous (approximately 80%) reserve capacity and a remarkable potential to regenerate. 4 symptoms occur only when progressive disease exhausts hepatic reserves. diseases often remain subclinical for lengthy periods of time; symptoms may be relatively mild and nonspecific because the liver reserve prevents overt abnormalities. symptoms such as lethargy, vomiting, or mild polyuria and polydipsia (pu/pd) may alert the clinician that a liver disorder could be developing. serious symptoms may indicate loss of hepatic reserves. the onset of symptoms may be acute, but they may be the end result of a disease that has been present for many weeks or months. because no specific physical abnormalities occur with most liver diseases, it is important to remember that liver disease may be present when symptoms of illness are unexplained or nonspecific. sensitive and specific laboratory tests may easily detect such liver diseases. 5 second, most liver diseases cause similar signs and symptoms (table 61 -2). one of these is acholic feces, which occurs nearly exclusively in dogs with common bile duct obstruction. 6 owners b documented in humans, may occur in dogs and cats. a more likely to present with chronic rather than acute liver disease. the clinical signs listed in table 61 -2 are candidates for having a primary hepatopathy. in all such cases, further diagnostic studies should be performed to confirm or exclude liver disease ( figure 61 -5). breed, sex, age, and drugs may predispose dogs and cats to hepatopathies. the presence of numerous risk factors should be a stimulus for an extended diagnostic workup; other diseases should be investigated in the absence of such suspicions. caused by hypersensitivity to sulfonamides, destructive cholangiolitis is the most common drug-induced liver disease. 14 a recent history of therapy with sulfonamides or other potentially hepatotoxic drugs, combined with icterus makes this condition likely and should prompt immediate discontinuation of the medication. breed associations may occur when a disease is (in part) determined by genetic factors. breeders may, by chance, increase the incidence of hepatic diseases by familial selection. because dog breeds may represent more or less closed populations in a country, breed predispositions may vary among countries. therefore this section only mentions generally applicable predispositions; locally, other breed associations may be more pertinent. chronic hepatitis and cirrhosis, both of which are, as a rule, different stages of one disease, occur more frequently in certain breeds. 15 hepatitis may develop at any age, but typically not before 2 years of age. only lobular dissecting hepatitis tends to occur at a young age (i.e., often before 1 year of age). 16 breeds associated with hepatitis are doberman pinschers, bedlington terriers, west highland white terriers, american and english cocker spaniels, labrador retrievers, and many other breeds. recent copper excretion studies have shown that hepatitis is caused by copper retention and not vice versa in doberman pinschers. 17 the cause of copper retention remains unclear; many of the tested candidate genes (including murr 1, the affected gene in bedlington terriers) were excluded as monogenetic causes for copper-associated subclinical hepatitis in may note the light-gray appearance of stool, which in combination with icterus is virtually diagnostic for extrahepatic cholestasis. different combinations of clinical signs and symptoms may occur with any liver disease. statistically, one disease may be associated with a typical pattern of signs and symptoms in dogs and cats. however, overlapping patterns are so great that it is useless to try to identify the exact disease based on clinical signs and symptoms alone (table . clinical signs and symptoms associated with liver diseases of cats are similar to those in dogs, except for pu/pd, which is not clinically overt in most cats. certain liver diseases cause neurobehavioral signs associated with hepatic encephalopathy, 7 and these signs may wax and wane in their frequency and severity. any medication given may appear to be effective because of the natural fluctuation of signs. therefore signs of hepatic origin may be easily missed. seizures alone are never caused by hepatic encephalopathy; if they do occur, they occur in combination with other signs seen with this syndrome. 5 furthermore, very few, if any, medications to treat liver diseases have been tested in double-blind, placebocontrolled studies, making decisions about the best therapeutic regimens difficult. 8 it is usually not possible to differentiate between hepatic diseases and diseases of other organs based on symptoms and clinical signs. signs associated with hepatic diseases are nonspecific; similar signs may occur in diseases of many other organ systems, most notably the gi, neurologic, renal, and hematologic systems (see table 61 -2). 9 of the gi tract-related symptoms, nausea expressed as vomiting in acute cases or reduced, irregular appetite with occasional vomiting and weight loss over time, is very common in liver and biliary diseases of dogs and cats. for biliary diseases these are always the most prominent symptoms. diarrhea, however, is not a major symptom of liver disease, and in cases in which diarrhea is the leading symptom the liver is only rarely the causative organ (except for rare cases with complete common bile duct obstruction). a rare sign (not included in table 61 -2) is an ulcerative form of dermatosis, so-called superficial necrolytic dermatitis, hepatodermal, or hepatocutaneous syndrome. this syndrome occurs rarely in dogs with liver cirrhosis and nodular hyperplasia and whose pathogenesis is poorly understood. 10 this symptom and the more common symptoms of lethargy, inappetence, vomiting, diarrhea, weight loss, pu/pd, and neurobehavioral symptoms are frequently associated with diseases of other organs. therefore the history often discloses symptoms that may suggest hepatic disease, but may also be caused by other disorders. two main reasons account for the nonspecificity of liver-related symptoms. first, the liver is the central organ for many metabolic and detoxifying pathways; consequently, failing liver function may cause dysfunction of other organs. one example is hepatic encephalopathy; metabolic dysfunctions of the liver cause neurotransmitter dysfunctions of the brain, resulting in neurobehavioral signs. 11 second, toxic factors resulting from diseases of other organ systems (especially from the gi tract) often secondarily affect the liver. examples include hepatic lipidosis in diabetes mellitus, steroidinduced hepatopathy in cushing syndrome, reactive hepatitis in gi diseases, and centrolobular liver necrosis in acute, severe anemia. 5 therefore signs and symptoms of liver disease may be hidden within signs of other organ dysfunction, and vice versa. because clinical and physical examination findings may be compatible with hepatic disease, and because laboratory tests to detect hepatic disease are also abnormal with primary and secondary hepatopathies, it is often necessary to make a histologic diagnosis of the liver disorder to resolve this dilemma. 12, 13 lack of specific physical examination findings may prevent recognition of a primary liver disease. most dogs with illnesses causing pathetic innervation; therefore dilation (e.g., extrahepatic cholestasis), cholecystitis, or cholelithiasis should be suspected in vomiting dogs and cats. 5 vomiting is also common in upper gi disease. in many gi diseases, translocation of bacteria and endotoxins may cause secondary, nonspecific, reactive hepatitis. 30 this occurs frequently in dogs, but rarely in cats. reactive hepatitis is characterized by intrahepatic canalicular cholestasis, liver cell necrosis, and an exudative inflammatory reaction. clinical signs, symptoms, and diagnostic results associated with primary liver disease and reactive hepatitis are similar; therefore, a further diagnostic workup is important to reveal the primary cause. small bowel-type diarrhea occurs frequently with hepatic diseases (see table 61 -2). two primary mechanisms may account for clinical signs. first, cholestatic diseases (intrahepatic or extrahepatic caused by common bile duct obstruction) disrupt the normal enterohepatic cycle of bile acids; therefore less bile reaches the duodenum. 30, 31 decreased resorption of dietary fat may cause hyperosmotic intestinal contents and diarrhea. however, studies in rats show that cholestasis must be severe before steatorrhea as a result of disruption of the enterohepatic bile acid cycle occurs. another mechanism for diarrhea in liver disease is increased resistance to portal blood flow, resulting in portal hypertension and congestion of splanchnic organs. intestinal vasculature congestion reduces intestinal water resorption and increases intestinal volume content. this is the predominant mechanism underlying diarrhea in diseases such as chronic hepatitis, lobular dissecting hepatitis, portal vein thrombosis, and portal vein hypoplasia. 5 alternatively, when the primary cause of diarrhea is intestinal disease, the liver may be affected secondarily. in those cases, the hepatic macrophage system should remove the increased absorption of (endo)toxins or bacteria by the affected intestinal wall. increased exposure, however, can lead to secondary, nonspecific, reactive hepatitis. endotoxins also effectively inhibit bile formation and flow, leading to cholestasis. it is therefore common to find increased plasma liver enzyme activities and bile acid levels in cases of reactive hepatitis; clinical icterus may even be apparent. the cause of diarrhea can be determined only by further diagnostic methods, including histologic evaluation of liver biopsy specimens. reactive hepatitis resolves rapidly when the primary disease is treated successfully. in the authors' experience it is very rare to find diarrhea as single or the leading symptom in cases of liver disease. if present, it is usually one of the less prominent symptoms in the spectrum of other more prominent symptoms such as apathy, pu/pd, or vomiting. one may therefore elect to follow liver laboratory values after treatment of the intestinal disease and perform a liver biopsy if liver parameters fail to improve within a few weeks. hepatic encephalopathy is a complex of neurobehavioral signs resulting from portosystemic shunting of blood in combination with a reduction of functional liver mass. 11 it may occur in animals with cpss or in those with apscapsc because of portal hypertension. diseases associated with the latter form are chronic hepatitis, cirrhosis, portal vein hypoplasia, lobular dissecting hepatitis, and portal vein thrombosis. 32 cats, because of their dependence on some essential amino acids (e.g., arginine), may develop hepatic encephalopathy without portosystemic shunting, especially when they have a severe form of hepatic lipidosis. 33 doberman pinschers. 17 the hepatitis in doberman pinschers is sex linked, confined to females, and aggressive. 18 it is responsive to medication with penicillamine, 19 but may terminate in micronodular cirrhosis. this form of cirrhosis, predominantly seen in copper toxicosis, differs from other forms of chronic hepatitis, in which patients typically develop macronodular cirrhosis with large hyperplastic nodules. hepatitis is overrepresented in female doberman pinschers by a factor of 10; a study in finland showed that approximately 10% of doberman pinschers may be affected. 20 inherited copper toxicosis is also a well described entity in bedlington terriers worldwide. 21 both sexes may be affected. clinical signs usually develop after 4 years of age as a result of the gradual accumulation of copper. it is caused by a defect in the murr 1 gene, leading to a severely decreased excretion of copper by hepatocytes. other affected breeds are west highland white terriers (particularly in the united states), skye terriers, dalmatians, anatolian shepherd dogs, and labrador retrievers. [22] [23] [24] [25] [26] siamese cats may also be predisposed to copper-associated hepatopathies. 9 although essential for life, copper is usually ingested to excess and must be eliminated by the liver to prevent toxicity. the central role of the liver in copper homeostasis makes it vulnerable if elimination processes fail. 27 furthermore, increased copper levels add to the oxidative stress, which is an important component in chronic inflammatory and cholestatic diseases in dogs. 25 spaniels seem to have the form of chronic hepatitis unrelated to copper toxicosis and develop macronodular cirrhosis when left untreated. no sex predisposition exists, but there seems to be a worldwide overrepresentation of hepatitis in this breed. congenital portosystemic shunts (cpss) are seen in both sexes in various breeds. intrahepatic shunts predominate in large breeds, whereas extrahepatic shunts predominate in small and toy breeds. although cpss are most likely inherited in some fashion in all affected breeds, this has only been proven in irish wolfhounds 28 and cairn terriers. 29 worldwide predispositions occur in irish wolfhounds, australian cattle dogs, labrador retrievers, dachshunds, yorkshire terriers, cairn terriers, maltese terriers, and miniature schnauzers. 7, 9 in the united states, an increased prevalence of shunts has also been reported to occur in german shepherd dogs, doberman pinschers, and golden retrievers. cpss occur most often in mixed-breed cats; however, persian and himalayan cats are frequently overrepresented. clinical signs are usually seen in young dogs and cats (<1 year old) with congenital shunts. 5, 9 pathogenesis of common symptoms of primary liver diseases vomiting is one of the most common symptoms noted in dogs and cats with liver disease. vomiting may be caused by direct stimulation of the vomiting center via the chemoreceptor trigger zone in the fourth ventricle by (endo)toxins that are not cleared by the liver. 30 this typically occurs when toxins from the gi system bypass the liver and access other body systems. vomiting is common in all conditions that share portosystemic shunting and liver dysfunction (e.g., congenital shunts and acquired shunts because of hepatitis, fibrosis, cirrhosis, and portal vein hypoplasia or thrombosis). hepatic diseases that cause an abnormal liver shape may reposition the upper gi tract and induce nausea and vomiting by vagal stimulation. causes include hepatic tumors, especially liver cell (or hepatocellular) carcinomas, and unilateral collapse and contralateral hypertrophy, which may occur with thrombosis of a main branch of the portal vein. the gallbladder and larger bile ducts have a rich sym-form ammonium urate. affected dogs usually have clinical signs related to shunting and liver dysfunction, such as hepatic encephalopathy, pu/pd, or vomiting. in the other category, the enzyme uricase, which forms allantoin, is inactive because of an inborn error affecting only this function. ammonium urate urolithiasis occurs commonly in dalmatians but may occur in other breeds. 43 affected dogs only have signs related to urolithiasis (e.g., pollakiuria, stranguria, dysuria). an owner may note acholic feces, which can provide a direct clue to the underlying diagnosis. steatorrheic feces that do not contain normal bile pigment are seen only when bile flow into the intestinal tract is completely disrupted, usually as a consequence of extrahepatic obstruction of the common bile duct. 6 destructive cholangiolitis is the only intrahepatic process severe enough to seriously disrupt bile flow. the latter disease is caused by a hypersensitivity reaction to sulfonamide-containing drugs. the smaller bile ductules become necrotic and liver lobuli may be disconnected from the biliary tract. affected dogs have a history of recent medication with sulfonamides. acholic feces contain excess fat because resorption is impaired. the lack of normal black-brown fecal pigments occurs because their precursor, bilirubin, does not reach the duodenum. therefore, the feces from affected animals are gray-white and soft. animals with this condition often are icteric. the presence of icterus reduces the likelihood of exocrine pancreatic insufficiency as a diagnosis. abdominal distention may occur in dogs and cats with liver disease for several reasons. first, ascites is a frequent finding associated with liver disease in dogs as a result of portal hypertension, but is less common in cats. abdominal distention may also result from organ enlargement, which in the case of liver disease may include the liver and, in the spleen in the presence of portal hypertension. in contrast to dogs, cats often have hepatomegaly with liver disease. nonspecific symptoms, such as apathy, reduced appetite or anorexia, and weight loss, may occur in dogs and cats with liver disease. retarded growth is common in young animals. these problems reflect the central role of the liver in many metabolic and detoxifying functions. in addition, nausea, inappetence, vomiting, and diarrhea can result in a catabolic state, which, in turn, may aggravate hepatic encephalopathy. signs of early hepatic encephalopathy include depression and other nonspecific problems. anemia, another common finding in liver disease (see below), can cause general malaise. dogs with liver cell carcinoma often are hypoglycemic, 44 which may be the primary problem underlying apathy and weakness. production of insulin-like growth factors by the tumor may be responsible. as with historical findings, physical examination findings rarely provide enough information to pinpoint the liver as definitive cause of the presenting problems. possible findings include icterus, hepatomegaly, splenomegaly, ascites, and pale mucous membranes. petechiae of the skin or mucous membranes occur infrequently. of these possible findings, only icterus and hepatomegaly are more or less specific for liver diseases; other abnormalities on the physical examination occur more frequently with diseases of other organ systems. hepatic encephalopathy is caused by derangement of neurotransmitter systems caused by defective metabolic processes in the liver. 34 inadequate metabolism of ammonia and aromatic amino acids by the liver may reduce the excitatory glutamatergic and monoaminergic neurotransmitter system tones, respectively. 35 in addition, there is an increased tone of the inhibitory γ-aminobutyric acid (gaba) system. 36 these neurotransmitter derangements make anesthesia risky in some animals with liver disease. the liver inactivates many anesthetics and the unforeseen delay of recovery from anesthesia may suggest an underlying liver disease as a cause for nonspecific clinical signs. this occurs especially in dog and cats with portosystemic shunting, either congenital or acquired. in addition to reduced hepatic clearance, anesthetics exert their action via various neurotransmitter systems in the brain, which may already be functioning abnormally as a consequence of hepatic encephalopathy. 34 this is especially true of drugs that act via the gaba-benzodiazepine pathway. that pathway is already overstimulated and may provoke an exaggerated and prolonged anesthetic effect. pu/pd is one of the most frequent signs (50% of cases) in dogs with liver disease, but is less common in cats. pu/pd is most common in diseases associated with congenital or acquired portosystemic shunting and, therefore, with hepatic encephalopathy. in affected dogs, abnormal neurotransmitter disturbances lead to increased secretion of adrenocorticotropic hormone (acth) from the anterior and intermediate pituitary lobes. 37 chronically elevated acth stimulates increased levels of free cortisol. increased levels of free cortisol, in turn, affect the posterior lobe of the pituitary creating an increased threshold for the release of arginine vasopressin. 38, 39 thus, a higher plasma osmolality is required to stimulate antidiuresis through arginine vasopressin, and before reaching that threshold, affected dogs become thirsty and start drinking. 38 pu/pd is not only present in cases with hepatic encephalopathy, but also frequently in all other liver diseases. this may be caused by certain bile acids, which are often increased in plasma of animals with liver diseases. bile acids may inhibit the activity of 11β-hydroxyl steroid dehydrogenase. 40 this enzyme protects the aldosterone receptor from occupation by cortisol, by converting cortisol into cortisone, which cannot bind to the receptor. present in plasma in tenfold excess compared with aldosterone, cortisol can occupy and stimulate the aldosterone receptor thereby inducing pseudohyperaldosteronism and pu. 41 reduced hepatic formation of urea is another possible but undocumented mechanism that may play a role in the pathogenesis of pu/ pd. in urea deficiency states, the kidney does not have sufficient urea available to build up an osmotic gradient in the medulla. apart from this mechanism, pd also occurs in liver diseases not associated with hepatic encephalopathy (e.g., extrahepatic cholestasis and liver tumors). the mechanism is unclear; however, nausea with an impulse to drink and compensation of water loss by vomiting and diarrhea may play a role. 5 dysuria may occur as a result of insufficient liver function when nonmetabolized uric acid is excreted by the kidneys and precipitates to form uroliths. such calculi are seen in dogs but rarely in cats. 42 there are two main categories of liver dysfunction that cause ammonium urate urolithiasis. most frequently it is caused by congenital portosystemic shunting, whereby the liver is underdeveloped and fails to metabolize uric acid into allantoin. in urine, uric acid flocculates easily in the presence of high ammonia concentrations to a result of congestive heart disease can, in most cases, be recognized easily by physical examination of the circulatory system. measurement of central venous pressure is diagnostic. the exception is liver congestion caused by a thrombus in the caudal vena cava proximal to the liver, which is assessed by other methods. when the liver is overtly enlarged because of congestion, ascites is usually present. ascitic fluid has the typical slightly hemorrhagic appearance of congestive fluid. dogs with enlarged livers and no signs of congestive disease often have liver cancer, which may be primary, metastatic, or a form of malignant lymphoma. with most tumors, the liver is diffusely enlarged, but primary hepatocellular carcinomas or adenomas may cause enlargement of the affected lobe only. bile duct carcinomas disseminate easily over the biliary system and usually cause pronounced icterus and hepatomegaly. most cats with hepatic disease develop pronounced enlargement of the liver. in cats, liver enlargement occurs with cholangitis, hepatic lipidosis, amyloidosis, hepatic tumors (primarily malignant lymphoma), and congestive disease. when the liver is involved in feline infectious peritonitis, it may not be enlarged. cats with cpss have small livers. splenomegaly and ascites in association with liver disease are nonspecific findings. they occur especially with hepatic diseases causing portal hypertension. both findings are frequent in dogs but rare in cats. there is a positive undulation test with distinct ascites; slight ascites can be found with ultrasonography rather than physical examination. the liver may be enlarged with central causes of venous congestion. canine liver diseases associated with portal hypertension include chronic hepatitis and cirrhosis, portal vein hypoplasia, and lobular dissecting hepatitis. portal hypertension is sometimes seen with cirrhosis because of advanced cholangiohepatitis in cats. in these diseases, hepatic encephalopathy is also common. portal vein thrombosis is a prehepatic cause of portal hypertension that usually causes ascites. although as a rule the liver is small in these cases, unilateral obstruction of a main branch of the portal vein may cause hypertrophy of the rest of the liver, which may be palpable. diseases of the hepatic parenchyma, hepatic vasculature, and biliary tract are relatively common in dogs and cats. because the symptoms and signs accompanying liver disease are quite nonspecific, and the liver may be secondarily involved in diseases of other organs, liver disease can easily go undetected. therefore, after taking a thorough history and performing a physical examination, it is critical to perform additional biochemical tests with the highest possible diagnostic accuracy whenever liver disease is included in the differential diagnosis. if liver disease cannot be ruled out based on these examinations, additional testing is necessary to define the type of liver disease, most notably ultrasonography of the cranial abdominal quadrant and examination of a liver biopsy specimen. the algorithm in figure 61 -5 summarizes this approach. diagnosis usually depends on histopathologic examination of liver tissue, especially for parenchymal liver diseases, many biliary tract diseases, and tumors of the liver or biliary tract. although biopsy methods are beyond the scope of this chapter, excellent sources exist. 13 one note of caution: blood coagulation testing is vital before collecting a liver biopsy specimen for histopathology. most animals, for example, have one or more abnormal coagulation tests. [47] [48] [49] [50] [51] [52] factors involved may include vitamin k deficiency, reduced ascites and hepato-and splenomegaly may have been noted by the owner as abdominal enlargement. 5 biochemistry analyses are an integral part of the diagnostic process for liver diseases. most of these analyses are not a decisive factor in the diagnosis of liver disease, but serve to rule out liver disease from the differential diagnosis. 8 icterus is the most frequently encountered specific abnormality noted on the physical examination in dogs and cats with liver disease. however, only approximately 20% of dogs with hepatobiliary diseases and 30% to 40% of cats are icteric. icterus results from bilirubin accumulation in the blood and extravascular space as a result of increased production, reduced clearance, impaired conjugation by the liver, and/or impaired bile flow. in most cases, a combination of these factors is involved. cholestasis is predominant; therefore conjugated bilirubin is the fraction present in greatest quantity. hemolysis alone does not result in icterus with normal liver function. when hemolysis is severe, however, it may result in such a degree of portal hypoxia that the centrolobular zones of the liver lobules become necrotic. in those cases, icterus results from the combination of increased production and reduced liver function and cholestasis. 45 if hemolysis is the primary cause of icterus, it must be severe, and the mucous membranes will be extremely pale. primary liver diseases that may cause icterus are commonly accompanied by hemolysis. whereas the erythrocyte lifetime is reduced to 6 to 10 days in dogs with severe primary hemolytic disease; it is 20 to 60 days (normally 100 days) in hepatobiliary disease. increased production of bilirubin and liver dysfunction with cholestasis result in a combined conjugated and unconjugated hyperbilirubinemia in dogs and cats with primary hepatic or hemolytic disease. 46 icterus caused by hemolytic disease is characterized by pale mucous membranes, whereas the mucous membranes in animals with primary liver disease are normal or only slightly pale. the combined evaluation of icterus and the color of the mucous membranes immediately reveal the nature of the underlying process. as previously discussed, most hepatobiliary diseases are accompanied by increased degradation of red blood cells. the mechanisms behind hemolysis in liver disease are not completely clear. hypersplenism and reduced portal blood flow due to portal hypertension may drastically prolong the transit time of erythrocytes through the spleen, with a greater chance that they will be trapped when they are slightly abnormal. increased fragility of red cell membranes may be a result of the high bile acid levels in most liver diseases, whereas a reduced clearance of enteral endotoxins and bacteria by the liver may also induce immune-mediated hemolysis. apart from hemolysis, nonregenerative anemia also may occur as part of the syndrome of anemia of chronic disease as an expression of catabolism and slight deficiencies of iron and b vitamins. although common in liver diseases, 12 anemia, in contrast to icterus, is nonspecific. like icterus, hepatic enlargement is a distinct sign of an abnormal liver. in dogs, most liver diseases do not cause hepatomegaly. exceptions include tumors of the liver, liver congestion, and secondary liver involvement in metabolic diseases. examples of the latter conditions are glycogen accumulation in the liver in cushing disease, fatty liver with diabetes mellitus, and rare cases of amyloidosis of the liver. the more chronic liver diseases of dogs tend to reduce liver size, and acute diseases cause little change in size. liver enlargement as hormones and drugs, leading to an increase in their serum activities in patients without primary hepatic disease. additionally, serum hepatic enzyme activities can be increased as a consequence of secondary hepatopathies. the magnitude of hepatic enzyme activity increases may aid in the assessment of the severity or the extent of hepatic injury but should not be considered to be prognostic. the liver has a large capacity for regeneration, so even in cases of severe hepatic injury, with dramatically raised hepatic enzyme activities, a full recovery is possible. this is especially true when the injury is acute. conversely, in cases of chronic end-stage liver disease, such as cirrhosis, serum hepatic enzyme activities may not be markedly increased, or may even be within the reference interval as a result of the replacement of hepatocytes with fibrous tissue. consequently, serial evaluation of serum hepatic enzyme activities is more useful for assessing prognosis than measurement at a single point in time. consistent decreases of a previously increased activity are considered a favorable sign in acute liver injury, whereas a decreasing hepatic enzyme activity in a patient with chronic liver disease that is clinically deteriorating suggests loss of hepatocytes because of fibrosis. it is important to note that serum hepatic enzyme activities do not provide an assessment of liver function. alt is an enzyme found primarily in the cytosol of hepatocytes. alt is released into the serum when hepatocyte membrane permeability is increased, or if there is hepatocellular necrosis. alt is considered to be the most liver-specific enzyme. alt is also produced by cardiac muscle, skeletal muscle, and the kidneys. 1 apart from the hepatic form, only the muscle isoenzyme is clinically significant. although uncommon, severe muscle injury can result in an increased serum alt activity. hepatic microsomal induction in response to some drugs can also produce small increases in alt activity. some controversy exists regarding the serum half-life for alt in dogs. the mean serum half-life of alt was reported as being 149 minutes in one study 1 and 59 hours in another. 2 the serum half-life of alt is generally believed to be shorter in the cat than in the dog. a mean serum half-life of 207 minutes was reported in an experiment involving three healthy cats. 3 the shorter half-life in cats means that increases in serum alt activity are considered more clinically important in this species. as alt is metabolized in the liver its serum half-life may be longer in patients with liver disease. 4 increased cell membrane permeability in the absence of hepatocyte destruction can cause a rapid increase in serum alt activity. because of this, alt activity is a considered to be a highly sensitive marker of hepatocyte injury. this also means that an increased alt activity does not imply severe or irreversible hepatocellular injury. the highest increases in alt activity are seen during acute hepatic inflammation or necrosis, but because of the capacity for the liver to regenerate these do not indicate irreversible damage. consequently, a single measurement of alt activity does not provide an accurate prognosis. however, the degree of the alt activity increase is believed to have some correlation with the number of hepatocytes that have been injured. cholestasis can also result in an increased serum alt activity because of hepatocellular damage caused by the accumulation of bile acids. certain drugs can lead to increases in serum alt activity. these are usually minor, for example, phenobarbital used at therapeutic doses frequently leads to small increases in serum alt activity, in the absence of hepatic insufficiency. these increases are thought to occur as a result of subclinical hepatic injury rather than induction of hepatic microsomal enzymes. 5 toxic doses production of clotting factors, some degree of disseminated intravascular coagulation (dic), and severe protein deficiency. 13, 52 it should be noted that taking a fine-needle aspiration biopsy should be considered safe, even if abnormalities in coagulation are present. however, its diagnostic value is limited as the liver architecture is lost. diagnosis of circulatory hepatic diseases depends on information obtained from laboratory results, ultrasonography (most reliable diagnostic test to date), and histopathology. 13 a recent paper found that fasting ammonia concentration was superior to fasting bile acids for diagnosing portosystemic shunting in dogs. 53 ammonia is easily measured in practice with dry chemical methods, some of which provide reliable results. 54 another recent publication showed ultrasonography to be a reliable diagnostic method to noninvasively characterize the underlying hepatic disease in dogs with hyperammonemia. 55 diagnostic evaluation of the hepatobiliary system has several aims: (a) to determine if hepatobiliary disease is present, (b) to assess liver function, (c) to determine if liver disease is primary or secondary, (d) to definitively diagnose hepatobiliary disease, and (e) to monitor response to treatment. despite the apparent clarity of these aims, hepatobiliary disease can present a diagnostic challenge for a number of reasons. first, as clinical signs can be nonspecific, hepatobiliary disease should be a consideration when evaluating any patient with signs of systemic disease. dogs and cats with hepatobiliary disease may not have any clinical signs. furthermore, because of the liver's central role in metabolism and detoxification of endogenous toxins and xenobiotic agents, a number of extrahepatic diseases can secondarily affect the liver. it is important to distinguish these secondary hepatopathies from diseases that originate in the liver (primary hepatopathies). additionally, serum markers of hepatocellular damage, cholestasis, and hepatic function can be abnormal in the absence of hepatobiliary disease. finally, the liver's large reserve capacity means that detectable loss of liver function often occurs late in the course of disease. thus, when assessing a patient with suspected hepatobiliary disease, it is important to consider the clinical presentation, results of laboratory testing, diagnostic imaging findings, and the results of cytologic and/or histopathologic evaluation together. hepatic enzymes can be divided into markers of hepatocellular damage and markers of cholestasis. serum alanine aminotransferase (alt) and aspartate transaminase (ast) activities are the two most commonly measured markers of hepatocellular leakage, while serum alkaline phosphatase (alp) and γ-glutamyltransferase (ggt) activities are the two most commonly measured markers of cholestasis. although increased serum hepatic enzyme activities are considered to be sensitive, they are not specific for primary liver disease because they are produced by extrahepatic tissues. the relative importance of these extrahepatic isoenzymes varies, but their extrahepatic release can lead to increased serum activities. also, the production of some hepatic enzymes can be induced by certain alp (b-alp), and kidney alp (k-alp) are transcribed from the tissue nonspecific alp gene. the other gene encodes intestinal alp (i-alp) and probably glucocorticoid-induced alp (g-alp). 13 in dogs the serum half-lives of placental alp, k-alp, and i-alp are less than 6 minutes. 14 in cats the serum half-life of i-alp is less than 2 minutes. the half-lives of placental alp and k-alp are also assumed to be short in the cat as they have similar structures to i-alp. because of this, only l-alp, b-alp, and, in the dog but not the cat, g-alp, are believed to contribute significantly to serum alp activity. the serum half-life of l-alp is approximately 70 hours in the dog 14, 15 and 6 hours in the cat. 16 l-alp is bound to the membranes of the hepatocytes by glycosylphosphatidylinositol linkages. cleavage of these links by glycosylphosphatidylinositol-phospholipase allows the enzyme to be released into the bloodstream. 17 as bile acids have detergent-like properties; accumulation of bile acids during cholestasis facilitates this process. cholestasis can also result in the induction of synthesis of l-alp (and g-alp in the dog). consequently, serum alp activity is often severely increased in patients with cholestatic disorders. in the dog, alp is considered to be a sensitive marker for cholestasis with a sensitivity of 85%. 18 the short half-life of l-alp in cats means that increases in alp during cholestasis are not as high as in the dog. consequently, alp is a less-sensitive marker of cholestasis in the cat than in the dog, with a reported sensitivity of only 48%. 19 however, the shorter half-life in cats and the absence of g-alp means that any increase in alp activity should be considered clinically important in this species. an increased serum alp activity does not differentiate between intrahepatic or extrahepatic cholestasis. a wide variety of liver diseases can cause intrahepatic cholestasis. this is generally caused by hepatocyte swelling, causing obstruction of the small bile canaliculi. the increase of alp following a hepatic insult is delayed compared to rises in markers of hepatocellular leakage. the reason for this is that it takes time for the enzyme to be synthesized and released into the systemic circulation. alp often remains increased for some time after the resolution of liver injury. b-alp is released into the bloodstream as a result of the activity of osteoblasts. therefore any condition that results in increased bone formation can lead to increased serum alp activity. in animals that are skeletally immature, mild increases in serum alp activity are commonly observed. animals with increased osteoblast activity, such as those with hyperparathyroidism, neoplasia involving bones, and osteomyelitis, may have mild to moderate increases in alp activity. these causes of increased b-alp activity are unlikely to be confused with primary liver disease because the increases are smaller than would be expected with cholestasis, and because bone diseases are often clinically apparent. finally, an increased serum activity of b-alp was reported in a family of asymptomatic huskies. 20 in dogs, but not in cats, g-alp and tissue-nonspecific alp may be induced by corticosteroids. g-alp is believed to be an isoform of i-alp with a prolonged serum half-life that is produced by the liver. 13 posttranslational glycosylation of the g-alp is believed to be responsible the prolonged half-life. induction of g-alp may cause an increase in total serum alp activity after administration of exogenous corticosteroids. synthesis of this isoenzyme can also be induced by the administration of anticonvulsant drugs, such as phenobarbital. similarly, hypercortisolemia frequently causes an increased serum alp activity because of induction of g-alp. however, in dogs with excess serum concentrations of endogenous or exogenous corticosteroids, hepatocyte swelling caused by glycogen accumulation (vacuolar hepatopathy) may lead to intrahepatic cholestasis, another potential contributor to increased serum alp of phenobarbital can cause dramatic increases of serum alt activity and hepatic insufficiency. prednisone and other glucocorticoids can cause an induction of alt (and steroid hepatopathy) and consequently small increases in serum alt activity. serum alt activity can also be increased with any secondary hepatopathy. however, a persistently increased serum alt activity, even with an apparently normal liver function, is an indication for further diagnostic testing. serial evaluation of serum alt activity can be helpful to prognosticate but must be done while considering the patient's clinical signs and other laboratory values. in general, a declining serum alt activity after acute liver injury is considered a good sign. ast is another aminotransferase enzyme that is used as a marker of hepatocellular leakage. ast is found in skeletal muscle, the brain, liver, kidney, cardiac muscle, and to a lesser extent within other tissues. 6 the extrahepatic isoenzymes of ast are relatively more important than they are for alt. muscle disease can cause an increase in serum ast activity. because of this, ast is considered less liver specific than alt. however, by looking at serum ast activity in conjunction with the activities of other hepatic enzymes and muscle enzymes, it is usually possible to differentiate increases caused by muscle damage from increases caused by hepatic damage. again, there is controversy regarding the serum half-life of ast. in dogs one study 1 reported the half-life to be a mean of 263 minutes; another study reported a mean of 22 hours. 2 one study reported the mean half-life to be 78 minutes for cats. 7 unlike alt, a considerable proportion of ast (approximately 30%) is found within hepatocyte mitochondria rather than the cytosol. 8 the cytosolic fraction of ast is released into the serum from hepatocytes when cell membrane permeability is increased, or in case of hepatocellular necrosis. in contrast, the mitochondrial fraction is only released during hepatocellular necrosis. release of ast from hepatocytes into the serum parallels the release of alt. therefore, like serum alt activity, serum ast activity is considered a sensitive marker for hepatocyte injury. it has been suggested that increased ast activity may be more sensitive than increased alt activity for the detection of hepatocellular injury in cats. 9 corticosteroids and phenobarbital may cause mild increases in serum ast activity. because of the considerable overlap in the information provided by the measurement of serum alt and ast activities, measurement of serum ast activity may be redundant. alp is an enzyme bound to the membranes of the hepatocytes that comprise the bile canaliculi and the sinusoidal membranes. it is considered a sensitive marker for cholestasis, especially in the dog, but is not liver specific. cholestasis, canalicular cell necrosis, and increased hepatic synthesis may lead to the release of this enzyme into the circulation. synthesis of this enzyme can be induced by certain drugs, most notably corticosteroids. the possibility that an increase in serum alp activity could be caused by extrahepatic disease, or could be induced by glucocorticoids in the dog, can make the interpretation of this finding challenging. in the dog a wide variety of tissues exhibit alp activity, including intestinal mucosa, kidney (cortex), bone marrow, pancreas, testicle, brain, lung, kidney (medulla), lymph node, liver, skin, spleen, skeletal muscle, and cardiac muscle. 6 there is disagreement in the literature regarding the relative contributions of alp activity from each of these tissues in cats. [10] [11] [12] there are two genes encoding alp in the dog. different forms of alp arising from the same gene are called isoforms. differences among these isoforms arise because of differing posttranslational processing. liver alp (l-alp), bone globulins are produced in the liver, but not exclusively so. the liver produces α-globulins and β-globulins, whereas lymphoid cells produce immunoglobulins (γ-globulins). hepatic insufficiency rarely leads to a decrease in serum globulin concentration. conversely, inflammatory liver disease may be associated with hyperglobulinemia because the nonimmunoglobulin fraction produced by the liver includes several acute-phase proteins (c-reactive protein, haptoglobin, and serum amyloid a). the hepatic synthesis of these proteins is increased during systemic inflammation [26] [27] [28] [29] possibly leading to a rise in the total serum globulin concentration. additionally, immunoglobulin production may be increased in infectious, neoplastic, or autoimmune diseases. coagulation factors (except factor viii), anticoagulation factors (antithrombin and protein c), and the fibrinolytic protein plasminogen, are all synthesized by the liver. the liver is also the site of activation of the vitamin k-dependent clotting factors: ii, vii, ix, x, and protein c. furthermore, as bile acids are needed to emulsify fat and aid in the absorption of vitamin k from the intestine, vitamin k malabsorption may develop secondary to cholestasis. consequently, hepatobiliary disease can affect hemostasis in more than one way. in canine and feline liver disease, coagulation parameter abnormalities have been reported in specific clotting factor activities, prothrombin time, aptt, [30] [31] [32] proteins induced in the absence of vitamin k, 33, 34 fibrin degradation products, fibrinogen, and protein-c activity. 35 these abnormalities of hemostasis are not specific for liver disease but may support its presence. patients with liver disease may develop dic, which can be difficult to distinguish from coagulopathy because of reduced hepatic synthesis of clotting factors alone. although spontaneous bleeding seldom occurs in patients with liver disease, the assessment of the coagulation status of these patients is important, especially when an invasive procedure such as a liver biopsy is being considered. a recent study investigated the diagnostic value of serum protein c as a marker for hepatobiliary disease and portosystemic shunting in dogs. serum protein-c measurement was reported to aid in the differentiation of portal vein hypoplasia without portal hypertension (formerly called microvascular dysplasia) from portosystemic shunt (pss). dogs with portal vein hypoplasia without portal hypertension had a significantly higher serum protein-c concentration than those with portosystemic shunting. 35 urea is produced from ammonia in the liver, released into the systemic circulation, and subsequently excreted by the kidneys. serum urea nitrogen concentration may be close to or below the lower limit of the reference interval in patients with hepatic insufficiency, pss, 36 or urea cycle enzyme deficiencies. however, serum urea nitrogen concentration may also be decreased because of medullary solute washout caused by diuresis, malnutrition, or a protein-restricted diet, and is a normal finding in neonates. in a patient with liver disease, a high fasting serum urea nitrogen concentration relative to the serum creatinine concentration suggests gi hemorrhage. ammonia (nh 3 ) is produced in small intestinal enterocytes from the catabolism of glutamine and in the colon as a consequence of bacterial deamination. ammonia is a highly diffusible gas and passes readily through the bowel wall into the bloodstream. in the blood, at a ph of 7.4, most of the ammonia exists in the form of ammonium ions (nh 4 + ). the ammonium is transported in the blood from the intestines through the hepatic portal circulation to the liver. the extraction of ammonia from the portal circulation is highly efficient. endogenous ammonia is produced from the breakdown of activity. before diagnosing primary liver disease in a dog with an increased serum alp activity, induction of g-alp by endogenous or exogenous steroids should be ruled out. recently a group of scottish terriers were found to have increased serum alp activity with no identifiable underlying cause. 21 it is technically possible to selectively measure the activity of g-alp using techniques such as levamisole inhibition. measurement of g-alp activity was initially investigated as a way to differentiate increases in alp caused by corticosteroids from those caused by cholestasis. unfortunately, measuring g-alp is not clinically useful as g-alp activity may be increased in a variety of conditions, including hepatic disease, diabetes mellitus, hypothyroidism, and pancreatitis. ggt is a glycoprotein enzyme that is bound to the membranes of those hepatocytes that form the bile canaliculi and bile ducts and also periportal hepatocytes. in comparison to alp its distribution includes more distal areas of the biliary tract, but measurement of serum ggt activity is not useful to distinguish between intrahepatic and extrahepatic cholestasis. ggt is also produced by a number of extrahepatic tissues. most of the ggt activity in serum is thought to be a result of the hepatic isoenzyme. colostrum also contains ggt, which is responsible for the mild increases in serum ggt activity that are seen in suckling animals. 22 changes in serum ggt activity generally parallel those in serum alp activity, in that activity is often increased in patients with cholestasis. because ggt is also induced by glucocorticoids, its activity may be increased in patients with hyperadrenocorticism or those exposed to exogenous steroids. in dogs, an increased serum ggt activity is considered to be more specific, but less sensitive than alp activity for the presence of liver disease. 18 in cats, measurement of serum ggt activity is more sensitive but less specific for the detection of liver disease than alp. cats with hepatic lipidosis may be an exception to this as they often have a normal serum ggt activity but an increased serum alp activity. 19 the liver plays a central role in protein metabolism. it is responsible for the synthesis of plasma proteins, deamination of amino acids, conversion of ammonia to urea, amino acid synthesis, and interconversion of amino acids. 23 consequently, in patients with hepatic disease these functions may be compromised. albumin is an important plasma protein that is produced exclusively by the liver. the rate of albumin synthesis must equal the rate of albumin loss to maintain serum albumin concentrations. mild decreases in serum albumin concentration can occur from a variety of conditions. however, the differential diagnoses for severe hypoalbuminemia (<2 g/dl) are limited to hepatic insufficiency, severe exudative skin disease, protein-losing enteropathy, and proteinlosing nephropathy. it is possible to determine the cause of severe hypoalbuminemia from a combination of clinical findings, measurement of the serum globulin concentration, urinalysis (including protein creatinine ratio), tests of gi protein loss, and tests of liver function. as albumin contributes significantly to colloid oncotic pressure, 24 severe hypoalbuminemia can lead to ascites, pleural effusion, and/or subcutaneous edema. the liver has a large reserve capacity for the synthesis of albumin and albumin has a serum halflife of approximately 7 days in dogs. 25 consequently, hypoalbuminemia is a relatively insensitive marker for hepatic insufficiency and is only likely to be seen in patients with advanced chronic liver disease or portosystemic shunts (psss). for hepatobiliary disease in dogs or cats. in patients with severe hepatic insufficiency or pss 49 serum cholesterol, concentration may be decreased as a consequence of impaired hepatic synthesis. hypocholesterolemia might also occur as a result of inadequate dietary intake, maldigestion, malabsorption, or hypoadrenocorticism. the serum cholesterol concentration of patients with hepatobiliary disease may be within the reference interval. patients with cholestatic disease can become hypercholesterolemic. 50 fasting hypercholesterolemia also may be observed in patients with various endocrinopathies, obesity, protein-losing nephropathy, pancreatitis, or primary hyperlipidemias. serum triglyceride concentration may be increased or normal in patients with liver disease. however, an increased fasting serum triglyceride concentration is not a sensitive or specific marker for hepatobiliary disease in dogs or cats. a mild increase in serum triglyceride concentration may develop in patients with cholestasis. there is some evidence that hypertriglyceridemia is associated with gallbladder mucocele formation. 51 hypertriglyceridemia is associated with increased serum hepatic enzyme activities in miniature schnauzers. 52 increased fasting serum triglyceride concentrations are also observed in patients with endocrinopathies, obesity, pancreatitis, and primary hyperlipidemias. the liver plays a central role in carbohydrate metabolism and is responsible glycogen storage, conversion of galactose and fructose into glucose, gluconeogenesis, and the synthesis of many compounds from carbohydrates. 23 blood glucose measurement is not a sensitive or specific marker for liver disease. the liver has a large reserve capacity for glucose production. consequently, hepatic insufficiency must be severe for hypoglycemia to occur. hypoglycemia occurs in a proportion of patients with congenital pss. 53 hepatic neoplasia can also lead to hypoglycemia. this is thought to be caused by release of insulin-like substances. 54 a variety of extrahepatic conditions can also lead to hypoglycemia. bilirubin is a yellow pigment produced from the breakdown of heme-containing compounds. measurement of serum bilirubin concentration can be used to assess liver function. hyperbilirubinemia can be the result of hepatobiliary or extrahepatic disease. icterus is the yellowish pigmentation caused by the retention of bilirubin in the soft tissues. laboratory assessment is the most sensitive way to detect increased serum bilirubin concentrations. hyperbilirubinemia is classified as prehepatic, hepatic, or posthepatic in origin. bilirubin may be artifactually increased by in vitro hemolysis or by the administration of synthetic hemoglobin polymers. when assessing a hyperbilirubinemic patient it is critical to localize the underlying cause. bilirubin is the major product of the degradation of hemecontaining compounds by cells of the mononuclear phagocyte system. bilirubin is released from the mononuclear phagocyte system and is transported in the plasma. the bilirubin is reversibly bound to albumin as it is water insoluble. the unconjugated bilirubin is absorbed through the hepatocyte cell membranes and is bound to glucuronic acid (conjugation). conjugated bilirubin is water soluble and is actively excreted from the hepatocytes into the bile canaliculi, eventually being excreted into the intestines. once in the intestine some of the bilirubin is converted into urobilinogen by bacteria. some of the urobilinogen is reabsorbed from the intestines, but most of this is immediately reexcreted by the liver. when nitrogenous substances in the body, especially glutamine. in the liver the ammonium is converted to urea by the enzymes of the urea cycle, or is used during the conversion of glutamate to glutamine. 37 urea enters the circulation and is excreted by the kidneys. ammonium that is not removed by the liver enters the systemic circulation. the liver has a large reserve capacity for the conversion of ammonia into urea. because of this, plasma ammonia measurement is a relatively insensitive marker for hepatic insufficiency. however, measurement of blood ammonia concentration is a sensitive test for congenital psss and apsc shunts (also known as acquired psss). this is because when portosystemic shunting occurs, the ammonia absorbed from the intestines bypasses the liver and reaches the systemic circulation directly. the sensitivity of plasma ammonia measurement for the detection of pss is reported to be between 81% and 100% in dogs [38] [39] [40] [41] and 83% in cats. 41 the measurement of postprandial venous ammonia is more sensitive than the measurement of fasting ammonia (sensitivities of 91% and 81%, respectively) for the detection of congenital pss. 42 however, the sensitivity for detecting dogs with hepatocellular disease is only 36%. generally, hyperammonemia is considered specific for hepatic insufficiency or pss. however, although they are uncommon, urea-cycle enzyme deficiencies may also cause an increased blood ammonia concentration. these enzyme deficiencies can be hereditary as a result of the absence of a particular enzyme 43 or secondary to cobalamin or arginine deficiency. 44, 45 arginine deficiency is especially relevant in cats with hepatic lipidosis. ammonia is one of the substances that cause hepatic encephalopathy (he). therefore blood ammonia measurement is a useful marker for he. however, other substances can also cause he and the plasma ammonia concentration of a patient with he may be within the reference interval. ammonium ions are labile in plasma, so sample handling is critical when measuring plasma ammonia concentration. samples should be collected, placed immediately on ice, and the plasma separated from the red blood cells as soon as possible. the plasma must be kept cooled and should be analyzed within 30 minutes of collection. these handling requirements have meant that ammonia measurement has been mainly confined to practices with immediate access to a commercial laboratory. measurement of plasma ammonia is available on an in-house dry chemistry analyzer (vettest, idexx laboratories, westbrook, me) although this method was only considered to reliably agree with a reference method for serum ammonia concentrations greater than 150 µm. 46 a recent study found that a point of care blood ammonia analyzer (pocketchem ba, menarini diagnostics, florence, italy) may be suitable for the measurement of blood ammonia concentrations in dogs and cats. 47 ammonia tolerance tests (atts) have been investigated in an attempt to increase the sensitivity of ammonia measurement for detecting hepatic insufficiency and pss. however, the oral administration of ammonium salts can cause vomiting and potentially worsen he signs. ammonium chloride or sulfate can also be given rectally, which is less likely to produce adverse. this method is sensitive for the detection of pss in dogs. 48 the liver plays a central role in lipid metabolism and is responsible for oxidation of fatty acids, synthesis of cholesterol, synthesis of lipoproteins, and synthesis of fatty acids from proteins and carbohydrates. 23 serum cholesterol concentrations may be increased, normal, or decreased in patients with liver disease. increased or decreased fasting serum cholesterol concentrations are not sensitive or specific (fasting or postprandial) suggest hepatic dysfunction, pss, or cholestasis, but they are not specific for any particular liver disease. bile acids are exclusively synthesized in the liver from cholesterol. nearly all of the bile acids that are produced by the hepatocytes are conjugated to an amino acid. in both dogs and cats conjugation is primarily to taurine, but dogs may also convert to a conjugation with glycine. 61 in contrast, even taurine depleted cats conjugate their bile acids almost exclusively to taurine. 62 the conjugated bile acids produced by the liver are called primary bile acids. these are secreted in the bile, and then stored in the gallbladder. cholecystokinin is released from endocrine cells in the small intestine. this hormone stimulates gallbladder contraction and the flow of bile into the duodenum. when the gallbladder contracts the bile acids are released into the intestines. spontaneous gallbladder contraction also occurs during the interdigestive phase. 63 bile acids act as ionic detergents, aiding the emulsification of dietary lipids and their subsequent intestinal absorption in micelles. 64 bile acids are recycled in a process known as enterohepatic circulation ( figure 61-6 ). primary bile acids are lipid insoluble and thus are only absorbed from the intestines when they bind to specific high affinity ileal mucosal receptors. 64 this ileal reabsorption is very efficient. the reabsorbed bile acids enter the portal circulation and upon reaching the liver they are efficiently extracted from the plasma and subsequently reexcreted. the total bile acids pool can be recirculated several times in a day. consequently, the rate of hepatic bile acid synthesis and the fasting serum bile acids concentration in dogs and cats with normal hepatic function is low. because of the increased release of stored bile acids during the postprandial period, small increases in total sba concentration occur in animals with normal hepatic function. primary conjugated bile acids can undergo bacterial deconjugation in the intestinal lumen. the resulting unconjugated bile acids are called secondary bile acids. these are readily absorbed from the colon by passive diffusion. first-pass extraction and reexcretion of secondary bile acids is less efficient than that for primary bile acids. consequently, secondary (unconjugated) bile acids are often present in postprandial serum samples. 65 hepatobiliary disease can cause increased sba concentrations by interfering with hepatocellular function, by causing decreased bile flow (cholestasis), or by altering the hepatoportal blood flow. the main clinical use of sba measurement is to assess hepatic function in patients suspected to have hepatic disease, with serum exposed to air the urobilinogen remaining in the intestines is altered and oxidized into the brown pigment sterocobilin. 23 prehepatic hyperbilirubinemia is caused by increased production of bilirubin as a result of hemolysis. the liver has a large reserve capacity for bilirubin excretion so, for hemolysis to cause hyperbilirubinemia, hepatic bilirubin clearance must be decreased. 55 this occurs if the hemolytic anemia results in hepatocyte dysfunction because of hypoxia. if hepatic hypoxia occurs serum hepatic enzymes activities are often increased. prehepatic hyperbilirubinemia is mainly distinguished from other causes of hyperbilirubinemia by the presence of severe anemia. other supportive evidence includes the presence of a regenerative erythroid response, characteristic changes in red blood cell morphology, and possibly the detection of red blood cell bound antibodies. 56 hepatic hyperbilirubinemia is caused by a decreased rate of hepatocyte bilirubin uptake, conjugation, or excretion (as a result of intrahepatic cholestasis). usually, hepatocyte dysfunction and intrahepatic cholestasis occur concurrently. hepatic enzyme activities (both hepatocellular leakage markers and cholestatic markers) are often increased, although they can also be increased with both prehepatic and posthepatic hyperbilirubinemia. because of the hepatic reserve capacity, hepatic disease must be severe in order to cause hyperbilirubinemia. a range of primary and secondary hepatopathies can cause hepatic hyperbilirubinemia. hepatic hyperbilirubinemia can usually be distinguished from prehepatic hyperbilirubinemia by assessment of the patient's hematocrit, and from posthepatic hyperbilirubinemia by abdominal ultrasound. other markers of hepatic insufficiency, when present, provide additional support for the presence of hepatic hyperbilirubinemia. posthepatic hyperbilirubinemia is a result of extrahepatic bile duct obstruction. this is often caused by pancreatic inflammation or, much less commonly, neoplasia. the main diagnostic tool for documenting extrahepatic bile duct obstruction is abdominal ultrasound. typically, extrahepatic bile duct obstruction leads to dramatic increases in serum cholestatic enzyme activities (compared to hepatocellular leakage enzyme activities) and hypercholesterolemia. when the bile duct is completely obstructed, acholic (pale-colored) feces may be noted. rupture of the biliary tract frequently leads to hyperbilirubinemia as bilirubin accumulates in the abdomen. it is possible to measure the concentration of serum conjugated bilirubin. however, this test is not considered to be clinically useful for distinguishing between prehepatic, hepatic, or posthepatic hyperbilirubinemia, and is thus rarely performed. bilirubin can covalently (nonreversibly) bind to albumin. this biliprotein cannot be cleared by the liver and thus persists in the plasma. biliprotein has a serum half-life comparable to albumin. this is of clinical importance because it means hyperbilirubinemia (and icterus) may persist for several weeks after the resolution of its cause. 57 bile acids (or bile salts when they are deionized) are formed from cholesterol in the liver and are the major constituent of bile. serum bile acids (sbas) measurement is a useful test of liver function in dogs and cats. sbas are either measured as a fasting sample (after withholding food for 12 hours) or by collecting paired fasting and 2-hour postprandial samples. 58 both of these tests are simple to perform and safe. enzymatic measurement of the concentration of total bile acids in serum has become widely available and has replaced other techniques such as radioimmunoassays. 59, 60 once collected, the samples of serum can be stored at room temperature, making it possible to send them to an outside laboratory for evaluation. lipemia and hemolysis of the blood samples should be avoided as both can interfere with the assay. increased sba concentrations recent study did not support the use of a 13 c-labeled galactose breath test for assessment of liver function in dogs. 80 the metabolism of endogenous substances has been investigated to find possible markers of hepatic cellular metabolism. dogs with hepatic disease (hepatitis and neoplasia) had significantly higher serum l-phenylalanine concentrations than did healthy dogs and those with nonhepatic diseases. 81 further investigations are needed to determine the utility of these tests for assessing liver function in veterinary patients. urine specific gravity can be decreased in patients with hepatic insufficiency or pss. this can be caused by an inability to fully concentrate urine, resulting in pu, or from primary pd. bilirubin is commonly measured semiquantitatively in canine and feline urine using urine dipsticks. bilirubinuria (<2+ on a dipstick) can be a normal finding in dogs (especially males). 82 bilirubinuria in dogs without hemolytic or hepatobiliary disease can occur as a consequence of the loss of unconjugated bilirubin that is bound to albumin in proteinuric patients and renal filtration of small amounts of conjugated bilirubin that has leaked from the liver. additionally, the renal tubular cells of male dogs have the enzymes needed to produce and conjugate bilirubin. as cats have a higher renal threshold for bilirubin than dogs, bilirubinuria should always be considered abnormal in cats. bilirubinuria in cats and excessive bilirubinuria in dogs implies hemolytic or hepatobiliary disease. because dogs have a relatively low renal threshold for bilirubin, bilirubinuria is often detected before bilirubinemia or jaundice. ammonium biurate crystals are detected in the urine sediment by light microscopy. uric acid is a product of purine catabolism and is converted to allantoic acid by hepatic urate oxidase. in cases with severe hepatic insufficiency or pss, the serum uric acid concentration may be higher than the renal threshold. this combined with hyperammonemia may lead to ammonium biurate precipitation in the urine. urate urolithiasis seems to be more common in patients with pss than those with other types of hepatic dysfunction. between 40% and 70% of dogs with pss were found to have urate crystalluria. 83 however, it should be noted that urate crystalluria is not specific for hepatobiliary disease. the erythrocyte series may be affected by hepatobiliary disease, resulting in erythrocyte dysmorphias and anemia. these abnormalities are suggestive of, but are not specific for, hepatobiliary disease. patients with hepatobiliary disease can be anemic as a result of blood loss, in which case signs of a regenerative response are normally present within 3 days of hemorrhage. acute, severe hemorrhage may occur in patients with hepatobiliary disease following invasive procedures such as liver biopsy or as a consequence of hemorrhage from a hepatic neoplasm or hepatic rupture. less-severe anemia may occur as a result of gi bleeding. 84 chronic gi blood loss may eventually lead to iron-deficiency anemia. this is characterized by microcytic hypochromic erythrocytes and a variable regenerative response. additionally, hepatobiliary disease may lead to anemia of chronic disease, which is typically nonregenerative with normocytic normochromic erythrocytes. red blood cell morphologic changes are sometimes observed in dogs with hepatobiliary disease. poikilocytosis, characterized by the presence of acanthocytes and target cells, may be seen in patients with chronic hepatic disease. this is thought to be a result of altered phospholipid metabolism. patients with pss can have microcytic red blood cells. this is more common in dogs than in bilirubin concentrations that are within the reference interval. measurement of postprandial sba concentration does not seem to have an advantage over fasting sba concentrations or vice versa. sensitivity can be increased by collecting paired preprandial and twohour postprandial samples. 66 numerous studies show that sba measurement is a useful test for diagnosing hepatobiliary disease, including pss in dogs and cats. 41, [66] [67] [68] [69] [70] a recent study found the sensitivity of fasting sba measurement for diagnosing pss (using a cutoff value of 20 µmol/l) to be 93% for dogs and 100% for cats. the reported specificities were 67% for dogs and 71% for cats. 41 however, the sensitivity of sba measurement for detecting hepatic insufficiency is lower than that for detecting pss. measurements of sba concentrations have several limitations. first, this test does not allow differentiation between various types of hepatobiliary disease. also, measurement of serum bile acids in a patient with proven cholestasis is of no clinical benefit. additionally, there is limited utility in measuring sbas concentrations in patients with hyperbilirubinemia, although potentially sba measurement could be useful in distinguishing prehepatic from hepatic or posthepatic causes of hyperbilirubinemia. with prehepatic causes of hyperbilirubinemia, the bile acid concentrations should be within the reference interval. however, in most cases prehepatic hyperbilirubinemia is easily distinguished by the presence of severe anemia. it should also be noted that the magnitude of increases of sba concentration are not correlated with prognosis or disease severity. it is important to note that fasting sba concentrations may be higher than the upper limit of the reference interval or higher than the postprandial value because of spontaneous contraction of the gallbladder in the fasting state, or because of delayed gastric emptying. this could result in an increased fasting sba concentration in the absence of hepatobiliary disease. increased fasting and postprandial serum bile acids concentrations can be the result of increased bacterial deconjugation of primary bile acids into secondary bile acids. 71 false-negative results may occur if enterohepatic circulation of bile acids does not occur from a lack of gallbladder contraction. this could be a problem if a patient is anorectic, does not eat enough food, consumes a diet with insufficient protein or fat, vomits the test meal, or has delayed gastric emptying. ceruletide is an injectable cholecystokinin analogue that has been used to stimulate gallbladder contraction when using sba measurement to diagnose hepatobiliary disease. 72, 73 this test circumvents many of the factors that influence postprandial sba concentrations. urine bile acids measurement has been described in dogs and cats. the diagnostic performance was similar to that of sba measurement in both species. this test does not offer any advantages over sba measurement. [74] [75] [76] [77] excretion of exogenous tracers, such as the anionic cholephilic dyes, bromsulphalein, and indocyanine green have been used historically to assess hepatic function in veterinary patients. however, these tests are considered unreliable and have been replaced by the measurement of sba concentrations. the metabolism of exogenous substances can be used to assess liver function. a variety of substances have been investigated as markers for hepatic metabolism in human medicine. assessment of the metabolism of aminopyrine has been investigated in dogs and to a lesser extent in cats. the 13 c-labeled aminopyrine demethylation blood test involves intravenous administration of 13 c-labeled aminopyrine to the subject. the aminopyrine is metabolized by the liver, resulting in the production of 13 co 2 . this is measured in the blood by fractional mass spectroscopy. 78, 79 further investigation of the utility of this test for assessment of liver function is needed. a in dogs with liver disease than in dogs with extrahepatic disease, and were higher in dogs with cirrhotic liver disease than in dogs with noncirrhotic liver disease. blood ha concentration may prove be a useful marker for hepatic fibrosis in dogs but further studies are necessary to evaluate its clinical utility. histopathologic analysis of liver biopsies or identification of a shunting blood vessel is often required to definitively diagnose hepatic disease. however, the pattern of laboratory test abnormalities, particularly when interpreted in conjunction with the patient's clinical presentation, and the results of diagnostic imaging, can increase or decrease a clinician's index of suspicion for specific liver diseases (table 61-3) . it is important to note that there is considerable overlap between the patterns for different diseases. to avoid misinterpretation and misdiagnosis when evaluating a patient for liver disease, it is essential to consider the limitations of the laboratory tests discussed above. diagnostic imaging is an important part of the investigation of hepatobiliary disease in dogs and cats. diagnostic imaging may help to determine whether or not hepatobiliary disease is present, identify the cause of a secondary hepatopathy, aid in the diagnosis of specific hepatobiliary diseases, and provide prognostic information. however, with the exception of diagnosis of a pss, imaging seldom yields a definitive diagnosis. radiography and abdominal ultrasound are the most frequently used imaging modalities for assessment of the hepatobiliary system in dogs and cats, but alternative imaging techniques are now being used more frequently. abdominal radiographs allow assessment of hepatic size, shape, opacity, and location in most patients. 91 radiography may also allow identification of extrahepatic abnormalities that affect the liver. however, radiographs provide limited information about the hepatic parenchyma. it is important to note that patients with hepatobiliary disease often have normal abdominal radiographs. radiography allows subjective assessment of liver size. cranial displacement of the gastric axis may be observed on lateral abdominal radiographs when microhepatia is present. however, subtle microhepatia is unlikely to be appreciated radiographically. psss and hepatic cirrhosis are the most common conditions causing microhepatia. mild bilateral renomegaly may also be cats. 83 microcytosis also occasionally occurs in patients with hepatocellular disease. altered iron metabolism is thought to lead to a delay in red blood cell precursors gaining a sufficient amount of hemoglobin to be released into the circulation. this delay leads to the precursors undergoing an extra cell division in the bone marrow, resulting in microcytosis. 85 microangiopathy can occur as a result of hepatic neoplasia or dic, and may lead to the formation of schistocytes. the leukocyte series may be affected by hepatobiliary disease in a variety of ways. the resultant abnormalities are inconsistent and are not specific for hepatobiliary disease. leukocytosis, leukopenia, and sometimes an inflammatory leukogram may be present when infectious and, less commonly, inflammatory or neoplastic processes affect the hepatobiliary system. a leukocytosis was found to be present in 44% of dogs with chronic hepatitis. 86 the thrombocyte series is occasionally affected by hepatobiliary disease, but changes are both inconsistent and nonspecific. mild to moderate thrombocytopenia may occur in patients with severe liver disease. 86 this may be the result of a decreased production of thrombopoietin by the liver. disseminated intravascular coagulopathy associated with liver disease also may lead to thrombocytopenia. additionally, infectious diseases affecting the liver, such as leptospirosis may result in thrombocytopenia. 87 genetic testing for copper hepatotoxicosis has been developed in bedlington terriers. affected bedlington terriers have an autosomal recessive defect of their commd1 gene. dogs with a homozygous affected genotype develop copper hepatopathy as a result of impaired biliary excretion of copper. initially, a microsatellite marker that is in linkage disequilibrium with the mutation was discovered and used to identify affected dogs and select dogs homozygous unaffected dogs for breeding. 88 subsequently, a mutation of the commd1 gene (a deletion of exon 2) was identified as the cause of the condition in the majority of bedlington terriers. 89 a genetic test for this disease has become commercially available (vetgen, ann arbor, mi). this test is run alongside the linked marker as a small proportion of bedlington terriers do not have the deletion but are nevertheless affected by the disease. these dogs are likely to have a rare second mutation of their commd1 gene which the linkage markers may track. hyaluronic acid is a major constituent of the ecm and hyaluronic acid (ha) concentration in blood has been used as a marker for hepatic fibrosis in humans. a recent study investigated the use of ha concentration as a marker for hepatic disease in dogs. 90 this study found that blood ha concentrations were significantly higher the size of the liver can be subjectively assessed by abdominal ultrasonography. the findings of a small liver and cranial displacement of the stomach suggest microhepatia. hepatomegaly is another subjective finding and can be generalized or focal. the finding of rounded liver lobe margins suggests hepatomegaly. hepatic parenchymal changes can be classified as being diffuse, multifocal, or focal. a wide variety of disease processes can cause diffuse changes to the hepatic parenchyma. these changes can be isoechoic, hypoechoic, hyperechoic, or of mixed echogenicity. in some cases the architecture of the liver will not be altered, but in other cases changes will occur. examples of diseases in which the echogenicity of the hepatic parenchyma is diffusely changed but no changes in architecture occur include cholangitis, neoplasia, hepatic lipidosis, other vacuolar hepatopathies, toxic hepatopathy, and early micronodular hyperplasia with various degrees of fibrosis. 100 hyperechogenicity of the liver compared to the falciform fat, poor visualization of the intrahepatic blood vessels, and increased attenuation of the ultrasound beam have been used as criteria for the sonographic diagnosis of feline hepatic lipidosis. 101 diseases where the hepatic architecture is altered are easier to detect sonographically. these include neoplasia, micronodular hyperplasia, and chronic hepatitis with fibrosis. cystic structures, abscesses, hematomas, and granulomas are examples of focal parenchymal liver disease. these lesions are usually easily detected sonographically. sonography seldom allows a definitive diagnosis of hepatic parenchymal disease to be made. in one study the overall accuracy of ultrasound for discrimination among different categories of diffuse liver disease was 36.5% for dogs and 54.6% for cats. hepatic lipidosis in cats could be diagnosed slightly more accurately than other diffuse hepatic diseases. 102 cytologic or histologic evaluation of a hepatic tissue sample is usually needed to make a definitive diagnosis. hepatic neoplasia, whether primary or metastatic, can be diffuse, multifocal, or focal in its distribution. round cell tumors are the most likely tumor type to diffusely infiltrate the liver. these tumors can cause hypoechoic, hyperechoic, or mixed-echoic changes, or appreciated radiographically for patients with pss. urate uroliths can be radiolucent so they might not be visible on plain abdominal radiographs. hepatomegaly can be generalized or focal. generalized hepatomegaly can be caused by a number of conditions including neoplasia, vacuolar hepatopathies, congestion, or amyloidosis. focal hepatomegaly can be caused by neoplasia, abscesses, granulomas, or a liver lobe torsion. radiographic signs associated with hepatomegaly are rounded hepatic borders, caudal displacement of the gastric axis, and extension of the hepatic silhouette beyond the costal arch. radiography does not allow appreciation of mild hepatomegaly. additionally, it can be normal for the hepatic silhouette to extend beyond the costal arch in brachycephalic breeds, chondrodystrophic breeds, neonatal animals, or geriatric animals. 91 the liver is normally appreciated as an area of homogenous softtissue opacity on radiographs. radiolucent areas within the liver indicate accumulation of gas within the hepatic parenchyma, biliary tract, or portal vasculature. gas in the parenchyma of the liver can be associated with an hepatic abscess. 92 gas in or around the gallbladder has been reported in dogs with emphysematous cholecystitis. 93 although uncommon in dogs and cats, if choleliths or choledocholiths contain enough calcium, they may be appreciated as mineral opacities within the hepatic silhouette. 94 mineralization of the gallbladder wall can be associated with a biliary adenocarcinoma in the dog. 95 parenchymal mineralization can be associated with granulomas, abscesses, 96 hematomas, neoplasia, or hepatic necrosis. 91 angiography allows the visualization of the hepatoportal vasculature, including abnormal vessels. this often provides a definitive diagnosis of pss and is indicated in patients that are suspected of having a pss, where the shunt cannot be adequately evaluated by abdominal ultrasound. anatomical characterization of congenital pss is important when planning attenuation, and angiographic procedures often allow this. there are several techniques for mesenteric portography. operative mesenteric portography is commonly performed immediately prior to surgical attenuation of a congenital pss and involves catheterization of a mesenteric vein and injection of a contrast agent. operative contrast portography allows evaluation of the portal vasculature before and after shunt attenuation. this technique has the disadvantage of being relatively invasive. cranial mesenteric portography can be accomplished less invasively by using ultrasound guidance to percutaneously catheterize the splenic vein. 97 however, this can be technically demanding and may not be possible in smaller patients. transvenous retrograde portography has been described and involves the catheterization of the jugular vein. 98 this technique allows selective catheterization of the shunting vessel and measurement of portal pressures (figure 61-7) . transvenous retrograde portography has been applied during percutaneous transjugular coil embolization of intrahepatic shunts. 99 percutaneous splenoportography involves percutaneous injection of contrast media into the spleen. this technique is simple to perform, but there is a risk of complications such as splenic infarction or hemorrhage. abdominal ultrasonography is the most commonly used imaging modality for evaluating small animal patients with suspected hepatobiliary disease. ultrasonography allows assessment of the hepatic parenchyma and biliary tract. evidence of extrahepatic disease causing a secondary hepatopathy may also be detected. additionally, ultrasound guidance is often used when collecting samples for cytologic and histologic evaluation of the liver. gallbladder mucoceles can also lead to biliary obstruction, which might also be appreciated sonographically. the sensitivity of ultrasound for the detection of a gallbladder wall rupture in dogs with gallbladder mucoceles is reported to be 85.7%. 109 abdominal ultrasound can be used to assess the liver for vascular disease. congenital pss is classified as being intrahepatic or extrahepatic. although angiographic techniques are considered to be the gold standard for the detection and characterization of pss, abdominal ultrasound is being used increasingly for this purpose. sonographic assessment of the portal vasculature is time-consuming and highly operator dependent. because of this there should be a high index of suspicion for pss before performing these studies. secondary findings consistent with pss include mild bilateral renomegaly, urolithiasis (because of urate crystalluria), and microhepatia. ascites and hepatic parenchymal changes are not consistent with congenital pss. extrahepatic shunts typically occur in small-breed dogs and arise from the splenic vein or the right gastric vein while intrahepatic shunts typically occur in larger breeds of dogs and arise from the right or left portal branch. an intrahepatic pss is usually easier to detect sonographically than an extrahepatic pss in dogs. cats typically have single extrahepatic shunts with a wider degree of anatomical variation than in the dog. ultrasonography has been reported to have a sensitivity of 92% and a specificity of 98% for detecting pss in dogs. 110 portal hypertension can develop as a result of chronic hepatitis with fibrosis, hepatic arterioportal fistulas, portal vein thrombosis, primary portal vein hypoplasia, extraluminal compression of the portal vein, or after ligation of a congenital pss. the finding of hepatofugal or reduced velocity hepatopetal blood flow using doppler ultrasound is consistent with portal hypertension. 111 however, not all patients with portal hypertension will have these changes. ascites frequently, but not always, develops secondary to portal hypertension and this can be readily detected on abdominal ultrasound examination. acquired portosystemic collaterals (also known as acquired pss) may develop when sustained prehepatic or hepatic portal hypertension is present. 112 sonography may allow detection of portal hypertension and apscapsc although apsc vessels are more difficult to identify than congenital pss. posthepatic portal hypertension does not result in the development of may not affect the echogenicity of the liver at all. neoplasia can also lead to the appearance of nodules within the hepatic parenchyma. malignant liver nodules have a variable appearance and size and can be difficult to distinguish from nonmalignant conditions such as cysts, hematomas, benign hyperplastic nodules, granulomas, or abscesses. the finding of one or more target lesions in the liver or spleen had a positive predictive value of 74% for detecting malignancy, and thus should not be considered a specific finding. 103 cytologic or histologic evaluation of a tissue sample is needed to differentiate between malignant and benign liver nodules. tumors, such as hepatoma or hepatocellular carcinoma, can also focally infiltrate the liver. contrast-enhanced harmonic ultrasound allows assessment of tissue perfusion patterns. gas-filled microbubbles are administered intravenously to the patient. the microbubbles are relatively echogenic. when they reach the tissue of interest, they produce a more potent harmonic signal than the surrounding tissue. this technique allows enhanced differentiation between tissues with varying perfusion patterns. in one study the sensitivity of contrast enhanced ultrasound for differentiation between benign and malignant liver nodules in dogs was reported to be 100% and the specificity was reported to be 94.1%. 104 sonography is also a valuable tool for the evaluation of the biliary system. biliary disease can be classified as being obstructive or nonobstructive. the term cholangitis refers to a group of nonobstructive biliary diseases, which are more common in cats than in dogs. typical ultrasound findings in cats include a hypoechoic hepatic parenchyma and prominent portal vasculature. 105 additional findings can include evidence of pancreatic inflammation, thickening of the gallbladder wall, and dilation of the intrahepatic and extrahepatic biliary system. it is important to note these changes are not always present. cytologic or histologic confirmation and bacterial culture are needed to confirm this diagnosis. generalized gallbladder wall thickening can occur as a result of cholecystitis, cholangitis, or hepatitis. however, the gallbladder wall can also appear to be thickened when peritoneal effusion or hypoproteinemia are present. gall bladder wall masses can be identified sonographically as a focal thickening of the gallbladder wall. sonography has also been used to assess gallbladder motility in dogs. 106 it should be noted that gravity dependent gallbladder sludge can be found in dogs without hepatobiliary disease, so this finding should be considered incidental. 107 abdominal ultrasound is the most commonly used imaging modality for the detection of biliary obstruction in dogs and cats. findings consistent with biliary obstruction include common bile duct distention, intra-and extrahepatic bile duct distention, and/or gallbladder dilation. a retrospective study showed that common bile duct dilation greater than 4 mm was 97% sensitive for the detection of biliary obstruction in cats. 108 sonography can also aid in identifying the cause of biliary obstruction. biliary obstruction can be classified as being luminal or extraluminal. extraluminal causes include nonneoplastic pancreatic disease, abdominal adhesions, and, rarely, pancreatic neoplasia. luminal causes include gallbladder mucocele, biliary neoplasia, inflammation, and cholecystolithiasis. biliary tract obstruction can progress to biliary rupture and bile peritonitis. sonographic signs of biliary rupture include loss of gallbladder wall continuity, free peritoneal fluid, and signs of localized peritonitis. gallbladder mucoceles occur in the dog, but have not been described in cats. mucoceles have a variable sonographic appearance; typical findings include a stellate or finely striated bile pattern with a hypoechoic rim, which is not gravity dependent, and gb lt specificity of 100%. 119 the disadvantages of ct and mri include their limited availability, cost, and the need for anesthesia. although cytologic evaluation of the liver provides a definitive diagnosis, often histologic examination is also required. there are a variety of techniques to collect cytologic samples of the hepatobiliary system. abdominal effusion, when present, can be collected percutaneously. fine-needle aspirates (fna) of the liver can be collected percutaneously under ultrasound guidance. cholecystocentesis can also be performed percutaneously with ultrasound apsc vessels, but can lead to a distention of hepatic veins and ascites. nuclear scintigraphy involves administering a radioactive tracer substance (radiopharmaceutical) to the patient, which localizes to a specific organ or tissue. the radioactive decay of this substance is detected by a gamma camera and used to form images. scintigraphy has been used to detect pss and to assess gallbladder emptying in small animals. however, specialized equipment and a license for the use of radioisotopes are required. consequently, availability of this imaging modality is currently limited to academic institutions and specialty referral hospitals. technetium-99m pertechnetate is the most commonly used radiopharmaceutical for assessing the portal circulation of small animal patients. two techniques have been described: per-rectal portal scintigraphy and transsplenic portal scintigraphy. by analyzing the radiation emitted from regions of interest drawn over the patient's liver and heart, pss can be detected and a shunt fraction can be calculated. this allows for the minimally invasive diagnosis of pss, differentiation of pss from portal vein hypoplasia without portal hypotension (previously known as microvascular dysplasia), and comparison of the degree of shunting before and after shunt attenuation. transsplenic portal scintigraphy is preferred over perrectal portal scintigraphy as it is simpler to perform, uses lower doses of the radiopharmaceutical, and is more sensitive (figure 61-9 ). transsplenic portal scintigraphy is 100% sensitive and specific for the diagnosis of congenital pss, and significantly more likely than per-rectal portal scintigraphy to detect shunt number and termination in dogs. 113 nuclear scintigraphy has been used to quantify liver function and to assess biliary tract patency in dogs. in a retrospective study hepatobiliary scintigraphy was found to be 83% sensitive and 94% specific for the detection of extrahepatic biliary obstruction in dogs and cats. 114 computed tomography (ct) (figure 61 -10) and magnetic resonance imaging (mri) have been used to detect hepatic parenchymal neoplasia in humans. compared to abdominal ultrasound these techniques have an improved accuracy for the diagnosis of hepatic neoplasia in humans. however, there is limited data in the veterinary literature evaluating their diagnostic performance. in one study the diagnostic accuracy of ct for detecting hepatic masses was not found to be significantly different from that of abdominal ultrasound in dogs. 115 in another study mri was found to have a sensitivity of 100% and a specificity of 86% for the differentiation between benign and malignant liver lesions in dogs. 116 ct angiography is being used increasingly in dogs for the diagnosis of congenital pss and other hepatic vascular diseases. it offers the advantage of being less invasive than operative angiography, allows for improved assessment of the portal vasculature, and allows the creation of a three-dimensional reconstruction. the vasculature detail afforded by ct angiography is particularly useful when planning attenuation of a congenital pss. the diagnostic utility of ct angiography for detecting and characterizing pss in dogs was shown to compare favorably to that of other techniques, including surgical exploration. 117 transsplenic ct portography has been described in dogs without pss. this technique offers more intense enhancement of the splenic and portal veins than ct angiography. 118 mri angiography diagnoses pss in dogs with a sensitivity of 80% and a the findings above may aid in making a diagnosis of liver disease but are not a substitute for histopathologic analysis, as cytologic specimens do not allow assessment of the hepatic architecture. furthermore, only a tiny proportion of the liver is sampled when cytologic samples are examined. these limitations are reflected by the results of a retrospective study that found the overall agreement between the histopathologic and cytologic diagnosis of liver disease to be 30.3% for dogs and 51.2% for cats. 123 cytologic evaluation of bile can also be useful for the diagnosis of biliary disorders, particularly in cats. world small animal veterinary association (wsava) standards for the clinical and histological diagnosis of canine and feline liver disease suggest that the cytologic evaluation of bile forms part of the minimum diagnostic requirement for cats with extrahepatic cholestasis and for dogs guidance. these techniques are minimally invasive and the risk of complications is relatively low but caution should be exercised in patients with bleeding disorders. liver disease can cause abdominal effusion by several mechanisms. in cases with hepatic insufficiency, severe hypoalbuminemia (<1.5 g/dl) can occur. this can lead to the formation of a pure transudate as the result of a reduced plasma colloid oncotic pressure. increased capillary hydrostatic pressure because of portal hypertension may lead to formation of a pure or modified transudate. hepatic neoplasia can also lead to a formation of a modified transudate. biliary tract rupture can lead to bile peritonitis and abdominal effusion. an exudate with a bilirubin concentration greater than twice that of the plasma is suggestive of bile peritonitis. 120 cytologic evaluation of hepatic fna can aid in making a diagnosis of liver disease. suppurative, mixed inflammatory, lymphocytic and, more rarely, eosinophilic patterns of inflammation can be appreciated cytologically. each pattern of inflammation suggests a group of possible diagnoses. the finding of dark green or black bile casts suggests cholestasis. infectious diseases such as histoplasmosis can be definitively diagnosed based on the cytologic finding of the infectious agent ( figure 61-11 ). hepatocellular vacuolation can be classified as being caused by lipid or not. lipid vacuolation of hepatocytes is characterized by colorless cytoplasmic vacuoles. severe lipid vacuolation is suggestive of hepatic lipidosis in cats ( figure 61 -12). however, feline hepatic lipidosis often occurs secondary to another disease process. a group of cats with cytologic findings suggestive of hepatic lipidosis were reported to have underlying infiltrative liver disease. 121 nonlipid vacuolation is characterized by generalized hepatocyte swelling and lacy vacuolation ( figure 61-13 ). vacuolar hepatopathy occurs secondary to a wide variety of extrahepatic disease processes in dogs. 122 metastatic tumors and round cell tumors, such as lymphoma ( figure 61-14) , affecting the liver can often be diagnosed cytologically. additionally, cytologic evaluation can aid in distinguishing liver nodules because of extramedullary hematopoiesis from those caused by neoplasia. however, it is not possible to distinguish hepatic nodular hyperplasia from hepatic adenoma or well-differentiated carcinoma cytologically. some cases of hepatocellular carcinoma can be diagnosed cytologically if criteria for malignancy are present. and cats suspected to have cholangitis. 124 the finding of neutrophils and bacteria on bile cytology supports a diagnosis of feline neutrophilic cholangitis. cytology is essential for the diagnosis of this disease, as cats with neutrophilic cholangitis may not have typical hepatic histopathologic changes and it can be difficult to distinguish these cats from those with lymphocytic cholangitis. bile should also be submitted for aerobic and anaerobic bacteriologic culture. histopathologic evaluation is required to make a definitive diagnosis of most liver diseases. histopathologic evaluation of the liver allows a morphologic and sometimes an etiologic diagnosis to be made (see chapter 29) . in addition to routine staining with hematoxylin and eosin, a variety of other staining techniques can be employed to demonstrate hepatic pathology. to optimize the value of histopathologic evaluation of the liver, particular attention should be paid to specimen collection, specimen handling, and communication between the clinician and the pathologist. although liver biopsy is considered to be relatively safe, the patient should be assessed for bleeding disorders before this procedure. this assessment should include a platelet count, coagulation times, and a buccal mucosal bleeding time. liver biopsies can be collected in a number of ways. each method has advantages and disadvantages, and there is controversy in the veterinary literature as to which technique is optimal. laparotomy allows collection of relatively large wedge biopsies, with direct visualization. this technique does not require specialized equipment or training, and excessive bleeding can be readily identified. however, laparotomy requires general anesthesia and is the most invasive biopsy technique. percutaneous needle biopsy techniques have been described. these techniques may be possible under heavy sedation and are the leastinvasive method for collecting liver biopsies. ultrasound guidance is often used, allowing biopsy of focal lesions. it is also possible to biopsy tissue that is deeper within the hepatic parenchyma than is possible with other techniques. however, the specimens that are collected are relatively small and may be inadequate for accurate assessment in some patients. a prospective study showed that there was agreement between the histomorphologic diagnoses made upon examination of needle biopsies and those made on wedge biopsies collected during laparotomy or necropsy for only 48% of dogs and cats. 125 excessive hemorrhage after biopsy may not be identified immediately. laparoscopy allows collection of biopsies using forceps with laparoscopic guidance. this technique requires general anesthesia, but is less invasive than laparotomy. the biopsies collected are larger than needle biopsies and excessive bleeding can be visualized. however, laparoscopy requires specialized equipment and training. the use of biopsy forceps may result in crushing artifact and the tissue collected may be too superficial to identify lesions that lie deeper within the hepatic parenchyma. 126 regardless of the technique used, a tiny proportion of the organ is sampled and, because liver disease can affect the hepatic parenchyma in a heterogeneous manner, sampling error is possible. to reduce the effect of sampling error, several biopsies from different areas of the liver should be collected and focal lesions should be specifically biopsied. the clinician should provide the pathologist with all the pertinent information from the patient's history, physical examination findings, the results of laboratory testing, and the findings from diagnostic imaging. in turn the histomorphologic diagnosis that the pathologist makes should be interpreted by the clinician along with the other clinical data. when the histopathologic diagnosis does not fit the clinical picture, the pathologist should be consulted and when necessary a second opinion should be requested. variation in the assessment of hepatic pathology between pathologists was highlighted by a study that found agreement between examiners for only 44% of needle biopsies and 65% of wedge biopsies examined. 125 hopefully, the adoption of wsava standards for the clinical and histological diagnosis of canine and feline liver diseases since the aforementioned study will reduce this interobserver variation. quantification of hepatic metal concentrations requires submission of tissue for flame atomic absorption spectroscopy. although zinc has a role as an antioxidant, hepatic copper and iron retention can lead to oxidative liver injury. copper is the most frequently course of the evaluation. in patients that are asymptomatic and have abnormal biochemical testing, repeat evaluation is sometimes warranted. a general guideline is to obtain a liver biopsy in asymptomatic patients if there are moderate to severe elevations in serum hepatic enzyme activities that persist for at least 3 months, or if there are mild to moderate elevations in serum hepatic enzyme activities that persist for at least 6 months. if clinical signs of hepatic disease develop, then biopsy should not be unreasonably delayed. if there are concurrent elevations in serum bile acids, biopsy should also not be delayed. other indications for hepatic biopsy are ultrasound imaging abnormalities. if there are focal hepatic masses or diffuse echotextural changes, a biopsy may be warranted, depending on results of laboratory testing. in one study, abdominal ultrasound findings alone were not reliable for obtaining a diagnosis of infiltrative hepatic disease with diffuse changes in echogenicity (either hypoechoic or hyperechoic, uniform or mottled). 1 in another study, sonographic detection of a hepatic mass greater than or equal to 3 cm, ascites, abnormal hepatic lymph node(s), and abnormal spleen were predictive of liver neoplasia based on cytology. 2 conversely, sonographic detection of hepatic nodules less than 3 cm was predictive of vacuolar hepatopathy on cytology. thus several sonographic findings, alone or combined, may be predictive of liver ultrasound-guided fine-needle aspiration cytology results. in light of the fact that ultrasound-guided fine-needle aspiration cytology of the liver has limitations , the results of ultrasound and cytology should be adjuncts to other findings. another indication for hepatic biopsy is the need to assess response to therapy. in cases of chronic hepatitis in dogs, it is often difficult to determine if there is ongoing inflammation and resolution/ progression of fibrosis during long-term therapy. this is particularly true when the patient is receiving glucocorticoid therapy as these medications cause variable increases in serum alp and transaminase activities independent of the underlying disease. repeat or serial hepatic biopsy analysis is often helpful to guide therapeutic decisions in these cases. among the most serious complications of liver biopsies are hemorrhage, infections, and injury to the adjacent viscera. consequently the clinician must take into account the clinical question, the appropriate invasive biopsy method, and methods of managing postbiopsy complications. postbiopsy hemorrhage is often the first concern, although it is unclear as to what the best predictor of hemorrhage is in patients about to undergo hepatic sampling. in one study of 200 human patients in which bleeding was evaluated laparoscopically, there was no correlation between any in vitro coagulation test and "liver bleeding time." 3 other studies in man have used laparoscopy and ultrasonography to assess hepatic bleeding time following needle biopsy, and most have shown similar poor correlation between coagulopathies and hepatic bleeding times. similar studies have not been reported in veterinary medicine. there also have been studies in human and veterinary medicine evaluating risk factors for bleeding complications (as opposed to "hepatic bleeding times"). 4,5 bigge et al. correlated coagulation profile findings and bleeding complications after ultrasound-guided biopsies in 310 dogs and 124 cats. 5 there was no apparent correlation between coagulation parameters and major complications following liver biopsy. studies show that clotting times assessing proteins induced by vitamin k antagonism are more sensitive in detecting coagulopathies in patients with hepatic disease. 6, 7 the measured of these metals and quantification is essential for the diagnosis of hepatic copper retention. these measurements are usually performed on freeze-dried pieces of liver. specimens for metal measurement should not be stored in saline and should be kept in metal-free containers. recently, it was shown that measurements of the concentration of copper and iron, but not zinc, can be ascertained from deparaffinized-archived liver tissue. 127 hepatobiliary diseases can be challenging to diagnose. although diagnostic tests that employ biochemical, molecular biologic, serologic, functional, as well as imaging techniques are capable of establishing the etiology of some chronic or acute liver diseases, in most instances the gold standard for definitive diagnosis and the assessment of stage and severity of liver diseases is the histologic evaluation of a liver sample. recent advances in imaging technology, the use of multiple imaging modalities, and newer biopsy methods have resulted in improvement in the ability to safely procure hepatic tissue for evaluation. there are several means of obtaining hepatic samples including fine-needle aspiration, ultrasound-guided biopsy, laparoscopy, and laparotomy. all techniques have both advantages and disadvantages, which should be carefully considered before choosing the appropriate sampling method. many biochemical tests are available to evaluate the anabolic and/ or catabolic function of the liver and the hepatic circulation. these include measurement of concentrations of bile acids, ammonia, bilirubin, and the ability to excrete organic dyes. other tests of hepatic function include measurement of serum albumin, glucose, urea nitrogen, and clotting factor analysis. hepatic function can be markedly abnormal despite maintenance of the hepatocellular membrane and therefore normal serum activities of hepatic enzymes. examples include psss, terminal cirrhosis, and metastatic hepatic neoplasia. likewise, the liver can continue normal anabolic or catabolic function despite severe hepatocyte leakage of intracellular enzymes because of its marked reserve capacity. this can occur, for example, in certain cases of hepatocellular necrosis, blunt abdominal trauma, or primary hepatic neoplasia. thus, the limitations of serum hepatic enzyme activities must be taken into consideration. hepatocellular leakage enzyme activities include alt and ast. enzyme activities that increase with biliary tract obstruction include serum alp and ggt. no laboratory test identifies a specific problem, helps determine specific therapeutic management, or predicts an outcome. this is because different diseases produce similar alterations in hepatic function or in laboratory tests. once biochemical tests identify the presence of hepatic disease, the diagnosis must be pursued further. in some instances, diagnostic imaging can reveal specific abnormalities (e.g., psss and extrahepatic bile duct obstruction). when results of imaging do not give a specific etiology, the next step is often to pursue a morphologic diagnosis obtained by analysis of a biopsy specimen. often it is a judgment call as to when to pursue hepatic biopsy. in cases with severe clinical signs and/or severe biochemical abnormalities, biopsy is usually warranted early in the fine-needle aspiration has several advantages. little to no sedation is usually required. because the size of the needle is so small, there is little risk of hemorrhage. therefore multiple sites can easily be sampled. the procedure is rapid and can usually be performed on an outpatient basis. there is also less cost to the client. the primary disadvantage of fine-needle aspiration is its questionable accuracy. the sample size often limits the number of available cells to obtain an accurate diagnosis, and hemodilution makes it difficult to assess whether inflammatory cells were present in the liver or peripheral blood. there are several important elements used to interpret pathologic information including lobular architecture, presence and location of inflammation within a lobule, presence and severity of fibrosis, metal accumulation, vascular abnormalities, and lobule heterogeneity. these criteria cannot be accurately determined using a cytologic sample obtained using fna. several studies have compared fine-needle aspiration cytology with biopsy with histopathology. [9] [10] [11] [12] in one study with a total of 34 cases, there was good correlation in 35% of cases, partial correlation in 35% of cases, and no correlation in 30% of cases. 9 poor correlation was found with a variety of histologic changes, including vacuolar change, lipidosis, cholestasis, inflammation, and neoplasia. in a similar study with 97 cases, complete agreement between fineneedle aspiration and histopathology was seen in only 30% of cases in dogs: 25% agreement with inflammation, 14% agreement with neoplasia (mainly carcinoma), and 64% agreement with vacuolar hepatopathy. 10 in cats, there was overall agreement in 51% of cases: 27% agreement with inflammation, 33% agreement with neoplasia (lymphoma), and 64% agreement with vacuolar hepatopathy. although vacuolar hepatopathy was the most sensitive diagnosis, it was also the most common misdiagnosis using cytology. in another study, the best correlation between hepatic cytology and biopsy was seen with lipidosis, lymphoma, and carcinoma, whereas the worst performance was seen with inflammatory and fibrotic disorders. 11 another study found high sensitivity and specificity with fine-needle aspiration in detecting inflammatory hepatic disease in dogs. 12 however, further information was not provided such as the severity of the inflammation or other histopathologic features. additionally, for noninflammatory hepatic disease, cytology was inaccurate in 76% of cases. proteins-induced-by-vitamin-k-antagonism test is more than twice as sensitive in dogs and more than three times as sensitive in cats in detecting coagulopathies compared with prothrombin time (pt) and aptt. 6, 7 however, in a pilot study performed by me, hepatic bleeding times assessed via laparoscopy did not correlate with proteins induced by vitamin k antagonism times. thus it appears that indices of coagulation in the peripheral blood are generally unreliable guides of the risk of bleeding after liver biopsy, and hence, are of limited value in determining contraindications to this procedure. this lack of correlation may be explained by the high concentration of clotting factors in the hepatic parenchyma and by mechanical compression of the needle tract by the elastic tissue within the liver. in most cases of significant hemorrhage, technical errors such as damaging a large vessel are the cause rather than persistent oozing from a needle biopsy site. controlled studies in veterinary patients will be necessary to make final conclusions regarding postbiopsy hemorrhage in the patient with a coagulopathy. in one study of normal dogs, biopsies taken from the left lateral hepatic lobe using a biopsy punch, biopsy needle, ligature method, laparoscopic biopsy forceps, and ultrasonically activated scalpel resulted in minimal hemorrhage (<2 ml). 8 however, this investigation did not assess the risk of hemorrhage in dogs or cats with hepatic disease. these risks will be discussed later under each sampling method. with the exception of fine-needle aspirations, each patient should have a prebiopsy packed cell volume and 3 and 6 hours postbiopsy packed cell volume for close monitoring of potential hemorrhage. fine-needle aspiration involves obtaining a small amount of hepatic tissue for cytologic analysis, and is typically performed in conjunction with, and guided by, ultrasound. ultrasound imaging helps determine if there is a diffuse abnormality (e.g., increased or decreased echogenicity, diffuse mottling) or if there are focal abnormalities (e.g., discrete nodules, cysts, masses, or focal areas of heterogenous mottling). an appropriate site to be sampled is chosen. often multiple sites are chosen to represent different lobes, and in the case of focal lesions, to sample more than one area of abnormal tissue and sample seemingly normal tissue. the sites are also chosen based on accessibility. for example, a solitary nodule in the dorsocranial aspect of the liver in a large deep-chested dog would be impossible to reach with a 1.5-inch needle. a lesion adjacent to the gallbladder or caudal vena cava would involve considerable risk. the clinician would need to decide whether the relative risk of sampling such lesions is the appropriate decision, or whether other methods of sampling would be more appropriate such as laparoscopy or laparotomy. figure 61 -15 depicts a typical setup for fine-needle aspiration. usually a 22-gauge, 1.5-inch needle is used. for most patients, the procedure is performed without sedation or local anesthetic. if it is determined that the animal is moving too much during the initial ultrasound examination, a sedative may be necessary (or an anesthetic in extreme cases). the needle is inserted without a syringe using ultrasound guidance. the needle is rapidly agitated in and out (sometimes referred to as mimicking the action of a sewing machine) and simultaneously twisted multiple times for a few seconds to obtain a sample. this method relies on capillary action rather than suction to get tissue into the needle, resulting in less hemodilution. after removing the needle from the liver, a syringe is attached and cells are expelled onto a glass slide for cytologic examination. often three to five separate attempts are made to increase the sample size and diversity. examination is performed prior to biopsy. this allows planning of the procedure based on echo pattern, lesion size, proximity to other organs, proximity to blood vessels, determination of cystic or solid tissue, and optimal approach of the needle path. care must be taken prior to taking samples to ensure that vessels and other organs are not within the path of the needle. for diffuse lesions, the transducer is typically placed caudal and to the left of the xiphoid, and aimed at the left medial or lateral lobes. in patients with a small liver, it may be difficult to adequately visualize the needle without gastric gas interference. placing these animals in a 45-degree right lateral oblique position can reduce this interference. if the animal is under general anesthesia, an assistant can compress a rebreathing bag to hold the animal in deep inspiration, which serves to move the diaphragm and liver caudally to improve visualization. the area is surgically prepared. the ultrasound transducer is covered with sterile wrap and sterile lubricant is used to enhance skin contact. a small stab incision is made in the skin at the desired needle insertion site. while one hand maneuvers the transducer, the other hand advances the needle into the liver under direct ultrasound visualization. the image should be optimized to maximize the chance of recognizing the needle within the liver. to allow distinction of the needle from other echogenic structures, the needle can be gently moved in and out with minimal movements (attempting to move the liver within the abdominal cavity rather than the needle within the liver). occasionally the needle cannot be seen, and indirect evidence of organ penetration must be used such as movement of the liver or visualization of movement at the liver border. the needle is then directed so the trajectory will avoid other structures when it is fired. the needle is then fired, and immediately removed. for most cases, four to five samples are obtained, and are submitted for aerobic/anaerobic culture, histopathologic evaluation, and metal (copper, zinc, and iron) quantification. in one study, liver tissues with high metal concentrations had significantly lower copper and iron in needle-core versus wedge biopsy specimens. 13 consequently the value of needle-core biopsy specimens for measurement of metal concentrations is questionable. careful examination for post-biopsy hemorrhage is then performed. external digital pressure may be used to help control hemorrhage in smaller patients. usually an abdominal compression wrap is ineffective for controlling hemorrhage. ultrasound-guided biopsy has many of the advantages of fine-needle aspiration, including the need for minimal sedation in some patients, the ability to sample multiple sites, and low to moderate cost to the client. additionally, tissue is obtained for histopathology. one disadvantage of ultrasound-guided biopsy is the risk of bleeding (especially when multiple sites are sampled and largergauge needles are used). in one study, 96 percutaneous transabdominal hepatic needle biopsy samples were obtained with no adverse consequences noted 14 ; however, this study was performed in normal dogs, and still carries high risk. additional disadvantages of ultrasound-guided biopsy include the needing sedation or anesthesia in some patients, difficulty of imaging small livers, difficulty of obtaining liver tissue in patients with fibrosis, and, most importantly, the obtaining of samples that have a questionable representation of the underlying hepatic pathology. the diagnostic accuracy of needle biopsy has been questioned by many clinicians, observing that results of needle biopsy analysis often do not adequately reflect the clinical and laboratory features of the patient. this questionable accuracy is in most part a result of potential for sampling error. this method still results in a relatively small sample size, possible although fine-needle aspiration is easy to perform, involves little risk, and little to no sedation, the information is of little value if it is inaccurate as often as it is accurate. there is institutional bias regarding its accuracy, which may relate to the experience and expertise of the cytologists. given its clear limitations, fine-needle aspiration is best used as an adjunctive diagnostic modality in conjunction with other techniques or clinical findings, and does not replace histopathology. the clinician must be aware of its inherent inaccuracy before undertaking fine-needle aspiration and relying on the cytologic findings. ultrasound-guided hepatic biopsy uses a cutting-type needle as a sampling tool. automated needles are preferred and should be either completely automated or semiautomated. these are spring-loaded needles similar in style to the manual tru-cut needle. completely automated needles thrust the inner obturator (containing the biopsy tray or specimen notch) followed by the outer cutting sheath into the liver in a fraction of a second. these needles can be operated with one hand while the other hand operates an ultrasound transducer to allow precise placement of the biopsy instrument. there is minimal displacement of the liver, a shorter intraparenchymal phase, and a more reliable yield of tissue. this allows a smaller diameter needle to be used and a lighter degree of sedation in some cases. using the rapid cutting action, the hepatic tissue tends to be less fragmented. semiautomated needles require manual placement of the internal obturator into the liver, followed by an automatic thrusting of the outer cutting sheath by a spring-loaded mechanism. these needles have the additional advantage of control over the final needle position, as the tip of the needle can be precisely localized before the outer cutting sheath is deployed. i generally use a 16-gauge needle for ultrasound-guided hepatic biopsy. figure 61 -16 depicts a typical setup for ultrasound-guided biopsy. in most dogs, the liver can be biopsied using local anesthesia and minimal sedation. most cats require general anesthesia to safely obtain tissue. it must be emphasized that the degree of sedation must be tailored to each individual patient. a careful ultrasound peritoneal space through a stab incision using a number 11 scalpel blade. after ensuring no obstruction and negative pressure using the infusion and aspiration of saline, the abdomen is insufflated with carbon dioxide gas and maintained at a pressure of approximately 12 mm hg. a scope port (cannula) is then placed 4 cm right lateral to the veress needle. the veress needle is then removed and replaced with an instrument port. hepatic sampling is achieved using a "spoon" or oval cup biopsy forceps. multiple samples are obtained under direct visualization, and samples are submitted for aerobic/anaerobic culture, histopathologic evaluation, and metal (copper, zinc, and iron) quantification. following procurement of all the biopsy specimens, the sites are inspected for hemorrhage. the abdomen is then decompressed, and lidocaine and bupivacaine are infused into the peritoneal cavity through either port. both the instrument and scope ports are removed, and the port site incisions are closed using either a cruciate or simple interrupted pattern in the body wall, subcutaneous tissue, and skin. this technique enables gross evaluation of the entire liver, extrahepatic biliary system, and surrounding structures while obtaining multiple large specimens of liver. the ability to obtain multiple samples decreases the risk for sampling artifact in cases of regional diversity within the liver. additionally, by directly visualizing the hepatic parenchyma, the clinician can correlate the histopathologic findings and clinical data with the gross appearance of the liver to render the most accurate diagnosis. this method also enables the visualization of smaller masses and irregularities that may not be evident with ultrasonographic imaging. these masses can also be individually sampled. laparoscopy also gives the clinician an excellent view of the liver regardless of the hepatic size or conformation of the patient, making it an easy method to sample the liver in patients that are difficult to image with ultrasound. there is generally minimal bleeding during this procedure, even in patients with in vitro coagulopathies. using a "spoon" or oval-cup biopsy forceps typically results in a marked decrease in the amount of hemorrhage when compared with needle biopsies. any hemorrhage can be directly visualized for adequate clot formation. if hemorrhage persists, direct pressure using a blunt probe for 5 minutes can be used. if the site continues to bleed, electrocautery can be applied to the biopsy site or a topical hemostatic agent (gelfoam) can be placed directly on the biopsy site using laparoscopic forceps. disadvantages of laparoscopy include the need for expensive equipment, the need for extensive training, the need for general anesthesia in most cases, and higher cost to the client. laparoscopy gives the clinician the advantages of a laparotomy (large sample size, ability to best direct sampling, and ability to take multiple samples, thus resulting in the highest diagnostic accuracy), though with a relatively minimally invasive procedure. the complication rate (especially hemorrhage) is far less than with ultrasoundguided biopsy in my practice. for these reasons, it is my method of choice for obtaining hepatic biopsy specimens in most cases. wedge biopsy via laparotomy is another potential method for obtaining hepatic biopsies. if a random liver biopsy is needed and a section of liver is protruding, a guillotine suture can be used. a preformed encircling ligature of 4-0 monofilament absorbable suture material is placed around the protruding section of liver. fragmentation of fibrous tissue, and may not enable sampling of abnormalities located in other lobes (the left medial or lateral lobes are generally sampled because of their ease of imaging). in one study, percutaneous hepatic sampling using core biopsies resulted in 92% diagnostic quality samples, however these were not compared with large wedge biopsy to assess the accuracy of this method. 15 in another study, the diagnostic accuracy of the tru-cut-type needle biopsy was compared with the gold standard of surgical wedge biopsy of the liver in 124 patients. 16 the overall discordance between the two methods was 53% in dogs and 50% in cats, with a greater than 60% discrepancy occurring with chronic hepatitis or cirrhosis, cholangitis/ cholangiohepatitis, portosystemic vascular anomalies, microvascular dysplasia, fibrosis, and miscellaneous disorders. these disorders are the most commonly seen among dogs and cats with hepatobiliary disease. the greatest accuracy was with neoplasia (80% concordance). figure 61 -17 is an example of a mass amenable to an ultrasound-guided needle biopsy. use of a 14-gauge versus 18-gauge needle may reduce this discordance as it raises the number of portal triads sampled from an approximate mean of four to seven, though larger needles carry the risk of increased hemorrhage. in summary, ultrasound-guided hepatic biopsy is relatively easy to perform, but involves more risk to the patient (primarily bleeding). like fine-needle aspiration, ultrasound-guided biopsy has questionable accuracy. the accuracy may be increased by using a larger-gauge needle, but this carries a greater risk of postbiopsy hemorrhage. if the patient is suspected of having inflammatory disease, vascular abnormalities, or significant fibrosis, or is at risk for hemorrhage, laparoscopy or laparotomy should be considered. chapter 28 provides a detailed description of laparoscopic liver biopsy. briefly, laparoscopy is performed under general anesthesia with the patient in dorsal recumbency tilted 45 degrees to the left. a veress needle is placed at the level of the umbilicus into the acute hepatitis and necrosis are common morphologic hepatic lesions in dogs and cats presenting with acute liver disease caused by infectious, toxic, metabolic, and ischemic disorders (box 61-1). however, acute liver disease can also be associated with other pathologic processes such as severe hepatic lipidosis (cats), granulomatous hepatitis (fungal infections), intrahepatic cholestasis (bacterial cholangitis, leptospirosis), and malignant infiltration (lymphoma, malignant histiocytosis). canine adenovirus i, canine and feline herpesvirus in the neonate, clostridium piliforme, and toxoplasma gondii are specific examples of infectious agents that cause acute hepatic necrosis (with variable inflammation), often as part of a multisystemic disorder. 2 although leptospirosis is a well-recognized infectious cause of acute liver disease in dogs, hepatic necrosis is an uncommon histologic feature and hepatic lesions are typically characterized by cholestasis, liver cell dissociation, and nonspecific reactive hepatitis. 3 despite the large number of potential causes of acute hepatitis and necrosis, a specific etiology is often not determined. 4, 5 in a recent case series of 101 dogs with primary hepatitis (acute and chronic hepatitis) that were presented to a referral clinic, 21 dogs were diagnosed with morphologic features of acute hepatitis. 4 a cause could not be determined in the majority of these cases, although increased hepatic copper was detected in five dogs with acute hepatitis, suggesting that copper accumulation could be a significant contributing factor. 4 despite numerous potential causes of hepatocyte death, two general mechanisms are recognized: apoptosis and necrosis. 2 these two mechanisms have traditionally been considered to be distinct events. however, it now appears that apoptosis and necrosis are alternate outcomes of the same initiating causes and signaling pathways. 1 apoptosis is adenosine triphosphate-dependent (caspasedependent) programmed cell death that causes shrinkage of the cell (apoptotic bodies or acidophil bodies) with orderly resorption of cellular contents, minimal leakage of cellular components, and minimal secondary inflammation. 1,2 necrosis occurs when depletion of adenosine triphosphate results in cellular swelling, loss of integrity of the cell membrane and cell lysis, with release of cell contents and secondary inflammation. 1, 2 diffuse hepatic necrosis is the most consistent histological lesion detected in dogs and cats with acute liver failure. 5 acute liver failure (alf) is a rare clinical syndrome (usually fatal) that occurs when a sudden severe insult to the liver compromises at least 70% of functional hepatic mass. liver cell death exceeds hepatic regenerative capacity, resulting in clinical signs of liver failure. 5 the clinical and laboratory features of alf are not specific for the inciting cause but reflect disruption of one or more major hepatic functions. once hepatocellular injury has occurred (and assuming the patient survives), the morphologic hepatic response to injury may include parenchymal regeneration, fibrosis, and ductular proliferation. 2 nearly complete hepatic regeneration is possible if hepatocyte injury is limited and the reticulin network remains intact. 2 with severe parenchymal destruction or extensive loss of hepatocytes, periportal ductular proliferation, hepatic fibrosis, postnecrotic scarring, and regenerative hepatic nodules are more likely. 2 dogs with acute hepatitis may also progress to chronic hepatitis. 4 the clinical presentation of dogs and cats with acute hepatitis and necrosis varies with the underlying cause and the extent and severity of the hepatic lesions. the spectrum of hepatic involvement may the ligature is then tightened until it has crushed the hepatic parenchyma. after completing several throws in the knot, the sample is excised 1 to 2 mm distal to the ligature using metzenbaum scissors or a scalpel blade. if a specific area of liver is needed, a sample can be obtained using the transfixation method or a biopsy punch. the transfixation method entails placing a ligature through the liver lobe approximately 8 to 10 mm from its edge. the ligature is tightened to crush through the hepatic parenchyma along one border of the desired biopsy specimen. an additional throw is made at a right angle to the first ligature, and this throw is tightened to crush the parenchyma of the second border of the specimen. the sample is removed 1 to 2 mm distal to the crushed area using a scalpel blade or metzenbaum scissors. if the desired area does not lie near the edge of a liver lobe, a 6-mm biopsy punch can be used. the biopsy punch should be inserted into the hepatic parenchyma ensuring not to penetrate the opposite surface. if the biopsy site is close to the hilus, extra caution must be used so that no more than half of the thickness of the liver is penetrated. the biopsy sample is removed from the liver using scissors. hemorrhage can be controlled by filling the defect with a topical hemostatic agent (gel foam) and applying digital pressure for 3 to 5 minutes, or by suturing the hepatic capsule with fine, absorbable monofilament suture in a cruciate pattern. akin to laparoscopy, this method has similar advantages and disadvantages as listed previously. although it is more invasive, it allows easier biopsy of other abdominal organs (such as intestine and mesenteric lymph node) and the ability to perform therapeutic maneuvers (such as hepatic mass removal or biliary diversion). etiology hepatocyte death (necrosis and apoptosis) in dogs and cats occurs secondary to a broad variety of insults, including infectious agents, drugs and toxins, hypoxia, immunologic events, and metabolic disorders. hepatic necrosis and acute inflammation often occur together and the relationship between these two processes is complex. acute inflammation may be the primary event, or necrosis of hepatocytes can be followed by a substantial inflammatory response, the "hallmark" of necrotic cell death. 1 the term acute hepatitis traditionally has been used when infectious agents cause hepatocellular necrosis, even though in the early stages, hepatic inflammation can be minimal or absent. 2 controversy exists among veterinary pathologists regarding the preferred terminology (acute hepatitis versus acute hepatic necrosis), when necrosis predominates and is caused by noninfectious insults such as toxins or ischemia. 2 for the purposes of this discussion, lesions of acute hepatitis and acute hepatic necrosis are discussed together, recognizing that the primary contributions of each lesion may be variable, depending on the cause, host response, and passage of time. acute hepatitis, a form of primary hepatitis, should be differentiated from "nonspecific reactive hepatitis," a response of the liver to a variety of extrahepatic disorders that is characterized by focal inflammation without necrosis. 2 nonspecific reactive hepatitis is discussed in a later section of this chapter. synthesis. 7 increased activity of the cholestatic liver enzymes, alkaline phosphatase, and ggt, also commonly occur with acute hepatitis and necrosis, but the magnitude of the increase is much less than for the alt and ast. abnormalities in biochemical tests such as hyperbilirubinemia, increased sbas, hypoglycemia, and hyperammonemia indicate compromised hepatic function. hyperbilirubinemia and bilirubinuria support more significant hepatic injury once prehepatic (hemolytic) causes have been discounted. primary biliary tract disorders including posthepatic mechanisms of hyperbilirubinemia should also be considered in the differential diagnosis. other considerations for hypoglycemia in conjunction with acute liver disease include xylitol toxicity (excess insulin release) and sepsis. hypoalbuminemia usually suggests chronic rather than acute liver disease, because of the long serum half-life of albumin. if azotemia is detected, dehydration, gi blood loss, and concurrent renal damage (e.g., leptospirosis, nonsteroidal antiinflammatory drugs [nsaids]) should be considered. interpretation of azotemia is facilitated by concurrent urinalysis. renal injury is supported by findings of cellular or granular casts, glucosuria, isosthenuria, and proteinuria. the complete blood cell count may reveal an inflammatory response suggesting underlying infectious or inflammatory disorders, and it is also useful for ruling out hemolytic anemia as cause of jaundice. documentation of a coagulopathy is required for the clinical diagnosis of alf. laboratory findings indicative of a coagulopathy include prolonged pt and activated partial thromboplastin time (aptt), decreased fibrinogen, increased fibrin degradation products, and thrombocytopenia. abdominal radiographs are often unremarkable in dogs and cats with acute hepatitis and necrosis. the liver may appear normal or increased in size. on abdominal ultrasound, the liver may appear normal or hypoechoic. thoracic and abdominal imaging may be helpful to evaluate for other causes of acute hepatic disease, and biliary tract disorders. because dogs and cats with acute hepatitis and necrosis present with nonspecific signs of acute liver disease, the clinician should maintain a broad perspective regarding the many potential diseases and processes that can acutely affect the liver. prior to obtaining a liver biopsy, ancillary testing (cytology or biopsy of more accessible lesions, infectious disease titers or molecular tests, diagnostic imaging) should be performed to evaluate for systemic disorders with secondary hepatic effects or multisystemic infections, thus providing a diagnosis of other causes of acute liver disease in a less-invasive manner. when acute hepatitis or hepatic necrosis is suspected (or confirmed by liver biopsy), a thorough history is essential to identify exposure to potential hepatotoxins and infectious agents. the owner should be questioned regarding recent medications, including prescription and over-the-counter drugs, and alternative medicines such as herbal and dietary supplements. the potential for exposure to chemicals or hepatotoxins (amanita mushrooms, blue-green algae, sago palms, aflatoxins, or xylitol) should be assessed (for more details, see "drug and toxin-induced liver injury" section). other pertinent historical questions include current vaccination history (canine adenovirus, leptospirosis), travel history (fungal infections or tick-borne diseases), and exposure to other animals (infectious causes). liver biopsy is required to document the presence of acute hepatitis and necrosis; evaluate for specific causes; and differentiate acute from chronic disease. in patients with mild (or absent) clinical signs and liver enzyme elevations that correspond to recent medication administration, a liver biopsy may be postponed, the medication discontinued, and clinical signs and liver enzymes monitored for include (a) subclinical (biochemical abnormalities only), (b) clinical signs of acute liver disease, or (c) the clinical syndrome of alf. when liver injury is mild (focal necrosis and inflammation), clinical signs may be absent, mild, or related to an underlying cause in another organ system. in this setting, hepatic involvement may not be recognized until biochemical evaluation reveals increased liver enzyme activity or mild hyperbilirubinemia. it has been suggested that many dogs with acute hepatitis are not recognized clinically, because signs are mild and self-limiting, and dogs recover spontaneously regardless of treatment. 6 clinical signs of acute hepatitis include acute onset of lethargy, anorexia, vomiting, diarrhea, pu, and pd, in a previously healthy animal. these are nonspecific findings of acute liver disease, which overlap those of other systemic disorders. the finding of icterus on the physical examination is a more specific indicator of hepatobiliary disease, especially in the absence of anemia. dogs and cats with acute diffuse hepatic necrosis often present with alf. 5 in addition to the signs of acute liver disease described above, animals in alf show signs of he (depression, behavioral changes, dementia, ataxia, pacing, circling, blindness, hypersalivation, seizures, and coma) and clinical evidence of a bleeding tendency (melena, hematemesis, or cutaneous and mucosal hemorrhages), which suggest severe hepatic dysfunction. 5 signs of alf are rapidly progressive (over hours to days) and this clinical syndrome is often fatal, with reported mortality varying from 25% to 100%. 5 with acute hepatic disease, the history typically reveals acute onset of signs in a previously healthy animal. however, liver failure that is recently recognized may not necessarily be recent in onset. with occult chronic liver disease, clinical signs may be vague and go unrecognized by the owner until a final phase of hepatic decompensation. the owner should be questioned about any subtle signs of chronic illness that would suggest the underlying liver disease may be chronic rather than acute, and that the current illness may be an exacerbation or decompensation of chronic liver disease. dogs and cats with alf are generally in good nutritional status compared with those with chronic hepatic disease. findings of cachexia, emaciation, ascites, or edema suggest a more protracted illness and are characteristic of chronic rather than acute liver disease. it is important to make a distinction between acute and chronic liver disease as the intensive supportive care indicated in alf might not be warranted in chronic end-stage liver disease. the long-term prognosis is better for acute hepatitis than chronic hepatitis. 4 an initial database consisting of complete blood cell count, serum chemistry, and urinalysis should be obtained in dogs and cats with acute liver disease. liver enzyme elevations are a common finding in dogs and cats with acute hepatitis and necrosis. with mild hepatic injury or focal hepatic necrosis, increased alt activity may be the only finding on an otherwise unremarkable biochemical profile. alt and ast activities are moderately to markedly increased, because of enzyme leakage from damaged hepatocytes. 7 although alt activity increases with many hepatic diseases, the largest magnitude of increase is seen with acute hepatic necrosis and roughly correlates with the number of involved cells. 7 alt activity may be increased as much as 100 times the upper range of normal, with increases in ast activity that parallel but are generally lower (30 times the upper limit of normal) than the alt. it should be noted that some recognized hepatotoxins (aflatoxin and microcystin in blue-green algae) are not associated with severe or protracted increases in alt activity because of toxin-suppressed transaminase improvement over a 2-to 3-week period. for patients with alf and coagulopathy, the clinician must carefully weigh the benefits of histologic characterization versus the risk of excessive bleeding from the procedure. acute hepatitis is characterized histologically by a mononuclear or mixed inflammatory pattern, accompanied by hepatocellular apoptosis or necrosis. 2 necrosis should be further characterized by the pathologist as to the morphologic pattern of injury (focal, multifocal, confluent, bridging, massive, or piecemeal) because the pattern of necrosis may provide insight into the pathogenesis of the lesion. 2 for example, because centrilobular hepatocytes have an abundance of cytochrome p450 enzymes, these hepatocytes are preferentially affected in drug-induced hepatotoxicity, when cytochrome p450 metabolism of the parent drug results in toxic metabolites. 8 quantitative copper analysis and histochemical staining for copper are recommended, as copper accumulation may be an underappreciated cause of acute hepatitis in dogs. 4 infectious causes of acute hepatitis may be diagnosed on liver biopsy, or by additional tests performed on liver tissue (culture, immunohistochemistry, polymerase chain reaction [pcr], virus isolation; table 61 -4). unfortunately, in most cases, routine liver biopsy is unlikely to reveal a specific cause of acute hepatitis. 2, 4 findings of inflammation and necrosis/apoptosis accompanied by nodular regeneration and fibrosis suggests chronic rather than acute hepatitis. the long-term prognosis is better for acute hepatitis than for chronic hepatitis. 4 if a probable cause of acute hepatitis and hepatic necrosis can be determined, then specific treatment is directed at the primary etiology (e.g., discontinuing potentially hepatotoxic medications, treating for leptospirosis with doxycycline, or chelating hepatic copper with penicillamine). in most cases specific therapy is unavailable and treatment is directed at more general supportive and symptomatic treatment of liver disease. glucocorticoid therapy is not typically indicated in the treatment of acute hepatitis. 4 empirical treatment with antioxidants such as s-adenosylmethionine (same; 20 mg/ kg po q24h), milk thistle (siliphos; 3 to 6 mg/kg po q24h), or vitamin e (10 to 15 iu/kg q24h) may be warranted, as oxidative stress is believed to play a role in drug (carprofen, potentiated sulfonamides, diazepam, methimazole, lomustine, others), and toxin (aflatoxin, organic solvents, and heavy metal toxicity) induced hepatic injury. 9 same and milk thistle have additional cytoprotective properties that could be beneficial in necroinflammatory hepatopathies and hepatotoxicity. antioxidants and cytoprotective agents are discussed in more detail in chapters 40 and 46, respectively. liver biochemistries should be monitored to assess patient response to therapy. repeat liver biopsy performed 6 to 8 weeks after the initial diagnosis has been recommended, to confirm that acute hepatitis has improved or resolved, or to document a progression toward chronic hepatitis. 4, 6 it has been suggested that most dogs with mild idiopathic acute hepatitis (not in alf) recover after several days, regardless of treatment. 6 for patients with alf, aggressive supportive treatment is required. goals of therapy are to treat the underlying cause when possible, allow adequate time for hepatic regeneration and repair, and prevent or control complications of liver failure, such as hypoglycemia, coagulopathy and anemia, he, gi ulcers, and septicemia. intravenous n-acetylcysteine (nac), a glutathione source/ antioxidant, is the antidote of choice for treatment of acetaminophen toxicity. nac also appears to have additional potential benefits (improved systemic hemodynamics and tissue oxygen delivery), and should be considered for use in any dog or cat with alf. 9 the optimal dose regimen when nac is used for this purpose has not been determined. treatment of complications of liver failure are discussed in "complications of liver disease" section. the prognosis for recovery in dogs with acute hepatitis is good, as most dogs recover uneventfully. 4 however, there is a potential for dogs with acute hepatitis to develop chronic disease. 4 if animals present with signs of advanced liver failure (e.g., he, coagulopathy, hypoglycemia), the prognosis is guarded. if the animal survives, hepatic lesions such as periportal ductular proliferation, hepatic fibrosis, postnecrotic scarring, and regenerative hepatic nodules are likely. 2 if a hepatic drug reaction is suspected, reexposure of the patient to the suspect drug should be avoided. etiology hepatic abscesses from bacterial infection of the liver occur uncommonly in dogs and cats. [10] [11] [12] [13] abscesses may form as solitary or multiple macroscopic masses or microabscesses. in newborn animals, gram-positive and gram-negative bacteria cause hepatic abscesses, presumably related to postpartum umbilical infections. 14 in adult animals, gram-negative enteric bacteria (especially escherichia coli) and anaerobes (especially clostridia spp.) are most commonly identified; multiagent infections are frequent. 10, 12 other organisms such as yersinia spp., actinomyces spp., nocardia asteroides can also cause hepatic abscesses as part of a systemic infection. 14 the pathogenesis of hepatic abscesses in dogs and cats is unclear. hepatic abscesses are usually associated with extrahepatic infections or regional hepatic parenchymal damage. small numbers of bacteria, including clostridium spp., can be cultured from liver tissue of healthy dogs. hypoxia of hepatic tissue caused by hepatic neoplasia, liver lobe torsion, or trauma may predispose to abscess formation, because small numbers of existing anaerobes (e.g., clostridium spp.) can proliferate under these conditions. other potential sources of bacteria include hematogenous spread (via the umbilical vein, hepatic artery, or translocation of intestinal bacteria into the portal blood), ascension via bile ducts, penetrating abdominal and caudal thoracic wounds, and direct extension from local suppurative diseases. concurrent diseases or potential predisposing factors in dogs include systemic infections (pneumonia, pyelonephritis, prostatitis, pyometra, endocarditis), gallbladder rupture, pancreatitis, diabetes mellitus, liver lobe torsion, coexisting hepatic disease such as hepatic neoplasia (infected necrosis), longterm phenobarbital administration, long-term corticosteroid administration, and previous surgical biopsy. 10, 11 concurrent diseases in cats include cholecystitis, pyothorax, and hepatic neoplasia. 12 solitary abscesses are more common in dogs, whereas cats are more likely to be septic and have multiple hepatic abscesses. 11, 12 no association with feline leukemia virus or feline immunodeficiency virus infection has been made. 12 solitary liver abscesses are more likely to involve the right liver lobe in cats and the left liver lobe in dogs. 11, 12 clinical examination when adult dogs and cats are diagnosed with hepatic abscesses, they are usually older than 8 years of age. [10] [11] [12] clinical signs are nonspecific and can be attributed to sepsis, inflammation, and hepatic dysfunction. the most common signs are anorexia, lethargy, vomiting, and diarrhea. 10, 11 clinical signs of hepatic involvement may be overshadowed by signs of the associated disease process (e.g., neoplasia, pyelonephritis, pancreatitis). dogs with hepatic abscess may have a history of failure to respond to antibiotics or improvement that relapsed when antibiotics are discontinued. 10 physical examination findings are often vague and include depression, dehydration, fever, abdominal pain, hepatomegaly, abdominal mass, and abdominal effusion. [10] [11] [12] hypothermia is a more common finding than fever in cats with hepatic abscesses. 12 because the clinical findings are vague and nonspecific, hepatic abscesses often go undetected until an abdominal ultrasound is performed or they rupture and are discovered during laparotomy. rupture of a hepatic abscess leads rapidly to peritonitis, septic shock, and death. clinicopathologic abnormalities are consistent with an inflammatory hepatic disease. potential findings on the complete blood count include neutrophilia with a left shift (or neutropenia and degenerative left shift if rupture occurs), mild anemia, and thrombocytopenia. 10, 12 increased alt and alp activity are common findings although the alt may be in the normal range. 10 liver enzyme elevations are a less-consistent finding in cats with hepatic abscesses and 79% in cats. 12 the survival rate appears to be better when solitary abscesses are detected. 12, 13 granulomatous hepatitis is characterized histologically by focal or multifocal aggregates of activated macrophages with an epithelioid appearance, usually accompanied by lymphocytes and plasma cells. 14 this inflammatory response is distinct from that encountered in canine chronic hepatitis. systemic infectious diseases are an important cause of granulomatous hepatitis and this lesion has been described with fungal infections (histoplasmosis, coccidioidomycosis, many others), bacterial infections (mycobacteria, bartonella, nocardia, actinomyces, rhodococcus), protozoal diseases (cytauxzoonosis, leishmaniasis); parasitic diseases (visceral larval migrans, schistosomiasis, alveolar echinococcus, hepatozoon americana), and disseminated protothecosis (see table 61 -4). 15, 16 in cats, feline infectious peritonitis (coronavirus) is an important cause of multisystemic granulomatous or pyogranulomatous inflammation. other causes of granulomatous inflammation include a local response to foreign material (crystalline material, sutures, plant material) or a drug reaction. in humans, granulomatous liver lesions have been associated with administration of diltiazem, sulfonamides, quinidine, allopurinol, interferon-α, and phenytoin. 17, 18 however, drug therapy as a cause of granulomatous hepatitis in dogs and cats has not been specifically reported. granulomatous lesions in the liver has been described in a small number of dogs with lymphangiectasia, lymphosarcoma, and histiocytosis. 19 many cases of granulomatous hepatitis are idiopathic. 16 hepatic lipogranulomas ("fatty cysts"), which are often found in dogs with congenital portosystemic shunt, are aggregates of pigment-laden foamy macrophages and should not be confused with granulomatous hepatitis. clinical findings with granulomatous hepatitis are highly variable, depending on the underlying cause. when granulomatous hepatitis is identified on liver biopsy, special stains for fungal and mycobacterial organisms should be performed. other diagnostics to either identify an organism (cytology, culture, fecal exam, pcr) or detect antibodies against the organism (serology) vary widely with the underlying agent (see table 61 -4). if a cause cannot be found after a thorough diagnostic evaluation, consideration should be given to presumptive treatment for undiscovered infectious agents such as atypical mycobacteria, bartonella spp., or systemic fungal infection. corticosteroids or other immunosuppressant agents should only be used when diagnostic testing and empirical treatment have been unsuccessful, as steroid-induced immunosuppression may exacerbate an underlying infection. 19 eosinophilic hepatitis occurs rarely in dogs and cats. 2 potential causes include visceral larval migrans (toxocara), schistosomiasis, liver fluke infections, sarcocystis canis, and possibly, fungal infections (see table 61 -4). 2 with parasitic causes, eosinophils are often located at or near the site of the parasitic lesion in the liver. dogs and cats with systemic allergic, parasitic (heartworms), or hypereosinophilic syndromes, may also have scattered eosinophils in the liver, a variant of nonspecific reactive hepatitis. 2 hepatic druginduced liver injury should also be considered. phenytoin and minocycline are associated with eosinophilic infiltrates in humans with drug-induced liver injury. 18, 20 potentiated sulfonamides have been suggested to cause drug-induced eosinophilic hepatitis in dogs, 2 although a more typical pattern is acute hepatocellular necrosis or a cholestatic hepatopathy. 21 when eosinophilic infiltrates are identified, efforts should be directed at diagnosing parasitic causes (fecal, (increased alt and alp activity occurred in less than 50% of cats). 12 other potential biochemical findings include hyperglobulinemia, mild hyperbilirubinemia, and hypoglycemia (sepsis). laboratory abnormalities may also reflect the associated disease processes (e.g., hyperglycemia with diabetes mellitus, increased pancreatic lipase immunoreactivity with acute pancreatitis). if an abscess ruptures, cytology of the abdominal infusion reveals septic suppurative inflammation. abdominal radiographs may be normal or reveal hepatomegaly, hepatic mass lesion, or decreased abdominal detail or effusion associated with secondary peritonitis. with proliferation of gas-producing organisms, radiolucent areas may be seen in the liver. ultrasonographic examination permits earlier detection of hepatic abscesses. 11 ultrasonographically, a liver abscess appears as a hypoechoic or anechoic structure with irregular, hyperechoic margins. 11, 13 the ultrasonographic pattern is similar to that seen with hepatic hematomas, cysts, neoplasia, and biliary cystadenoma. gas may be seen within the abscess. 11 if abscess rupture has occurred, concurrent abdominal effusion may be detected. additional ultrasonographic findings may reflect associated disorders such as pancreatitis, cholecystitis, or pyelonephritis. ultrasound-guided fine-needle aspiration of a suspected liver abscess can be safely performed to obtain samples for cytology and culture to confirm the diagnosis. 11 if ultrasonography is not available, the diagnosis of hepatic abscesses is usually established during exploratory laparotomy (or at necropsy). an attempt should be made to isolate and identify the organism(s) associated with abscessation so that appropriate antibiotic therapy can be instituted based on sensitivity testing. aerobic and anaerobic cultures can be performed on abscess contents (by fine-needle aspiration), abdominal exudate, blood or hepatic tissues. treatment of hepatic abscesses consists of surgical resection or drainage of focal lesions, administration of appropriate antibiotics, correction of associated fluid, electrolyte, and acid-base imbalances, and identification and treatment of any underlying disease process. treatment of large unifocal hepatic abscesses has typically involved surgical resection of affected tissue, which may necessitate partial or full lobectomy. 10, 12 if perforation and peritonitis are present, surgical abdominal drainage and lavage are indicated. ultrasound-guided percutaneous drainage of a solitary abscess may resolve the abscess or allow stabilization until surgical resection can be performed. 11 the successful management of focal hepatic abscesses (up to 8 cm in diameter) by ultrasound-guided percutaneous drainage and alcoholization has been described in five dogs and one cat. 13 broad-spectrum combination antibiotic therapy (directed toward both aerobic and anaerobic bacteria) should be initiated as soon as cultures have been obtained. results of a gram stain on the exudate may provide preliminary information as to type of organism and guide the empirical choice of potentially effective antibiotics. recommendations for broad-spectrum antimicrobial coverage of hepatobiliary infections include either a fluoroquinolone combined with amoxicillin/clavulanate or a fluoroquinolone combined with penicillin and metronidazole, until culture results are available. the dose of metronidazole should be adjusted in animals with hepatic dysfunction (7.5 mg/kg po q8-12h). antibiotic therapy should be continued for at least 6 to 8 weeks. response to treatment can be monitored with serial ultrasound examinations and repeated blood work. historically, hepatic abscesses have carried a grave prognosis, with an overall reported mortality rate of approximately 50% in dogs 10 case series (see chapter 62) . breeds of dogs at increased risk for chronic hepatitis include the bedlington terrier, 22, 23 west highland white terrier, 24, 25 doberman pinscher, [26] [27] [28] american and english cocker spaniel, [29] [30] [31] [32] [33] skye terrier, 34 dalmatian, 35 labrador retriever, [36] [37] [38] and english springer spaniel. 39 unfortunately, with the exception of hereditary copper-associated liver disease in bedlington terriers, information is lacking for most of the breed-related disorders. female dogs appear to be at increased risk in some studies, 4,40 while others report that male and female dogs are equally affected. 41, 42 within particular breeds, sex differences have been noted (female doberman pinschers, labrador retrievers, and english springer spaniels; male cocker spaniels). 26, 29, 36, 38, 39 dogs diagnosed with chronic hepatitis are generally 4 to 7 years of age, but adult dogs of any age (or breed) can be affected. 4, 29, 41 etiology and pathogenesis ideally, canine chronic hepatitis should be classified on an etiologic basis. however, with the exception of copper-associated liver disease in bedlington terriers, the cause, pathogenesis, natural history, optimal treatment, and prognosis of these disorders are unknown (table 61 -5) . idiopathic chronic hepatitis is the most common clinical diagnosis. 4, 32, 40, 41 infectious causes. viral infections are a common cause of chronic hepatitis in humans, but are not currently recognized as an important etiology in dogs. in humans, viruses have the potential heartworm test; see table 61 -4), systemic eosinophilia, and hypersensitivity reactions. if no specific cause can be determined, empirical treatment with fenbendazole should be considered, followed by corticosteroid therapy as described for idiopathic chronic hepatitis. the term nonspecific reactive hepatitis is used to describe the slight to moderate widespread inflammatory infiltrates of the liver that occur secondary to a spectrum of extrahepatic disease processes. 2 lesions of nonspecific reactive hepatitis are associated with febrile and inflammatory disorders, especially those involving the gi tract and pancreas, or they may represent residual evidence of a previous intrahepatic inflammatory disorder. 2 inflammation occurs in portal or parenchymal areas and necrosis is absent. neutrophils predominate with acute extrahepatic disorders, whereas mononuclear inflammation occurs with chronic extrahepatic disorders or residual hepatic inflammation. the liver may be secondarily affected by systemic disorders because of changes in liver blood flow, portal blood delivery of bacteria, drugs, hormones, cytokines, or other substances from the gi tract, or activation of intrahepatic kupffer cells (monocyte-macrophage system) involved in the hepatic immune response. it may be challenging to differentiate nonspecific reactive hepatitis from resolving acute hepatitis or mild chronic hepatitis, without supportive clinical information. clinical signs in dogs and cats with nonspecific reactive hepatitis are usually referable to the extrahepatic disorder. liver enzyme elevations (alt-two times the upper limit of normal; alpthree-to fourfold increases) are common, thus mimicking primary hepatic disease. however, tests that reflect liver function, including serum bile acids, are usually normal. it is important to consider extrahepatic disorders that can secondary affect the liver, prior to focusing on primary hepatic disease. treatment is directed at the underlying extrahepatic disorder. chronic hepatitis, a heterogeneous group of inflammatorynecrotizing diseases of the liver, occurs commonly in dogs, but is rare in cats. cholangitis, which is inflammatory liver disease that targets the biliary tract, rather than hepatocytes, is more common in cats but also occurs in dogs.the term chronic hepatitis, rather than chronic active hepatitis or chronic persistent hepatitis, is recommended. 2, 15 if the etiology is known, it should be included as an adjective, such as "drug-induced chronic hepatitis," or "copper-associated chronic hepatitis"; otherwise, it is considered "idiopathic chronic hepatitis." chronic hepatitis in dogs is defined based on histopathologic features of hepatocellular necrosis or apoptosis associated with inflammation and evidence of regeneration and fibrosis. 2 lymphoplasmacytic inflammation is characteristic, but a neutrophilic component may be present. 2 the histopathologic features of chronic hepatitis are similar, regardless of the underlying cause. chronic hepatitis has the potential to progress to cirrhosis. 15, 16 recommendations have been made to include a clinical component to the definition of chronic hepatitis, such as documenting an increase in alt activity along with histologic evidence of hepatic inflammation for a minimum of 4 months. however, many dogs with chronic liver disease are not clinically apparent until the advanced stages, so duration can be difficult to evaluate. the early stages may not be recognized unless biochemistries are monitored for hepatic injury. a familial predisposition to develop chronic hepatitis has been suggested by demographic studies, pathologic surveys, and clinical hepatitis (acute and chronic) accounted for one-third of all dogs with primary hepatitis. 4 hepatic copper accumulation and hepatopathy have been described in cats but appears to be rare. 58, 59 the severity of hepatic injury correlates with the amount of hepatic copper, but subcellular localization of molecules and the molecular association also plays a role. 54 serum copper levels do not accurately reflect hepatic copper content and quantitative analysis of copper in the liver is required. 56 hepatic copper concentration in normal dogs is between 150 and 400 µg/g dry weight (parts per million). 28, 57 inflammatory hepatic injury does not consistently occur until copper concentrations exceed 2000 µg/g dry weight. 60, 61 however, there may be breed variations; for example, in doberman pinschers hepatic inflammation is present with copper concentrations of less than 2000 µg/g. 27, 57 transient acquired fanconi syndrome has been described in dogs with excess hepatic copper accumulation. 62, 63 copper granules were demonstrated on renal biopsy in some but not all dogs. potential mechanisms for hepatic copper accumulation include primary metabolic defects in hepatic copper metabolism, cholestasis causing impaired biliary excretion of copper, and excess copper absorption. 54, 56 a primary defect in hepatic copper metabolism occurs in bedlington terriers with a genetic mutation in the gene encoding the copper transport protein, commd1 (formerly murr1), resulting in a defect in biliary copper excretion. 64, 65 in to cause hepatitis either because of a persistent hepatic infection or as a transient infection that triggers an immune response because of a cross-reaction between the virus and liver antigens. 43 in an attempt to identify infectious causes of canine hepatitis, pcr screening of liver tissue was performed in 98 dogs with various stages of hepatitis to look for canine adenovirus type 1, hepadnaviridae, hepatitis a virus, hepatitis c virus, hepatitis e virus, helicobacter spp., leptospira spp., and borrelia spp. 44 based on negative results, the authors concluded that canine hepatitis is not typically caused by these infectious agents. 44 however, dogs that are experimentally infected with canine adenovirus type i, but are partially immune, can develop chronic hepatitis that progresses to cirrhosis. 45 the virus could not be detected beyond the first week postinfection, although the disease progressed over a period of months. 45 canine adenovirus antigen has been demonstrated by immunohistochemical techniques in formalin-fixed liver sections from five of 53 dogs with various hepatic inflammatory lesions, suggesting that canine adenovirus 1 (cav-1) may play a role in spontaneous chronic hepatitis. 46 in contrast, pcr and immunohistochemistry failed to detect canine adenovirus in liver tissue of 45 dogs with chronic liver disease. 47 whether cav-1 is a significant cause of chronic hepatitis under natural conditions is unknown. another proposed viral cause of chronic hepatitis and cirrhosis is the "canine acidophil cell hepatitis virus," reported from great britain in the 1980s. 48, 49 this transmissible agent, most likely a virus, is distinct from cav-1. it was transmitted experimentally by subcutaneous injection of serum or liver extracts from affected dogs, resulting in experimentally induced acute and chronic hepatitis. no further studies have been published to clarify the nature of this infectious agent or the associated hepatitis. canine leptospirosis is typically associated with acute cholestatic hepatic disease and acute renal failure. however, persistent infection can cause chronic hepatitis in the absence of azotemia. 50, 51 leptospira serovar grippotyphosa was incriminated as a cause of chronic hepatitis in a kennel of american foxhounds, based on serologic evidence and demonstration of spirochetes in the liver. 50 leptospira serogroup australis (serovars australis, bratislava, and muenchen) infection was suspected to cause chronic hepatitis in 16 young beagle dogs in a breeding colony routinely vaccinated against leptospirosis serogroups canicola and icterohaemorrhagica. 51 canine leishmaniasis has been associated with histologic evidence of chronic hepatitis, 52 but clinical features suggestive of hepatic involvement (hepatomegaly, ascites, or icterus) were absent. histologic findings revealed granulomatous hepatitis in most dogs, but some dogs had marked portal infiltration with lymphocytes and plasma cells, and mild portal fibrosis. leishmania amastigotes were routinely identified in macrophages in liver or other affected tissues. bartonella clarridgeiae dna was amplified from a liver biopsy of a doberman pinscher with copper-associated chronic hepatitis, although the significance of this finding is unclear. 53 copper accumulation. copper is an essential trace element in diets and is required for a number of physiologically important enzymes. cells have highly specialized and complex systems for maintaining intracellular copper concentrations. at toxic concentrations, free intracellular copper initiates oxidative damage causing hepatocellular necrosis and inflammation. 54, 55 normal copper metabolism has been reviewed in detail elsewhere. 54, 56 copper accumulation in the liver can be associated with significant hepatic injury resulting in acute hepatitis, chronic hepatitis, and cirrhosis ( figure 61-18) . 4, 54, 57 it is one of the few well-documented causes of canine chronic hepatitis. in one study, copper-associated a b liver of 37 dogs but was not identified in any control samples from healthy livers. none of the dogs had decreased serum levels of α 1antitrypsin. positive α 1 -antitrypsin staining was a more consistent finding in english and american cocker spaniels with chronic liver disease, than in other breeds. the authors concluded that accumulation of α 1 -antitrypsin might play a role, but it could not be determined if it was the cause or a result of chronic liver disease. 31 drugs and toxins. drug or toxin exposure is a potential cause of canine chronic hepatic disease. drugs that have been incriminated include anticonvulsants (phenobarbital, primidone, phenytoin), oxibendazole-diethylcarbamazine, lomustine, and possibly carprofen. [70] [71] [72] [73] chronic hepatitis and cirrhosis from long-term phenobarbital therapy is most widely recognized. 70, 71 exposure to aflatoxin from contaminated commercial dog food is usually associated with alf, but low-level long-term exposure in dogs can result in chronic hepatic injury (biliary hyperplasia, fibrosis, nodular regeneration). a breeding colony of german shepherd dogs developed chronic hepatitis and cirrhosis that was suspected (but never confirmed) to be a result of exposure to a porphyrinogenic substance, based on the finding of aggregates of crystalline pigments with orange birefringence with polarized light. 74 early recognition of drug-or toxin-induced chronic hepatic injury requires biochemical monitoring of liver enzymes, as dogs are clinically asymptomatic in the early stages. autoimmune/immune mechanisms. autoimmune hepatitis has not been documented in dogs. however, some dogs with chronic hepatitis appear to respond to corticosteroid therapy and thus may correspond to autoimmune hepatitis in humans. 41 autoimmune hepatitis in humans is a progressive chronic hepatitis of unknown cause that is believed to occur when an environmental agent (viruses, medications) triggers a cascade of t-cell-mediated events directed at liver antigens, in a genetically predisposed individual. 43 women are more commonly affected than men. hyperglobulinemia is a common finding. an infectious cause is difficult to document, as exposure may have occurred many years prior to the overt autoimmune disease. 43 certain drugs may induce or unmask an autoimmune hepatitis, or simply cause hepatocellular injury that mimics autoimmune hepatitis. 43 an autoimmune component to doberman pinscher hepatitis has been speculated, because of the breed's predisposition, high female predominance, and the finding that expression of mhc class ii antigens on hepatocytes of affected dogs correlates with degree of inflammation and decreases after treatment with prednisolone. 75 dogs with chronic hepatitis may have concurrent disorders associated with immune aberrations (immune hemolytic anemia, hypothyroidism, atopy, glomerulonephritis), but whether this is coincidental or indicative of the presence of multiple immune disorders as seen with autoimmune hepatitis in humans is unknown. 37, 76, 77 autoimmune hepatitis in humans is diagnosed when other causes of acute or chronic hepatitis have been excluded and serum autoantibodies (antinuclear, antismooth muscle, antibody to liver/ kidney microsomes type 1, antibody to liver cytosol type 1) are detected. 43 a number of studies have evaluated the role of liverassociated antibodies and cell-mediated response in dogs with chronic hepatitis, but none answers the question of whether the immune response is the primary cause of the hepatitis or a secondary phenomenon. twenty-four dogs with chronic hepatitis were evaluated for circulating autoantibodies (against cell nuclei, smooth muscle, liver membrane, and mitochondria) by indirect immunofluorescence. 77 antibodies to cell nuclei and liver membranes were the early stages, copper is sequestered in hepatic lysosomes and hepatic damage is minimal. however, with progressive accumulation of copper, hepatic injury becomes significant. the average copper concentration in bedlington terriers with chronic hepatitis is approximately 6000 µg/g dry weight and values up to 12,000 µg/g dry weight have been reported. 23, 57 inherited copper-associated liver disease is also described in the west highland white terrier, skye terrier, doberman pinscher, dalmatian, and labrador retriever, but with the possible exception of dalmatians, the hepatic copper levels are much lower than in bedlington terriers. [25] [26] [27] 34, 35, 38 the pathogenesis of copper accumulation and the relationship to chronic liver disease in these breeds is poorly understood. it seems likely that these breeds have a hereditary disorder of copper handling, but it is unlikely to be the same as described for the bedlington terrier. hepatic copper accumulation in the liver may also be a consequence rather than the cause of chronic hepatitis. because copper is normally excreted in the bile, chronic cholestasis and impaired bile flow can result in secondary copper accumulation. 57, 66 secondary copper accumulation is predominantly periportal and is usually less than 2000 µg/g dry weight. 57, 66 the effect of cholestasis on hepatic copper content was evaluated in three groups of dogs: bedlington terriers with copper toxicity, dogs with extrahepatic biliary obstruction (the prototype example of a cholestatic disorder) and chronic hepatitis in breeds not known to be at risk for copper-associated liver disease. 66 hepatic copper content was evaluated by a semiquantitative method based on copper staining of liver tissue with rubeanic acid, using a scale of 0 (no copper) to 5. 67 copper staining revealed absent to mild increases (scores of 0 to 2+) in dogs with biliary obstruction and chronic hepatitis when compared with bedlington terriers (scores of 5+). it was concluded that copper scores of 3+ or higher were suggestive of a primary copper storage disease. 66 unfortunately, quantitative copper analysis was not evaluated. markers of oxidative injury and altered defense mechanisms were similar in the three groups, consistent with the concept that copper, inflammation, and cholestasis can all contribute to oxidative injury. 66 high dietary copper intake appears to be an unlikely explanation for hepatic copper accumulation and liver disease in dogs. 56 however, the copper content of commercial dog foods ranges from 12 to 16 mg/kg dry matter, which is relatively high compared with recommended minimum daily copper requirements in dogs. 56 there is speculation that the recent increase in pathologically elevated hepatic copper concentrations (specifically evaluated in labrador retrievers), may coincide with a pet food industry recommendation to replace cupric/cuprous oxide in feed formulations because of its low bioavailability. 68 many dogs with copper-associated chronic hepatitis also have increased hepatic iron concentrations. 69 hepatic iron accumulation usually correlates with degree of inflammation. 40, 69 whether iron, as an oxidant, interacts with copper to contribute to lesions seen in copper-associated hepatitis remains to be determined. α 1 -antitrypsin deficiency. inherited α 1 -antitrypsin deficiency is a well-recognized cause of chronic hepatitis and cirrhosis in humans, and may play a role in the pathogenesis of chronic hepatitis in some dogs. 31 α 1 -antitrypsin is a circulating protease inhibitor that is synthesized and secreted by the liver. α 1 -antitrypsin deficiency in affected humans results in defective formation and impaired hepatic secretion of α 1 -antitrypsin, resulting in hepatic accumulation of α 1 -antitrypsin and hepatic injury. serum levels of α 1 -antitrypsin are typically low. in a study of 57 dogs with chronic liver disease, α 1antitrypsin was detected by immunohistochemical staining in the early stages of chronic hepatitis, ultrasonography of the liver may be normal or reveal nonspecific changes in echogenicity. when chronic hepatitis has advanced to cirrhosis, potential ultrasonographic findings include microhepatia, irregular hepatic margins, focal lesions representing regenerative nodules, increased parenchymal echogenicity associated with increased fibrous tissue, and ascites. splenomegaly and acquired psss may also be detected. a liver biopsy is essential for the diagnosis of chronic hepatitis. wedge biopsies are preferred over needle biopsies because they provide more tissue and are more likely to represent pathologic process(es) in the liver. when cirrhosis is present, laparotomy or laparoscopy often provide a better appreciation for the gross nodularity of the liver than can be ascertained from blind percutaneous needle biopsy (figure 61-19 ). chronic hepatitis is characterized histologically by moderate to severe inflammation (usually combinations of lymphocytes and plasma cells) associated with piecemeal necrosis. piecemeal necrosis, also referred to as interface hepatitis, is necrosis involving the layer of hepatocytes adjacent to the portal tract or "limiting plate." 2 the term bridging necrosis is used when necrosis and inflammation dissect across the hepatic lobule from portal areas to central veins or to adjacent hepatic lobules and suggests a severe form of chronic hepatitis. 2 histopathologic evaluation of the liver should not only consider etiology, but the pathologist should also comment on the activity (amount of inflammation, extent of apoptosis and necrosis) and the stage of disease (extent and pattern of fibrosis; architectural distortion suggestive of cirrhosis). 2 biopsies from dogs with chronic hepatitis should routinely be evaluated for copper accumulation. on hematoxylin and eosin staining, excess copper appears as golden brown refractile granules. 28 histochemical stains, such as rhodanine or rubeanic acid, can be used to semiquantitatively evaluate for copper in the liver (see figure 61 -19) . these stains consistently detect copper when amounts exceed 400 µg/g dry weight. 60 values obtained by quantitative copper analysis have a strong correlation with the number and size of granules seen with histochemical stains within the range of 400 to 1000 µg/g of liver tissue. 60 zonal distribution of copper detected, but were also found in dogs with other types of hepatic disease, suggesting a nonspecific secondary response. patterns of circulating autoantibodies found in dogs differed significantly from those found in humans with chronic liver disease. 77 in another study, serum anti-liver-membrane-protein antibody-positive dogs (1 : 40 to >1 : 1600) had higher alt activity, total bilirubin concentration, and more severe hepatic lesions than did anti-liver-membraneprotein antibody-negative dogs, but it was not determined whether autoantibodies were primary or secondary. 78 cd3+ lymphocytes are the most common hepatic lymphoid cells in dogs with chronic hepatitis and are associated with hepatic necrosis, 79-81 but also account for 54% of hepatic lymphocytes in normal dogs. 82 historical and physical examination findings in dogs with chronic hepatitis are indicative of chronic hepatic disease, and are similar regardless of the underlying cause. signs are often initially vague and nonspecific, such as anorexia, lethargy, vomiting, diarrhea, weight loss, pu, and pd. 4, 32 with increased severity of hepatic dysfunction, signs of overt liver failure develop, such as ascites, jaundice, and he. the presence of ascites and he suggest that chronic hepatitis has progressed to cirrhosis, and ascites is a negative prognostic indicator. 4, 42 melena associated with gastroduodenal ulceration or coagulopathy is also more likely with advanced liver disease. 32 because of the large functional reserve capacity of the liver, the onset of signs may appear very recent, initially suggesting an acute rather than chronic hepatic disorder. clinicopathologic features that support chronicity include poor body condition, ascites, microhepatia, hypoalbuminemia, and histologic evidence of fibrosis. in the early (subclinical) stages, dogs are asymptomatic and only identified by biochemical screening for liver enzyme elevations. increased serum alt activity, reflecting ongoing hepatic injury, is reported in 75% to 95% of dogs with chronic hepatitis. 4,7 serum alt activity may exceed 10 times the upper normal limit. 7 periods of normal alt activity may reflect cyclic disease activity and the varying severity of necrosis. 7 serum alp activity is also commonly increased, but the magnitude of the increase is generally lower than seen with alt activity. when chronic hepatitis advances to cirrhosis, liver enzyme activity may be normal, indicating decreased viable parenchymal mass. 7 abnormalities in biochemical tests such as hyperbilirubinemia, hypoalbuminemia, decreased blood urea nitrogen (bun), hypoglycemia, and increased sba indicate hepatic dysfunction and a more advanced stage of disease. 32 hyperglobulinemia can be seen in dogs with cirrhosis, but it remains to be determined whether this corresponds with increased autoantibodies as occurs in humans with autoimmune hepatitis, or whether it reflects nonspecific systemic antibody production in response to antigens from the portal blood which bypass the liver through acquired psss. 83 mild nonregenerative anemia may be a reflection of chronic disease. regenerative anemia can occur from blood loss secondary to a coagulopathy or bleeding gi ulcers. copper-associated hemolytic anemia has only been documented in bedlington terriers. abnormal hemostatic parameters (prolonged aptt and pt) are indicative of severe hepatic dysfunction or dic. a prolonged pt and thrombocytopenia may be negative prognostic indicators. 16, 37 analysis of ascitic fluid reveals a transudate or modified transudate. 32, 42 abdominal radiographs are unremarkable except when advanced stages of disease are accompanied by microhepatia or ascites. in the kg every 48 hours is most often recommended for treatment of canine chronic hepatitis. complications of corticosteroid therapy include gi bleeding (which may precipitate he), secondary infections, iatrogenic cushing disease, and worsening of ascites. dexamethasone (0.2 mg/kg po q24h) may be preferred in dogs with ascites or edema, because it lacks mineralocorticoid activity, which could exacerbate these signs. prednisone is often used in combination with azathioprine, especially if side effects of prednisone become objectionable. azathioprine is an antimetabolite with antiinflammatory and immunemodulating effects, and is commonly used in combination with prednisone in humans with autoimmune hepatitis. 43 the dose of azathioprine in dogs is 1 to 2 mg/kg/day po every 24 hours for 1 to 2 weeks, then tapered to every 48 hours for maintenance therapy. prednisone (0.5 to 1.0 mg/kg/day) is given on the alternate days. because azathioprine may cause bone marrow suppression and acute hepatotoxicity, the complete blood count and biochemical profile should be monitored. antiinflammatory agents and immunosuppressive drugs are discussed in more detail in chapters 38 and 49, respectively. because glucocorticoids increase liver enzyme activity (especially serum alp activity), response to therapy is best evaluated by a followup liver biopsy performed 3 to 6 months after starting therapy. if glucocorticoid therapy is eventually discontinued, clinical and biochemical parameters should be periodically monitored to detect a relapse. dogs with hepatic copper concentrations greater than 1500 µg/g, should be treated with the copper chelator penicillamine at a dose of 10 to 15 mg/kg po every 12 hours. 57 treatment usually requires months to years to produce significant decreases in hepatic copper. a mean decrease in copper of approximately 1500 µg/g was achieved in bedlington terriers treated for 6 months. 86 dogs with secondary copper accumulation appear to respond more rapidly, possibly because hepatic copper content is lower in these breeds. 86 doberman pinschers with subclinical hepatitis treated with penicillamine for 4 months had a mean decrease in copper from 1036 µg/g to 407 µg/g. 87 penicillamine has additional effects beyond copper chelation, which may be beneficial in dogs with chronic hepatitis, including inhibition of collagen deposition, stimulation of collagenase activity, immunosuppression, and immunomodulation. 54 common side effects of penicillamine therapy include anorexia, nausea, and vomiting, which can be minimized by giving the medication with a small amount of food. the copper chelator, trientine (10 to 15 mg/kg po q12h), is also effective for reducing hepatic copper concentrations. 86 it has fewer side effects than penicillamine and is effective in dogs with hemolytic anemia caused by copper release from necrotic hepatocytes. iatrogenic copper deficiency (microcytosis and hepatic dysfunction) has been described in a dog treated with long-term copper chelation therapy (trientine) and a copper-restricted diet. 88 decisions on duration of chelator therapy are based on followup liver biopsies with periodic monitoring of quantitative hepatic copper content. oral zinc salts can be used for maintenance therapy after copper chelation, or as initial therapy in dogs with hepatic copper concentrations between 400 µg/g dry weight and 1500 µg/g dry weight. zinc supplementation is typically used in conjunction with dietary copper restriction. zinc decreases intestinal copper absorption by inducing the intestinal copper-binding protein, metallothionein, within intestinal epithelial cells, which preferentially binds dietary copper and prevents its absorption. zinc acetate is given at a dose of 100 mg po bid for 2 to 3 months, then at a maintenance dose of 50 mg po bid. 89 a minimum of 3 months of zinc therapy is required before copper uptake from the intestinal tract is blocked. 89 accumulation should be noted, as copper accumulation starting in the centrilobular area is more likely with a primary metabolic defect in copper metabolism. 54 copper granules can also be detected on cytology of hepatic aspirates or impression smears stained with rhodanine or rubeanic acid. quantitative analysis for copper, by atomic absorption analysis on fresh hepatic tissue, is the definitive method to document increased hepatic copper content. needle core biopsy specimens may not be reliable for metal analysis, as copper and iron values are consistently lower in needle core versus wedge biopsy samples. 84 formalin-fixed tissues should be avoided, because formalin may contain copper or leach copper from the tissue. 57 hepatic copper can be reliably determined retrospectively on deparaffinizedarchived liver biopsy specimens. 84 once chronic hepatitis has been confirmed, a careful consideration of known causes of chronic hepatitis is essential (see table 61 -5) . findings that would support a primary metabolic defect in copper metabolism include a previously recognized breed predisposition, copper accumulation that precedes cholestasis or inflammation, centrilobular (zone 3) distribution of copper, histochemical score for copper of 3+ or greater, or quantitative copper measurements that exceed 2000 µg/g dry weight. 54, 57, 66 special stains of the liver should be requested to evaluate for infectious agents such as leptospirosis; serum antibody titers for leptospirosis may be indicated. a history of chronic drug therapy should be sought, especially long-term anticonvulsant therapy or other drugs listed in table 61 -5. recommendations for treatment of chronic hepatitis are empirical at best, because of the lack of controlled therapeutic studies on a well-defined population of dogs with this disorder. if a probable cause or category of injury can be determined, then specific treatment is directed at the primary etiology, for example, discontinuation of phenobarbital, treatment of leptospirosis, or chelation of hepatic copper with penicillamine. in most cases, specific therapy will be unavailable. treatment of chronic hepatitis in dogs has traditionally centered on the use of corticosteroids, presuming that, as in humans with the autoimmune form of hepatitis, immunologic mechanisms (inflammatory cells and mediators, local cytokines), contribute to hepatic inflammation and progression to cirrhosis. corticosteroids have antiinflammatory, immune-modulating, and antifibrotic effects, which may be beneficial in chronic hepatitis. a large retrospective study suggested that corticosteroid therapy at initial immunosuppressive doses (2.2 mg/kg/day; eventually tapered to 0.6 mg/kg/day) improved survival in dogs with chronic hepatitis. 41 however, many concurrent drugs were given and, undoubtedly, a heterogeneous group of disorders were included under the diagnosis of "chronic hepatitis." corticosteroid therapy appears warranted in dogs with histologic features of active inflammation and persistent increases in serum liver enzyme activity, for which known causes of chronic hepatitis (including infectious causes) have been excluded. 41, 85 glucocorticoid therapy is not indicated for treatment of chronic hepatitis caused by drug therapy, infectious agents, or primary hepatic copper accumulation. the optimal dose and duration of corticosteroid therapy for treatment of canine chronic hepatitis is unknown, including whether immunosuppressive doses are required, or whether lower, anti-inflammatory levels would suffice. 76 even in humans with autoimmune hepatitis, immunosuppressive doses of corticosteroids may not be required. 43 prednisone (or prednisolone) at an initial dose of 1 to 2 mg/kg/day po and then gradually tapered to 0.5 to 1.0 mg/ is correlated with the amount of hepatic copper. hepatic injury is believed to occur when progressive copper accumulation exceeds the storage capacity of the lysosomes; copper is released to the cytoplasm, damaging mitochondria, initiating lipid membrane peroxidation, and eventually causing cell death. affected dogs can be asymptomatic (in the early stages) or show signs of acute hepatic necrosis, chronic hepatitis, or cirrhosis. 54 in young dogs, copper accumulates in centrilobular (zone 3) hepatocytes and is sequestered in hepatic lysosomes. during this first stage, copper concentrations are between 400 and 1500 µg/g, dogs are asymptomatic, biochemical testing is within normal limits, and liver biopsy findings are unremarkable. in the second stage, when hepatic copper concentrations are between 1500 and 2000 µg/g, copper granules are also found in midzonal (zone 2) and periportal (zone 1) hepatocytes. although dogs are still asymptomatic, focal hepatic inflammation (centrilobular mixed cell foci, with necrotic hepatocytes, lymphoplasmacytic inflammation, and copper-laden macrophages) is seen on biopsy, and increased serum alt activity reflects hepatocellular injury. in the most advanced stage, when hepatic copper concentration exceeds 2000 µg/g, morphologic changes reveal chronic hepatitis that may progress to cirrhosis, and clinical and biochemical evidence of liver disease become apparent. clinical signs include anorexia, lethargy vomiting, and weight loss. with progression to cirrhosis, findings of jaundice, ascites, and he may develop. biochemical findings vary with the stage of disease. increased serum alt activity is the most sensitive laboratory indicator, although findings will be normal in young dogs in stage i, because of the lack of hepatic inflammation. other serum biochemical abnormalities typical of chronic hepatic dysfunction eventually develop. in some cases, acute hepatic necrosis and alf occur. hepatocellular necrosis may be associated with release of copper from necrotic hepatocytes, resulting in hemolytic anemia. during episodes of hemolysis, plasma copper levels are increased; other findings include low packed cell volume, hemoglobinemia, and hemoglobinuria. liver biopsy and quantitative analysis of hepatic copper concentrations is required for definitive diagnosis and staging of the disease. serum copper or ceruloplasmin concentrations are not helpful to make a diagnosis. 57 liver biopsies should be performed in all bedlington terriers considered for breeding, in order to identify and remove affected dogs from breeding programs. screening of asymptomatic dogs with a liver biopsy at 6 months and 15 months of age can determine if an affected dog is homozygous or heterozygous (a carrier). 15 affected dogs (both homozygous and heterozygous) typically have increased hepatic copper by 6 months of age. however, copper concentrations in dogs who are carriers (heterozygous) return to normal by 1 year of age, whereas copper concentrations in homozygous dogs continues to increase. 57 selective breeding programs in the netherlands has decreased the prevalence of bedlington terrier copper-associated liver disease from 46% (1976) (1977) (1978) (1979) (1980) (1981) (1982) (1983) (1984) (1985) (1986) to 11% (1990-1997) . 92 dna testing of bedlington terriers is available from vetgen (www.vetgen.com). this assay evaluates a linkage-based dna marker (co4107, allele 2) that is located in the chromosome close to the gene for copper toxicity. 93 the test can identify normal, affected, and carrier dogs with 90% accuracy. however, the marker can only be relied on for diagnosis of the genetic status of an individual dog when supported by a pedigree study. 93, 94 significant discrepancies were reported in 22 bedlington terriers, when comparing results of liver biopsy and the dna marker. 94 this may be attributed to different subpopulations of bedlington terriers with variations in the disease-causing mutation of the commd1 gene or a second mutant copper gene could play a role. 94 liver biopsy for quantitative zinc administration should be separated from meals by at least 1 hour and should theoretically not be prescribed at the same time as a copper chelator. 54 serum zinc concentrations should be monitored to achieve a level of 200 to 400 µg/dl. zinc concentrations greater than 500 µg/dl may be toxic (hemolytic anemia). low-copper diets are most beneficial for managing early (subclinical) copper accumulation in dogs affected with primary metabolic defects in hepatic copper metabolism. feeding a low-copper diet decreases hepatic copper content in labrador retrievers with subclinical copper-associated liver disease. 90 additional treatment with zinc does not appear to increase the copper-lowering effect of dietary management. 90 foods containing large amounts of copper (liver, other organ meats, shellfish, eggs, bean/legumes, chocolate, nuts, cereals, and copper-containing vitamin supplements) should be avoided. because oxidative stress is a significant mechanism for hepatic damage associated with copper accumulation and necroinflammatory hepatic disorders, 66,91 antioxidant therapy with vitamin e (10 to 15 iu/kg/day), or same (20 mg/kg/day) has been advocated. 9 other cytoprotective agents such as silymarin (milk thistle) and ursodeoxycholic acid may also be beneficial. 9 chapters 40 and 46 discuss cytoprotective agents used in the treatment of hepatobiliary disease in detail. when end-stage cirrhosis is diagnosed, treatment is mainly supportive, as cirrhosis itself is essentially irreversible. measures should also be instituted to control the complications of chronic liver failure, such as ascites, he, gastroduodenal ulcers, and coagulopathy, which are discussed in more detail in "complications of liver disease" section. the response to treatment of chronic hepatitis is variable, which is not unexpected as it is likely a heterogeneous group of diseases. some dogs can eventually be taken off medication and remain in remission, but more often, therapy must be continued indefinitely. other dogs fail to respond, especially those that have advanced disease with cirrhosis. 4, 32 in one study, the estimated median survival time in 42 dogs with idiopathic chronic hepatitis was 18 months (range: 0 to 49 months) and in 23 dogs with copper-associated chronic hepatitis it was 17 months (range: 7 to 27 months). 4 mean survival time in 20 dogs with cirrhosis was 1 week. 32 bedlington terriers develop chronic hepatitis and cirrhosis from copper toxicity, as a consequence of an inherited metabolic defect resulting in impaired biliary copper excretion. 23, 57, 76 the disorder is transmitted by autosomal recessive inheritance. the gene responsible for this metabolic disorder is commd1, which is different than that described for copper toxicity (wilson disease) in humans, in which the gene involved is atp7b. 64 there is no gender predilection. at one time, it was speculated that as many as 60% of the breed might be affected. 57 hepatic copper concentration in normal bedlington terriers ranges from 91 to 358 µg/g with a mean of 206 ± 56 µg/g dry weight. 23 bedlington terrier copper-associated liver disease is associated with progressive, hepatic copper accumulation (copper levels of up to 12,000 µg/g) unless treatment is instituted. the lowest hepatic concentrations of copper are found in the youngest dogs and concentrations increase with age, peaking at around 6 years. copper content usually declines thereafter in affected dogs, but not to normal. this decline may be a result of replacement of copper-containing hepatocytes by fibrous tissue or regenerative nodules that do not contain copper. the severity of hepatic disease recent efforts have focused on identification of affected doberman pinschers prior to advanced hepatic disease. in finland, a survey of 626 randomly selected, clinically healthy doberman pinschers, revealed that 8.8% of dogs had increased alt activity, and 3.4% had hepatitis (parenchymal and portal mononuclear inflammation and positive stains for copper). 99, 100 the mean age of dogs with subclinical hepatitis was 3.8 years, compared with clinically affected dogs (5.5 years). the asymptomatic period lasted an average of 19 months. the prevalence of subclinical doberman hepatitis was investigated in 106 randomly selected 3 year old doberman pinschers in the netherlands. 27 subclinical hepatitis was identified in 22 dogs (19 females and three males); hepatic copper concentration was higher in dogs with hepatitis (419 ± 414 µg/g dry weight) than those without liver disease (197 ±113 µg/g). 27 serial liver biopsies over at least a 2-year period, revealed that hepatitis persisted only in dogs with copper levels greater than 400 µg/g dry weight, and copper levels continued to increase in these dogs (939 ± 299 µg/g), supporting a relationship between copper, inflammation and hepatitis. 27 it has also been proposed that hepatic copper is incidental to chronic hepatitis in this breed, based on the findings that five of 35 doberman pinschers with chronic hepatitis had normal copper levels, and histologic changes were similar regardless of copper status. 81, 26, 75 an immune-mediated mechanism has been suggested, based on the finding that expression of mhc class ii antigens on hepatocytes of dogs with doberman hepatitis was correlated with degree of inflammation. 75 aberrant mhc class ii molecule expression on nonlymphoid cells could be a result of toxins, drugs, viral infection, or autoimmunity, and hepatocytes with mhc class ii expression might become a target as an antigen-presenting cell for cd4+ t cells. 75 dogs treated with low-dose prednisolone (0.1 to 0.5 mg/kg/day) for 4 to 5 months had significantly decreased expression of mhc class ii antigens. 75 chronic hepatitis should be suspected in any doberman pinscher (especially females) with clinical or biochemical evidence of hepatic disease. definitive diagnosis requires liver biopsy. other causes of chronic hepatitis should also be considered, since doberman pinschers appear to be at risk for drug-induced hepatitis. 71 early detection of chronic hepatitis provides the best opportunity for treatment. it has been recommended that all doberman pinschers older than 1 year of age be screened for alt activity. 99 persistent increases in alt activity suggest further evaluation including liver biopsy is warranted. the magnitude of increased alt activity is not different between subclinical and clinically affected dogs. hyperbilirubinemia is suggestive of more advanced disease. 28, 99 effective treatment for doberman pinschers with chronic hepatitis has not been established. however, a preliminary study showed that if diagnosed in the subclinical stage, treatment with penicillamine (200 mg total dose po bid for 4 months) lowered hepatic copper content and improved hepatic histopathology. 87 traditionally, antiinflammatory or immunosuppressive drugs such as prednisone with or without azathioprine have been instituted. the efficacy of this treatment remains to be determined but generally, the response is poor if dogs are presented in advanced stages of liver failure. the use of ursodeoxycholic acid (15 mg/kg po bid) deserves special consideration in this chronic cholestatic disorder, but has not yet been objectively evaluated. treatment of copperassociated hepatitis in doberman pinschers with advanced disease is usually unsuccessful. most dogs die within weeks to months. the prognosis appears more favorable if the disease is detected in the early stages, but the optimal therapeutic regimen remains to be determined. copper and morphologic examination remain the best option for diagnosis in the individual dog. 94 a database for certification of bedlington terriers is maintained on the web site (www.caninehealthinfo.org) of the canine health information center, which is sponsored by the akc/canine health foundation and the orthopedic foundation for animals. affected bedlington terriers who are asymptomatic (copper >400 µg/g dry weight but less than 1500 µg/g dry weight) should have dietary copper restriction and zinc supplementation. bedlington terriers with copper accumulation (copper >1500 µg/g dry weight) and chronic hepatitis should be treated with a copper chelator such as penicillamine or trientine. 57 early diagnosis and treatment with either zinc or copper chelators will allow most dogs to lead a normal life. 57 treatment of hemolytic anemia may require a blood transfusion. trientine dihydrochloride (but not penicillamine) may be effective in chelating circulating copper during a hemolytic episode. chapter 43 discusses copper-chelating agents in more detail. doberman pinschers are at increased risk for the development of severe chronic hepatitis and cirrhosis. 26, 28, 81, 95 doberman hepatitis accounted for 4% of all deaths in a dutch population of 340 dobermans. 96 middle-aged (4 to 7 years) female dogs are at increased risk, but males also may be affected. although a hereditary mechanism is suspected, the pathogenesis of this disorder is unclear. 28 copper accumulation appears to be associated with hepatic damage, but the pathogenesis is different from the bedlington terrier disorder. 27 immune mechanisms may also play a role. 75, 81 many doberman pinschers are diagnosed in the advanced stages of hepatic failure. 26 evidence of excessive bleeding (gingival bleeding, epistaxis, and melena) are common. signs of he often predominate in the terminal stages. common physical examination findings include ascites, jaundice, and weight loss. splenomegaly (associated with portal hypertension) is common. laboratory findings included increased alt and alp activity, hyperbilirubinemia, hypoalbuminemia, hyperammonemia, coagulopathy, and thrombocytopenia. 26 typical histologic lesions include portal inflammation (lymphocytes, plasma cells, and macrophages), piecemeal necrosis, bridging necrosis, bile duct proliferation, and portal fibrosis. hepatic copper concentrations are increased in most affected dogs and are typically between 1000 and 2000 µg/g dry weight, although values as high as 4700 µg/g have been reported. 28, 81 the significance of the increased hepatic copper concentration in this breed remains controversial. copper accumulation was originally attributed to secondary mechanisms, as doberman pinschers with advanced disease (chronic hepatitis and cirrhosis) have biochemical and histologic evidence of cholestasis. however, evaluation of affected dogs in the early (subclinical) stage, reveals that copper accumulation precedes cholestasis, 27, 95 and decreased biliary excretion of radiolabeled copper has been documented. 97 the hepatic distribution of copper and location of inflammation varies with the stage of disease. in the early stages, the copper (and focal inflammation) is centrilobular. 27, 81, 95 as the disorder progresses, copper accumulation and inflammation are more pronounced in periportal regions and areas of bridging necrosis. 95 although copper appears to be related to the hepatic inflammatory reaction, copper levels are typically less than 2000 µg/g dry weight, the minimum amount of copper that is believed to cause hepatocellular injury in bedlington terriers and west highland white terriers. 60,61 a primary copper retention disorder has been proposed, 28 but the genes associated with bedlington copper toxicity (commd1) and wilson disease in humans (atp7b) have been excluded. 98 same, milk thistle), and symptomatic therapies that are not designed to lower hepatic copper. 37 feeding a low-copper diet to 20 labrador retrievers with hepatic copper accumulation (seven of 20 dogs had varying degrees of hepatitis) was effective in decreasing hepatic copper concentrations, but severity of inflammation remained unchanged. 90 additional treatment with zinc did not appear to increase the copper-lowering effect of dietary management. 90 long-term survival appears variable. 36, 37 dogs who died within 2 months of diagnosis were more likely to have a prolonged pt and thrombocytopenia. 37 dalmatians are reported to have acute hepatic necrosis, chronic hepatitis, and cirrhosis associated with increased hepatic copper concentrations. 35, 102 cholestasis is not a prominent biochemical or histologic feature until later in the disease, suggesting that hepatic copper accumulation is more likely to be caused by a familial metabolic disorder rather than secondary to altered hepatic biliary copper excretion. most dogs presented initially with acute gi signs (anorexia, vomiting, and diarrhea). biochemical findings revealed markedly increased alt activity with lesser increases in alp activity. hyperbilirubinemia and hypoalbuminemia were seen with advanced disease. glucosuria (in the absence of hyperglycemia) and proteinuria were identified in some dogs. ultrasound findings were usually unremarkable. liver biopsy revealed piecemeal necrosis, bridging fibrosis, and inflammation (predominantly lymphocytes or neutrophils). the mean hepatic copper level was 3197 µg/g dry weight (normal <400 µg/g dry weight) with a range of 754 to 8390 µg/g dry weight. in five of nine dogs, copper exceeded 2000 µg/g. rapid progression of the disease was characteristic. copper chelation therapy may be beneficial if diagnosed before advanced liver disease occurs. chronic hepatitis and cirrhosis associated with hepatic copper accumulation (800 to 2200 µg/g dry weight) in genetically related skye terriers has been described. 34 in the early stages, copper accumulation is absent, and biopsy findings indicate hepatocellular degeneration with cholestasis and mild inflammation. chronic lesions are associated with intracanalicular cholestasis, chronic hepatitis, and cirrhosis. skye terrier hepatitis is speculated to be a disorder of disturbed bile secretion with subsequent accumulation of copper. american and english cocker spaniels have an increased incidence of chronic hepatitis and cirrhosis. 29, 30 the cause is unknown. hepatic copper accumulation does not appear to be a consistent feature. it is unclear whether accumulation of α 1 -antitrypsin in hepatocytes, a well-recognized cause of cirrhosis in humans, is important in the pathogenesis. 31 male cocker spaniels (average age: 5 years) are at increased risk. 29, 30 despite the chronicity and severity of the underlying hepatic lesions, most affected dogs have a short duration of clinical illness, usually less than 2 weeks. ascites is the most consistent presenting complaint. profound hypoalbuminemia (mean: 1.7 g/dl) is a consistent laboratory finding. total serum bilirubin concentration is normal or only mildly increased, supporting that cholestasis is not a key feature of the disorder. ascitic fluid analysis is consistent with a transudate or modified transudate. on liver biopsy, hepatic lesions are consistent with chronic hepatitis and cirrhosis. treatment of cocker spaniels with chronic hepatitis consists of general supportive therapy for the complications of liver failure. corticosteroid therapy prior to progression to cirrhosis may west highland white terrier west highland white terriers are at increased risk to develop chronic hepatitis and cirrhosis. 25, 76, 101 males and females are equally affected. the mode of inheritance for the familial copper-associated disorder has not been established. 24 decreased biliary excretion of radiolabeled copper occurs in affected dogs. 89 centrilobular (zone 3) copper accumulation occurs during the first year of life, but rarely exceeds 2000 µg/g dry weight. 24 in contrast to the bedlington terrier, west highland white terriers do not continuously accumulate copper over their lifetime; in fact, copper content may actually decrease with time. 56 in one report of 395 clinically normal west highland white terriers, most dogs had hepatic copper levels between 100 and 1500 µg/g dry weight with normal liver biopsies. 25 in west highland white terriers with chronic hepatitis, histologic lesions include multifocal hepatitis, subacute bridging necrosis, massive necrosis, and cirrhosis. 25 the relationship of hepatic copper to chronic hepatitis in the west highland white terriers is unclear. there appears to be at least two types of chronic hepatitis. 25 some dogs have copper-associated hepatitis with elevated copper content (>2000 µg/g) and multifocal centrilobular hepatitis. copper concentrations do not usually exceed 3500 µg/g dry weight. lesions of chronic hepatitis can also be seen in the absence of substantial copper accumulation, and have been described as "idiopathic chronic hepatitis." 25 quantitative copper analysis is necessary to determine if copper accumulation is a significant (>2000 µg/g dry weight) contributing factor. if chronic hepatitis and cirrhosis are associated with increased hepatic copper content (>2000 µg/g dry weight), treatment for hepatic copper accumulation should be instituted. mature west highland white terriers with chronic hepatitis and less than 2000 µg/g dry weight of copper may not require chelation therapy, as hepatic copper accumulation is not continuous throughout life. other therapeutic options for treatment of idiopathic chronic hepatitis, such as glucocorticoids, should be considered in these dogs. labrador retrievers are at increased risk for chronic hepatitis. 29, 37 age at presentation ranges from 2.5 to 14 years, with an average age of 7 to 9 years. [36] [37] [38] a female predisposition was noted in two studies, 36, 38 whereas in another study, males and females were equally affected. 37 most affected dogs have increased hepatic copper, which has been described as centrilobular (zone 3) or diffuse. [36] [37] [38] copper concentrations exceeded 2000 µg/g dry weight in 10 of 12 dogs (mean copper: 3369 µg/g; range: 2375 to 4972; reference interval: 120 to 400 µg/g). 38 most dogs also had elevated iron levels with a mean of 4117 µg/g (reference interval: 350 to 1750). 38 a genetic basis is suspected, based on the finding of increased copper concentrations in asymptomatic related dogs, but the genetic defect remains to be determined. 36 a retrospective survey of hepatic copper content in labrador retrievers during two time periods (1980-1997 and 1998-2008) , revealed significantly higher copper concentrations in the more recent period both in dogs with chronic hepatitis and in control dogs; no difference in age or gender was noted. 68 it was speculated that increased hepatic copper might reflect increased dietary copper bioavailability, because of pet food industry recommendations to replace cupric/cuprous oxide in feed formulations. 68 treatment with penicillamine (15 mg/kg po q12h) appears to be effective in decreasing hepatic copper content and inflammation. 36, 90 however, some dogs appear to respond to immunosuppressive (prednisone, azathioprine), supportive (ursodeoxycholic acid, clinical features of cirrhosis in dogs include ascites (portal hypertension, hypoalbuminemia), he (intrahepatic and extrahepatic portosystemic shunting of blood), and evidence of decreased hepatic function (hypoalbuminemia, increased sba, coagulopathy, hyperbilirubinemia). findings on hepatic ultrasonography (small nodular liver, splenomegaly, and acquired psss) are suggestive for cirrhosis, but liver biopsy is required for confirmation. because cirrhosis is essentially irreversible, treatment is mainly supportive, emphasizing measures that control complications of severe generalized liver failure, such as ascites, encephalopathy, gastric ulcers, coagulopathy, and infection (see "complications of liver disease" section). if clinical signs of liver failure are already present, the prognosis is poor. prevention of fibrosis, an important long-term goal, is best achieved by early specific treatment directed at the probable cause of injury (e.g., discontinuing a suspect drug, penicillamine for copper-associated liver disease, antiinflammatory drugs for idiopathic chronic hepatitis, surgical relief of extrahepatic biliary obstruction). many therapeutic agents used for treatment of liver disease, such as penicillamine, prednisone, azathioprine, milk thistle, ursodeoxycholic acid, and zinc, have potential antifibrotic properties, 9 and are discussed in more detail in other chapters. colchicine, a microtubule assembly inhibitor which increases collagenase activity, has been recommended for treatment of hepatic fibrosis, but its effectiveness in dogs has not been critically evaluated. the recommended dose in dogs is 0.025 to 0.03 mg/kg/day po. reported side effects include nausea, vomiting, and diarrhea. bone marrow toxicity and myoneuropathy have been reported in humans. lobular dissecting hepatitis is a specific histologic form of cirrhosis seen in neonatal or young adult dogs. 2, 4, [110] [111] [112] it is suggested to be a nonspecific response to a variety of hepatic insults. 111 the age at presentation is younger than for dogs with either acute or chronic hepatitis. 4 in 21 affected dogs, the median age was 11 months, with 12 dogs (54%) being 7 months or younger. 4, 110 females appear to be at increased risk. 4, 111 lobular dissecting hepatitis may occur in an isolated dog or in groups of dogs from the same litter or kennel. 111 standard poodles may be at increased risk. 110, 112 clinical features are those of advanced hepatic failure and portal hypertension. 111 the most consistent clinical finding is ascites. liver enzymes are typically increased and hypoalbuminemia and increased sba concentrations are common. 4, 111 liver biopsy is required for diagnosis and to differentiate it from other types of chronic hepatitis and cirrhosis. the lesion is characterized histologically by lobular hepatitis: inflammatory cells (lymphocytes, plasma cells, macrophages, and neutrophils) are scattered throughout the hepatic lobule rather than concentrated in periportal regions. bands of collagen and reticulin fibers dissect around single or small groups of hepatocytes and disrupt hepatic lobular architecture. 111 copper stains are negative or moderately positive, consistent with secondary copper accumulation. specific treatment has not been reported, but general measures for management of chronic liver failure are appropriate. 4 in a small group of dogs with lobular dissecting hepatitis, the mean survival time was approximately 3 months, which was significantly shorter than for dogs with acute or chronic hepatitis. 4 infection of the liver is an important cause of hepatic disease in dogs and cats. 113 the liver may be the primary target of infection (e.g., infectious canine hepatitis, bacterial cholangitis, hepatic abscess) or be beneficial. the prognosis is poor and most dogs die within a month of diagnosis. a recent report described seven american cocker spaniels with histologic features resembling lobular dissecting hepatitis. 33 males and females were equally affected. in contrast to previous reports of hepatitis in cocker spaniels, most dogs in this study improved with corticosteroid therapy. 33 english springer spaniel a preliminary report has described chronic hepatitis in 34 english springer spaniels from norway and the united kingdom. 39 female dogs were overrepresented. copper does not appear to play a role. the prognosis appears to be poor, with most dogs dying 4 to 7 months after diagnosis. 39 hepatic cirrhosis (end-stage liver disease), is characterized by fibrosis, regenerative nodules that alter liver architecture and intrahepatic (microscopic) psss (see figure 61 -19) . 2 hepatic fibrosis is not synonymous with cirrhosis. cirrhosis is common in dogs but less so in cats. 2 cirrhosis can result from postnecrotic scarring after acute massive necrosis or from chronic hepatic injury caused by a variety of insults such as infection (e.g., leptospirosis, cav-1), hepatotoxins (e.g., copper, phenobarbital, aflatoxin), inflammation (chronic hepatitis), or hypoxia. the common denominator is hepatocyte death, which leads to repair by fibrosis and nodular regeneration. when cirrhosis is fully developed, the histologic features of the original inciting injury often are obscured by the cirrhotic changes. substantial hepatic fibrosis (without "cirrhosis") can be seen with long-standing extrahepatic biliary obstruction, noninflammatory fibrosis, congenital hepatic fibrosis (a disorder of biliary system development), and congenital portal vein hypoplasia. 2,103-105 a unique form of macronodular cirrhosis, characterized by noninflammatory regenerative hyperplastic nodules and diffuse vacuolar hepatopathy, is seen in dogs with hepatocutaneous syndrome (superficial necrolytic dermatitis). hepatic fibrosis was once considered irreversible, but is now recognized to be a dynamic process, which exists in a balance between synthesis and degradation. a better understanding of the underlying mechanisms may provide potential therapeutic targets. 106 the major fibrogenic cell in the liver is the activated hsc (ito cell, vitamin a-storing cell), which is normally present in the perisinusoidal space. 106, 107 under the influence of fibrogenic stimuli (inflammation and the immune response, oxidative stress, apoptosis, hypoxia, steatosis), the hsc is activated to a myofibroblast, which produces collagen and other extracellular matrix (ecm) constituents. 106 the cytokine, tgf-β, appears to play a central role in fibrogenesis in humans and dogs. 106, 108 perisinusoidal fibrosis decreases the permeability of normal sinusoids, impairing metabolic exchange between hepatocytes and sinusoidal blood further compromising hepatic function. 106 excess fibrous tissue also limits the ability of vessels and sinusoids to distend, resulting in increased resistance to hepatic blood flow and portal hypertension. when fibrotic septae become vascularized, these microscopic communications (between portal vein or arterial artery and hepatic vein) lead to portosystemic shunting of blood. reversal of hepatic fibrosis and improvement in liver function can occur, especially if the underlying cause of injury is treated or removed. 106 examples in human medicine include antiviral drugs for hepatitis b and hepatitis c and prednisone for autoimmune hepatitis. although fibrosis is potentially reversible, cirrhosis for all practical purposes is not, because of the accompanying architectural changes and psss. 109 these ocular complications occur in approximately 20% of naturally infected dogs, and are caused by corneal endothelial damage and antigen-antibody complexes. ich should be suspected in any young, unvaccinated dog with evidence of alf. ich must be differentiated from diseases with similar clinical signs, such as canine distemper, parvoviral enteritis, and hepatotoxicity. abnormalities on the leukogram are common and vary with the clinical stage of infection. during viremia, neutropenia and lymphopenia are often present. neutropenia is also a common a feature of canine parvovirus, a much more prevalent disease of puppies. rebound lymphocytosis and neutrophilia occur in the recovery stages of ich (7 days after infection). biochemical findings are characteristic of acute hepatic necrosis and include increased serum alt and alp activity, and abnormal liver function tests. hyperbilirubinemia is a less-consistent finding. hypoglycemia may complicate the terminal stages of the disease. coagulation parameters are consistent with dic. other potential findings include proteinuria secondary to glomerular damage, abdominal fluid consistent with an exudate, and an increase in protein and mononuclear cells in the cerebrospinal fluid. the clinical diagnosis of ich is usually suspected on the basis of age, vaccination history, clinical signs, and laboratory findings, and is confirmed by liver biopsy or necropsy findings. additional diagnostic tests that are used less frequently include serologic testing, virus isolation, and direct immunofluorescence. therapy for alf caused by ich is primarily supportive care and control of complications that frequently occur such as dic, he, and hypoglycemia. the prognosis in dogs with ich depends on the severity of hepatic necrosis and the incidence of serious complications such as dic. hepatic regeneration and recovery is possible unless widespread coagulation necrosis destroys entire lobules. ich can be effectively prevented by vaccination. canine herpesvirus causes an acute, afebrile, rapidly fatal disease in neonatal puppies (1 to 3 weeks of age). 116 hepatic necrosis is one manifestation of the widespread multiorgan necrosis and hemorrhage that occurs in this systemic viral infection. clinical signs include acute onset of depression, diarrhea, failure to suckle, crying, and abdominal pain in previously healthy puppies. other findings include petechial hemorrhages and vesicles of the mucous membranes. jaundice is rare. seizures and loss of consciousness may be present in the terminal stages, and most pups die within 24 hours of onset of clinical signs. typical gross pathologic findings include focal areas of necrosis and hemorrhage in the liver, kidneys, lungs, and serosal surfaces of the intestines. microscopically, these areas are characterized by foci of necrosis with occasional intranuclear inclusions. neonates are infected by oronasal exposure to the virus in utero or by secretions from an infected bitch or littermates. neonates are particularly susceptible, possibly because of their low body temperature and immature mechanisms for temperature regulation. the diagnosis of canine herpesvirus is primarily based on the history, physical examination, and pathologic findings. laboratory findings are inconsistent but include neutrophilia or neutropenia, and increased serum alt activity. treatment of affected puppies is generally unsuccessful because of the acute fulminant nature of the disease. maintenance of body it may be one of several organ systems involved in a multisystemic disease process such as feline infectious peritonitis (coronavirus), toxoplasmosis, or histoplasmosis (see table 61 -4). infectious agents can be associated with widespread invasion of organs with a large mononuclear phagocytic component, including the liver, spleen, lymph nodes, and bone marrow, although clinically significant liver disease is uncommon. liver biopsy can be diagnostically useful for identification of these organisms in infected animals. etiology infectious canine hepatitis (ich) caused by cav-1 has long been recognized as a cause of acute hepatic necrosis in dogs. 114 this virus is genetically and antigenically distinct from cav-2, a cause of infectious canine respiratory disease. the incidence of clinical disease caused by cav-1 is now very low because of effective vaccination procedures. neutralizing antibodies to cav-1 are also found in mature, unvaccinated dogs, suggesting that natural exposure to the virus is widespread. cav-1 has a special tropism for vascular endothelial cells and hepatocytes. 114 dogs with sufficient immunity (neutralizing antibodies >1 : 500) do not develop clinical signs of disease. susceptible dogs (titer <1 : 4) develop widespread centrilobular to panlobular hepatic necrosis, which is often fatal. distinctive intranuclear inclusions are present in hepatocytes and the endothelium of other tissues. experimentally, dogs with an intermediate titer (between 1 : 16 and 1 : 500) develop chronic hepatitis that can progress to cirrhosis. 115 whether cav-1 is a significant cause of chronic hepatitis under natural conditions is unknown. cav-1 antigen was demonstrated in formalinfixed liver sections from five of 53 dogs with various naturally occurring hepatic inflammatory lesions, suggesting that cav-1 may play a role in spontaneous chronic hepatitis. 46 other attempts to identify cav-1 in dogs with chronic hepatitis have been negative. 44, 47 clinical examination ich is seen most commonly in unvaccinated dogs younger than 1 year of age. clinical signs vary with the stage of disease. dogs that are peracutely ill do not have clinical evidence of hepatic disease but simply become depressed and moribund, and die within a few hours. dogs with a more extended clinical course (5 to 7 days) have signs associated with acute hepatic necrosis that include vomiting, diarrhea, and abdominal pain. a hemorrhagic diathesis may occur during the viremic phase and is manifested by epistaxis, petechial or ecchymotic hemorrhages of the skin, or excessive bleeding from venipunctures. failure of the liver to clear activated clotting factors and impaired hepatic synthesis of clotting factors probably also contributes to development of dic. signs of central nervous system (cns) dysfunction include depression, disorientation, seizures, and coma and have been attributed to he or nonsuppurative encephalitis. common physical examination findings include fever, enlarged tonsils, pharyngitis, laryngitis, cervical lymphadenopathy, and subcutaneous edema of the head, neck, and trunk. hepatomegaly, abdominal pain, and abdominal effusion can occur. jaundice is rare but can develop in dogs that survive the acute fulminant stage of ich. an uncomplicated clinical course lasts approximately 5 to 7 days before recovery begins. unilateral or, less frequently, bilateral corneal edema and anterior uveitis ("hepatitis blue eye") are complications that may become evident during the recovery period. diagnosis of fip should be supported by cytologic or histologic evidence of pyogranulomatous inflammation. the currently recommended "gold standard" for fip diagnosis is immunohistochemistry performed on effusions or lesions containing infected macrophages. 117 the prognosis for recovery is poor. etiology canine leptospirosis is caused by leptospira interrogans sensu lato, with at least 10 serovars appearing to have clinical significance in dogs. 118 serovars canicola and icterohemorrhagica have been included in vaccines for more than 30 years and the incidence of clinical disease from these serovars has decreased accordingly. an epidemiologic shift in serovars causing clinical disease has since occurred, with increasing reports of disease associated with serovars grippotyphosa, pomona, and bratislava. 118 it has been suggested that serovars icterohemorrhagica and pomona are more likely to be associated with hepatic damage. 118 however, other reports have been unable to correlate serogroups with specific clinical features. 119, 120 chronic hepatitis has been associated with serovar grippotyphosa 50 and serogroup australis. 51 reports of clinical leptospirosis in cats are rare, although antibodies to several serovars have been demonstrated. 118 pathophysiology acute renal failure is the most common clinical disease syndrome in dogs with leptospirosis. 121 the liver can also be a target organ and alf may occur concurrently in 10% to 20% of dogs with acute renal failure or independent from renal involvement. 121 with acute hepatic involvement, the liver is enlarged, friable, and yellowbrown. tissues are often markedly jaundiced. microscopic changes in the liver include intrahepatic cholestasis, liver cell dissociation, and nonspecific reactive hepatitis. 3, 122 hepatic necrosis is an uncommon histologic feature. the liver may not show striking changes, presumably because hepatic dysfunction can be caused by a toxin that produces mainly subcellular damage. organisms can be identified in tissues with a warthin-starry stain. common clinical signs include anorexia, depression, and vomiting. hepatocellular involvement is suggested by jaundice. other findings may include arthralgia or myalgia, pu, pd, fever, and dehydration. widespread petechial and ecchymotic hemorrhages of the mucous membranes, sclera, and skin are caused by thrombocytopenia and dic. the terminal stages include signs of cardiovascular collapse, shock, coma, and death. a diagnosis of leptospirosis should be considered in dogs with acute cholestatic liver disease, especially when accompanied by acute renal failure. hematologic findings vary with the stage and severity of disease. leukocytosis and left shift are frequent, but in the early stages of leptospiremia, leukopenia is more likely. thrombocytopenia can also be seen. coagulation parameters are normal unless complicated by dic. routine serum chemistry and urinalysis findings reflect involvement of the liver or kidney. serum liver enzyme activity is usually increased with hepatic involvement, and the magnitude of the increase in serum alp activity is usually greater than that of serum alt activity owing to intrahepatic cholestasis. other findings include hyperbilirubinemia, bilirubinuria, and abnormal liver function tests. an increase in bun or creatinine may result from renal failure or prerenal uremia. the urinalysis is often compatible with acute nephritis with findings of proteinuria and increased temperature (36.7°c to 37.8°c [98°f to 100°f]) may be helpful. intraperitoneal infusion of 1 to 2 ml of hyperimmune serum obtained from bitches with previously infected litters may reduce mortality rates. vaccination for herpesvirus infection is not routinely performed because of the low incidence of disease. canine acidophil cell hepatitis, which encompasses a spectrum of hepatic lesions ranging from acute and chronic hepatitis to cirrhosis and liver failure, has been reported in great britain. 48, 114 it is caused by a transmissible agent, suspected to be a virus that is distinct from cav-1, although a specific virus has never been identified. the disease is experimentally transmissible by serum or liver extracts from affected dogs. in the experimentally induced disease, acute hepatitis can progress to chronic hepatitis in the absence of clinical signs. episodic increases in serum alt activity and fever spikes correspond with histologic evidence of acute hepatitis. the liver is enlarged and friable in the acute stages, and becomes progressively smaller and nodular with chronicity. the most notable histologic feature, regardless of the stage of disease, is the acidophil cell. acidophil cells are dying hepatocytes with an angular shape, reduced volume, hyperchromatic nucleus, and strongly acidophilic cytoplasm caused by small acidophilic coalescing granules. end-stage hepatic disease is accompanied by typical findings of cirrhosis. most dogs with spontaneous disease are presented with signs of chronic hepatic failure. the duration of clinical signs can exceed 1 year. based on experimental studies, it is speculated that the early mild stages may go unrecognized until advanced hepatic disease and failure is present. biochemical findings are consistent with hepatic inflammation and necrosis, evidenced as increased serum alt activity. with advanced disease, severe hepatic dysfunction is noted. the diagnosis requires liver biopsy. recommendations for specific therapy of acidophil cell hepatitis await further information on the causative agent. supportive measures should be instituted as needed. feline infectious peritonitis (fip) is a highly fatal coronaviral infection of both domestic and wild cats. the liver is one of many organs (kidneys, spleen, pancreas, mesenteric lymph nodes, cns, uveal tract, omentum, serosal surfaces) that can be affected by widespread immune complex vasculitis and granulomatous or pyogranulomatous inflammation (see table 61 -5). 117 clinical findings in cats with hepatic involvement are nonspecific and include lethargy, depression, anorexia, dehydration, weight loss, and fever. jaundice is a common finding. extrahepatic findings include nodular renomegaly, abdominal mass (lymph node), and ascites or dyspnea (pleural effusion). ophthalmoscopic examination may detect chorioretinitis or anterior uveitis, which must be differentiated from similar ocular changes seen with the other systemic disorders that involve the liver such as lymphosarcoma, toxoplasmosis, and the systemic mycoses. serum hepatic enzyme (alp and alt) activities are usually normal or only mildly increased. mild to moderate increase in serum bilirubin concentration is common. other findings indicating hepatic dysfunction include bilirubinuria and increased sba concentrations. hyperglobulinemia, neutrophilia, and mild to moderate nonregenerative anemia are other laboratory features of fip. abdominal and pleural effusions, when present, are usually pyogranulomatous exudates with greater than 3 g/dl protein. serologic detection of a high coronaviral antibody titer may support a diagnosis of fip, but is not definitive because of its lack of specificity. the serum bile acid concentration can be markedly increased (>200 µmol/l). 124 extrahepatic bacterial infection-induced hepatic damage should be considered when evidence of cholestatic hepatopathy is found concurrently with extensive bacterial infection in other organ systems (e.g., pyometra, peritonitis) or with extrahepatic disorders likely to be associated with endotoxemia (e.g., parvoviral enteritis). associated clinical findings that would be compatible with endotoxemic crisis include shock, fever or hypothermia, hypoglycemia, neutrophilia or neutropenia with left shift, toxic changes of the neutrophils, and hyperbilirubinemia that is disproportionately increased in comparison to serum alp activity. 124 it is important to recognize that jaundice can occur secondary to extrahepatic infection from a diagnostic standpoint, so that the clinician is not misled into considering that the cause is a primary hepatic or biliary disease. in jaundiced patients with evidence of an inflammatory process, key differential diagnoses include acute pancreatitis, extrahepatic bacterial infections, and primary hepatobiliary disorders such as leptospirosis (dogs only), cholangiohepatitis, cholecystitis, ruptured gallbladder mucocele, and hepatic abscesses. specific therapy for hepatic disease is not usually required, and hepatic damage is reversible with control of sepsis. morbidity is related to the underlying disease process and not overt hepatic failure. etiology hepatotoxicity can be caused by a variety of drugs (prescription or over-the-counter), herbal and dietary supplements, or biologic toxins or chemicals (see box 61-1). 17, 126, 127 the liver is uniquely susceptible to xenobiotic substances because it is directly exposed to them following absorption from the gi tract. the liver is also vulnerable to toxic injury because it plays a central role in the metabolism of many substances. hepatic metabolism renders lipophilic substances more hydrophilic, which promotes excretion via the urine or bile. 17 the process is controlled by phase i and phase ii reactions. phase i reactions are catalyzed by the cytochrome p450 enzyme systems, which activate or detoxify (oxidize, reduce, or hydrolyze) a drug or toxin. phase i reactions may lead to generation of unstable chemically reactive intermediates, which can be toxic. phase ii reactions conjugate drugs or metabolites and produce products that are nontoxic. during biotransformation, the liver can either reduce or enhance the toxicity of the parent compound. for example, after carbon tetrachloride ingestion, the liver converts the nontoxic parent compound into toxic metabolites, which subsequently cause severe hepatocellular damage. genetic polymorphisms of phases i and ii enzymes have the potential to influence drug metabolism in the individual animal. there has been an increased awareness and recognition that drug-induced liver injury can be a significant cause of liver disease in dogs and cats (see box 61-1). this information has been gained from isolated case reports, retrospective clinical studies, and experimental studies. unfortunately, for many of these drug reactions, characterization of the clinical and pathologic features are lacking because only small numbers of affected animals have been described, and liver biopsies are not typically obtained when drug withdrawal results in clinical and biochemical improvement. it is possible that drug-induced liver injury is underrecognized in dogs and cats, as in humans, drug-induced hepatic injury accounts for more than 50% of the cases of alf in the united states, and is the most frequent reason cited for withdrawal of an approved drug from the market. 17 leukocytes, erythrocytes, and granular casts. increased serum creatine kinase activity may indicate leptospiral-induced muscle damage. leptospirosis is most easily diagnosed in the clinical setting by demonstration of a fourfold rise in serum antibody titer (microscopic agglutination test) in paired samples taken at initial presentation and 2 to 4 weeks later. the rise in titer indicates recent or active infection and differentiates a titer from previous exposure or previous vaccination. the optimum treatment regimen for leptospirosis is unknown. traditionally, intravenous ampicillin, 25 mg/kg every 6 hours (with dose reduction in dogs with renal failure), or penicillin g (25,000 to 40,000 units/kg iv q12h), has been used for initial treatment of leptospirosis. 121 doxycycline (5 mg/kg po q12h for 2 to 4 weeks), was recommended as followup therapy to eliminate organisms from renal tubules. however, doxycycline, 5 mg/kg orally or intravenously every 12 hours for 2 weeks, appears to be effective in clearing all phases of leptospiral infection and may be the most effective treatment strategy. 121 management of fluid, electrolyte, and acidbase imbalances is important supportive therapy. the prognosis generally depends on the degree of renal dysfunction and is poor when oliguria develops. c. piliforme (formerly known as bacillus piliformis), a spore-forming gram-negative bacteria, is a rare cause of multifocal hepatic necrosis and necrotizing ileitis in dogs and cats. 123 the infection is mainly opportunistic in stressed or immunocompromised animals with a predisposing disorder (e.g., canine distemper, feline panleukopenia, feline leukemia) or familial hyperlipoproteinemia in kittens. 123 clinical signs include an acute onset of anorexia, lethargy, depression, and abdominal discomfort. jaundice may be observed, especially in cats. these signs rapidly progress to a moribund state; death occurs within 24 to 48 hours. marked increases in alt activity may be detected. histopathology reveals multifocal periportal hepatic necrosis and necrotic ileitis or colitis. bacilli are best seen with special staining techniques (warthin-starry or giemsa), or methylene blue-stained impression smears of fresh tissue. organisms appear as large, slender, intracellular filamentous organisms within hepatocytes surrounding areas of necrosis and in intestinal epithelial cells. routine culture techniques are ineffective for isolation of this organism. the disease is rapidly fatal, and successful therapy has not been reported. 123 extrahepatic bacterial infection associated with sepsis and endotoxemia is an important cause of acute functional cholestatic hepatopathy. however, morbidity is generally related to the underlying infection and not overt hepatic failure. 113, 124 studies in humans and experimentally in dogs, suggest that endotoxemia and the subsequent release of cytokines induces functional changes that interrupt the transport and excretion of conjugated bilirubin. 113, 125 microscopically, hepatic lesions are often mild and nonspecific. intrahepatic cholestasis, characterized by bile canalicular plugs and bile pigment accumulation in hepatocytes, is the most consistent finding. 124 a mild periportal lymphocytic infiltrate can be seen, with scattered foci of macrophages or neutrophils, and occasional individual necrotic hepatocytes. total serum bilirubin concentrations as high as 30 mg/dl can be seen and are disproportionately high compared to the mild to moderate increases in serum alp activity. increased serum alt activity is a less consistent finding. hepatic injury caused by drugs, herbal and dietary supplements, biologic toxins, or chemicals can occur via a number of mechanisms, which influence the histologic pattern of disease. 17, 20 although hepatic necrosis is the most common histologic response, drug-and toxin-induced liver injury can also potentially mimic the full spectrum of acquired hepatic disorders, including acute and chronic hepatitis, granulomatous hepatitis, cholestatic hepatopathy, vacuolar hepatopathy (lipid or glycogen accumulation), hepatic fibrosis and cirrhosis, and venoocclusive disease. hepatic necrosis occurs when covalent binding of a drug or toxin to intracellular proteins disrupts cellular functions, or formation of drug-enzyme adducts stimulates an immunologic response (antibody-or t-cell-mediated cytotoxicity). hepatic inflammation (typically lymphoplasmacytic, but may be granulomatous or eosinophilic), suggests an underlying immunoallergic mechanism. intrahepatic cholestasis occurs when drugs or toxins interfere with hepatic transport proteins at the canalicular membrane, which interrupts bile flow. hepatic lipid accumulation results when direct damage to mitochondria disrupts fatty acid oxidation and energy production, and is the predominant hepatic lesion seen with stanozolol hepatotoxicity in cats, 132 and tetracycline in dogs and cats. 126 hepatic vacuolation caused by glycogen accumulation occurs in dogs treated with corticosteroids, and typically does not cause significant clinical evidence of hepatic dysfunction. damage to the sinusoidal epithelium can result in peliosis hepatic or venoocclusive disease. mechanisms of drug-induced liver injury (and also injury from herbal and dietary supplements) can be characterized as either intrinsic (predictable) or idiosyncratic (unpredictable) reactions. 126 intrinsic hepatotoxic reactions are dose-related and occur shortly after a consistent threshold of toxicity is reached. because intrinsic hepatotoxins predictably damage the liver in an exposed population, they can be experimentally reproduced and studied. hepatic injury is caused by a direct toxic effect of the parent compound (or a reliably generated toxic metabolite) on vital cell targets. acetaminophen is an intrinsic hepatotoxin in dogs and cats and is discussed in more detail later in this section. with intrinsic hepatotoxins, lowering the dose, rather than stopping the drug can be tried. in many cases, most such drugs or chemicals, (e.g., carbon tetrachloride, phosphorus, and chloroform), are no longer used for therapeutic purposes once their intrinsic hepatotoxicity is recognized, but accidental exposures could still occur. in contrast, idiosyncratic hepatotoxic reactions occur at therapeutic doses in only a small number of individuals in the exposed population. these reactions are unpredictable and infrequent; most individuals treated with the drug do not have a reaction, even at high doses. however, toxicity may be more pronounced at higher doses in susceptible individuals. 72, 133 examples of drugs causing idiosyncratic drug-induced liver injury in dogs and cats that have a dose-related effect are phenobarbital, itraconazole, amiodarone, and lomustine (ccnu). 134 idiosyncratic reactions are characterized by a variable latency period (5 to 90 days, but may be longer for some drugs, such as phenobarbital, ccnu, amiodarone) from initial drug ingestion to recognition of hepatic injury. because of the infrequent occurrence (approximately 1 in 100,000), the potential for hepatotoxicity may not be recognized in preclinical screening of a new drug, and cannot usually be reproduced in an experimental setting. individual differences in susceptibility to idiosyncratic drug-induced liver injury may reflect genetic differences in either (a) alternate metabolic pathways by which a drug is converted to different (potentially hepatotoxic) metabolites, (b) the ability of the individual to detoxify the toxic intermediates, (c) an underlying the center for veterinary medicine of the food and drug administration, washington, dc, maintains a registry (http://www.fda.gov/ animalveterinary/default.htm) for reporting adverse drug reactions in animals. this service has been useful to accumulate data and to alert veterinarians to suspected drug effects, including hepatotoxicity. however, because reporting of adverse drug reactions is voluntary and the information obtained may be incomplete, only subjective trends can be identified. furthermore, because mild hepatic injury may not be associated with clinical signs, these cases will not be detected unless biochemical testing is performed while the animal is receiving the drug. evaluating the incidence of druginduced liver injury in dogs and cats is further clouded by the fact that there are no pathognomonic clinical, laboratory, or biopsy findings to distinguish drug-induced liver injury from other causes of liver disease. specific drugs that have been reported to cause druginduced liver injury are listed in box 61-1. herbal and dietary supplements (herbs or other botanicals, nutraceuticals, vitamins, minerals) have the potential to cause hepatotoxicity, similar to drug-induced injury. [127] [128] [129] in humans, herbal and dietary supplements are reported to account for 10% of patients with "drug-induced" liver injury. 127 the incidence of hepatotoxicity may be underrecognized, as only a third of humans taking herbal and dietary supplements reported their use of these products to their health care provider. 127 causality is difficult to prove, because herbal and dietary supplements are often dispensed without medical supervision, fda oversight of product quality is minimal, multiple active ingredients contribute to product variability, and product contamination with hepatotoxic substances (toxic herbs or heavy metals) may occur. [127] [128] [129] drug interactions between herbal and dietary supplements and prescription medications may also occur, because medicinal plants can have effects on hepatic p450 enzyme systems. 129 the rate of dietary supplement use in dogs and cats appears to be lower than that reported for humans. 130 however, because these products are often marketed as "all natural," pet owners may assume they are safe and underreport their use to the veterinarian. reports of herbal and dietary supplement hepatic injury in animals are rare. pennyroyal oil, a volatile oil derived from plants of the labiatae family (pennyroyal, squaw mint, or mosquito plant) was associated with fatal acute hepatic necrosis in a dog after topical application for use as a flea repellent. clinical signs occurred within 1 hour after application and included vomiting, diarrhea, hemorrhage, seizures, and death. other reports include the finding of increased liver enzyme activity in dogs consuming st. john's wort, and increased alt activity and hypoglycemia after accidental ingestion of high doses of α-lipoic acid in two dogs. 129, 131 the paucity of reported hepatic reactions does not necessarily mean herbal and dietary supplements are safe, and the clinician should maintain a high level of suspicion regarding their potential toxicity. box 61-1 lists the herbal and dietary supplements that have been incriminated as causing hepatic injury in humans with potential relevance to dogs and cats. for more detailed information on hepatic injury and herbal and dietary supplements, additional sources should be consulted. [127] [128] [129] hepatic injury may also occur after exposure to a wide variety of industrial chemicals, organic solvents, pesticides, heavy metals, and biologic toxins. most information on chemical hepatotoxins is derived from experimental studies in dogs or extrapolated from information in other species. very few clinical case reports are available in the veterinary literature. isolated reports and case series of clinical liver disease in dogs associated with exposure to biologic toxins, such as aflatoxin, amanita mushrooms, blue-green algae, and cycads (sago palms) have been published. selected hepatotoxins are listed in box 61-1. increased liver enzyme activity is a common finding with hepatotoxicity. increased alt activity (more than three times the upper limit of normal, but can be as high as 100 times the upper limit), suggests hepatocellular injury (often necrosis), and is of more concern than an isolated increase in alp activity (reflecting cholestasis), although mixed patterns commonly occur. progressive increases in alt activity or those accompanied by evidence of hepatic dysfunction (hyperbilirubinemia, increased serum bile acids, coagulopathy, hypoglycemia, hyperammonemia, hypoalbuminemia) are more likely to represent serious hepatic injury. when drug-induced liver injury is suspected, the diagnostic approach is determined by the clinical presentation. if clinical signs are absent or mild, a minimum database consisting of complete history and physical examination, complete blood cell count, serum chemistry, and urinalysis should be performed. if the only abnormality detected is increased liver enzyme activity, and these increases correspond to the recent administration of a drug (especially those listed in box 61-1), the drug should be discontinued and serum biochemistries should be repeated in 10 to 14 days. in many instances, clinical and biochemical abnormalities resolve after the suspected hepatotoxic drug is discontinued and a liver biopsy is not performed. further evaluation of the liver, including sba concentrations, abdominal radiographs, ultrasonography, and liver biopsy, may be warranted if biochemical abnormalities persist, or if initial clinical and biochemical findings suggest hepatic dysfunction. with suspected drug-or toxin-induced liver disease, a liver biopsy can be helpful to (a) characterize the histologic changes (are they consistent with previously described lesions caused by this particular drug or toxin?), (b) determine the severity (focal or diffuse necrosis?) or reversibility (is cirrhosis present?) of the lesions for prognostic purposes, and (c) rule out known causes of liver disease. histologic changes secondary to drug-and toxin-induced hepatic injury are nonspecific and similar to those seen with other nondrug-related causes of acute and chronic liver disease. 126 the most common pathophysiologic response is necrosis without inflammation. 126 hepatic necrosis may be centrilobular (zone 3) or panlobular (figure 61-20) . centrilobular hepatocytes have an abundance of p450 enzymes, and are preferentially affected in drug-induced hepatotoxicity when p450 metabolism of the parent drug results in toxic metabolites. 8 drugs or toxins can also cause a variety of other hepatic lesions, such as cholestasis, lipidosis, or mild inflammation. a chronic response to injury is reflected by findings of biliary immunologic or allergic reaction, or (d) an individual's tolerance or ability to "adapt" to hepatocellular injury (mechanisms unknown) with resolution of injury despite continued medication administration. 20, 135 oral medications with substantial hepatic metabolism are more likely to be associated with adverse hepatic events in humans, presumably because of hepatic generation of reactive toxic metabolites. 136 because of the unpredictability of an idiosyncratic reaction and the low incidence of occurrence, a cause-and-effect relationship is difficult to establish. if an idiosyncratic reaction occurs, the drug must be discontinued or it could result in death of the patient. an idiosyncratic mechanism is suspected for most of the drugs that cause hepatic injury in dogs and cats. susceptibility to hepatotoxicity in humans is influenced by a number of factors such as age, sex, nutritional status, and concurrent drugs. 20 the most important factor may be the effect of genetic polymorphisms on hepatic drug metabolism. 20 in humans, preexisting liver disease does not appear to enhance susceptibility to druginduced liver injury, but impacts the patient's ability to recover. 20 this may be because drug-metabolizing enzyme systems are remarkably preserved in hepatic disease. similar information on risk factors for hepatotoxicity in dogs and cats has not been determined. however, because many toxic metabolites are normally detoxified by glutathione, some metabolites may become more toxic when hepatic glutathione stores are depleted (e.g., animals with preexisting chronic necroinflammatory and cholestatic liver disease). 126 a breed predisposition has been suggested for doberman pinschers (sulfonamides, amiodarone, diethylcarbamazine/oxibendazole) and labrador retrievers (carprofen), which may be a reflection of a genetic predisposition. 21, 71, 73, 134, 137 clinical examination the spectrum of drug-and toxin-induced liver injury and, thus, the associated clinical presentation, can vary from subclinical hepatic injury with only increased serum liver enzyme activity, to severe liver damage manifested as alf, or chronic end-stage liver disease. acute rather than chronic liver injury is more likely for most of the drugs and toxins listed in box 61-1. clinical features often include acute onset of lethargy, anorexia, vomiting, diarrhea, pu, pd, or jaundice in a previously healthy animal, which corresponds to hepatotoxin exposure. alf (signs of acute liver disease plus he and coagulopathy) is most likely with drugs or toxins that cause diffuse hepatic necrosis. drug-and toxin-induced injury is an important diagnostic consideration in dogs and cats presenting for acute hepatitis and hepatic necrosis. drugs or toxins also have the potential to cause chronic hepatic disease, if the initial hepatic injury is mild and goes unrecognized, and exposure to the drug or toxin is continued. for example, phenobarbital, ccnu, or chronic aflatoxicosis can cause chronic liver injury in dogs. 70, 72, 138 diagnosis there are no pathognomonic clinical, laboratory, or biopsy findings that distinguish drug-or toxin-induced liver injury from other causes of liver disease. the diagnosis of drug-induced liver injury often relies on the clinician maintaining a high level of suspicion, and obtaining an accurate and thorough medication history (including prescription and over-the-counter drugs, and herbal and dietary supplements), in every animal with unexplained increases in liver enzyme activity or clinical liver disease. a definitive diagnosis of hepatic injury caused by biologic toxins or chemicals is rarely possible in a clinical setting, unless the owner specifically observes ingestion of a substance that is a known hepatotoxin. prevented. treatment of drug-induced hepatic disease consists of discontinuing the suspect drug. after a drug is discontinued, clinical (and biochemical) improvement usually occurs within a few weeks, even with chronic drug administration. however, exceptions can occur. for example, in dogs with amiodarone toxicity, liver enzyme elevations can transiently progress despite discontinuation of the drug, and biochemical abnormalities may not resolve for 6 to 8 weeks. 137 with the exception of nac for acetaminophen, and silymarin for amanita mushroom toxicity, no specific antidotes are available, and treatment of drug-and toxin-induced liver injury is primarily supportive and symptomatic. however, nonspecific hepatoprotective therapy with antioxidants (vitamin e), glutathione replacement (nac, same), or milk thistle (silymarin) may be helpful and are discussed in more detail in chapter 46. use of nac (or same) may be beneficial, as glutathione depletion may predispose to hepatotoxicity (e.g., methimazole) or impair metabolism of toxic metabolites to a nontoxic form. nac has been suggested to be beneficial for treatment of alf associated with toxicities such as diazepam, methimazole, carprofen, and trimethoprim-sulfa. 9 in addition to treating amanita mushroom hepatotoxicity, silymarin may be beneficial in the treatment of carbon tetrachloride and acetaminophen toxicity. 9,140,141 corticosteroids are not typically indicated for treatment of drug-and toxin-induced hepatotoxicity. it is important to consider a drug-induced cause of liver injury because rapid recognition and prompt discontinuation of an hepatotoxic drug can lead to improvement or complete resolution of hepatic disease, depending on the specific drug and the stage of the lesion. when drug-or toxin-induced hepatic injury causes severe or widespread hepatic necrosis, rapid deterioration and death in 3 to 4 days often occur. with less-severe hepatic injury, complete recovery is possible. acetaminophen acetaminophen is well known as an intrinsic hepatotoxin in dogs and cats. 142, 143 although acetaminophen is occasionally used as an analgesic in dogs (therapeutic doses up to 15 mg/kg tid), most toxicity occurs because of accidental ingestion of improperly stored medication (dogs) or owner administration without veterinary supervision (dogs and cats). 142 toxic metabolites of acetaminophen cause oxidative injury to erythrocytes and hepatocytes, resulting in methemoglobinemia, anemia, and hepatic necrosis. in therapeutic doses, acetaminophen is detoxified by a combination of hepatic glucuronidation and sulfation and renal excretion. 144 after acetaminophen overdosage, these pathways become saturated and a greater proportion of acetaminophen is metabolized through the p450 system, leading to production of the toxic metabolite, n-acetylp-benzoquinoneimine (napqi). glutathione detoxifies napqi and thus protects hepatic cellular constituents from its direct toxic effect. however, once glutathione levels are depleted by large amounts of napqi, centrilobular necrosis occurs. toxicity occurs in a dose-dependent manner. there are substantial species differences in both the metabolism of acetaminophen and the toxic manifestations. 142 cats are uniquely sensitive to acetaminophen because of a deficiency of glucuronyl transferase and limited sulfation capabilities. clinical signs in cats may develop after administration of as little as 162.5 mg ( 1 2 tablet). signs of methemoglobinemia usually dominate the clinical picture, such as cyanosis, dyspnea, facial edema, depression, hypothermia, hyperplasia, fibrosis, and cirrhosis. although individual drugs and drug classes may follow the same pattern, there is often not a consistent reaction for any given drug. for example, hepatic injury in dogs secondary to potentiated sulfonamides may cause hepatic necrosis, primary cholestasis, or marked inflammation. 21 for many of the potentially hepatotoxic drugs listed in box 61-1, histologic features have not been fully characterized. it should be emphasized that for most drug-induced disorders, the diagnosis is presumptive and cannot be proved. it can be especially difficult to pinpoint the causative agent when the patient is receiving a combination of drugs. a clinical diagnosis of drug-induced hepatic injury is easier to establish when the hepatotoxicity of the drug has been previously described and the associated clinical and pathologic features have been characterized (see discussion of specific drugs). the diagnosis may be less convincing when the suspected drug has not been previously incriminated as causing liver damage. however, a drug reaction should still be considered, since an idiosyncratic reaction could occur with any drug. the clinician should also maintain a level of suspicion regarding the potential for hepatotoxicity in newly marketed drugs that have not yet been used widely in the population, where idiosyncratic reactions are often first detected. the suspected hepatotoxic drug should be discontinued while a complete diagnostic evaluation is pursued for other causes of liver disease, for which a specific treatment might be available. a diagnosis of drug-induced injury is supported by the following: (a) evidence of liver injury that occurred within the first 3 months of drug therapy (especially if predrug liver enzyme activity was within normal limits); (b) clinical and biochemical improvement when the drug is discontinued; (c) exclusion of other causes of liver disease; (d) reccurrence of hepatic damage after a challenge dose of the same drug (or inadvertent reexposure). it should be emphasized that rechallenge with a suspected hepatotoxic drug is not recommended as a diagnostic consideration, because it is potentially dangerous, especially with a drug that causes acute hepatic necrosis. rechallenge should only be considered if the association of the drug with hepatic injury is highly questionable and there is no alternative drug available for a significant medical condition. with hypersensitivity or immunologic reactions, the hepatic reaction is more rapid and severe with repeated exposure. 20 hepatic injury because of a biologic toxin or chemical is suspected when exposure to a potential hepatotoxin has been documented. a clinical diagnosis of "toxic" hepatic injury is often made when an episode of acute hepatic injury occurs, hepatic biopsy indicates diffuse hepatic degeneration and necrosis, and no other cause for liver disease can be identified. in selected cases, tissues, blood, or food (aflatoxin) samples can be submitted to a toxicology lab to confirm a suspected toxin. toxin-induced injury should also be considered in the absence of known exposure to toxins, because potential hepatotoxins can be present in contaminated dog food or garbage (aflatoxins), pond water (blue-green algae), and many other unobserved sources. when ingestion of a potential hepatotoxin (e.g., toxic mushrooms, sago palms) has occurred within the preceding 8 hours, general procedures for gi decontamination are recommended, including induction of emesis or gastric lavage (within first 3 hours), followed by administration of activated charcoal (1 to 3 g/kg). 126, 139 induction of vomiting is contraindicated if the patient is comatose or debilitated in such a way that the gag reflex is diminished, which could predispose to aspiration pneumonia. whenever possible, the source of toxin exposure should be identified and further exposure or given as alternate-day therapy), with careful monitoring of liver parameters every 2 weeks. 146 hepatotoxicity appears to be at least partly dose related, as dogs receiving higher daily doses of itraconazole (10 mg/kg) are more likely to be affected. icterus and evidence of hepatic dysfunction suggests a more serious, potentially fatal hepatopathy, requiring discontinuation of medication and symptomatic and supportive care. it is recommended that liver enzymes be monitored on a monthly basis in all animals receiving ketoconazole or itraconazole. azathioprine, a purine analogue commonly used for treatment of immune-mediated disorders in dogs, is commonly listed as a potential hepatotoxin. 126 however, few clinical details regarding the hepatotoxic reaction are available. in a clinical study of 12 dogs with atopic dermatitis treated with azathioprine (2.2 mg/kg daily for 8 weeks) as a single agent, an increase in alt or alp activity was noted in the first 2 weeks in 10 (83%) of the dogs. 147 three dogs had clinical signs suggestive of liver disease, which resolved uneventfully when azathioprine was discontinued. in an experimental study of dogs given azathioprine at a dose of 2 to 4 mg/kg po daily for 40 days, all dogs had increased liver enzyme activity (alt >> alp) within 2 to 7 days of initiating therapy. 148 values peaked within the first 2 weeks and then declined, but not to normal, despite continued medication administration. hyperbilirubinemia was absent. liver biopsies in most dogs revealed centrilobular degeneration and necrosis, with intrahepatic cholestasis but no inflammation. 148 these findings raise the possibility that azathioprine may be an intrinsic (dose related) hepatotoxin in dogs, with possible adaptive tolerance to liver injury. it should be noted that doses used in this study exceeded current clinical recommendations of 1 to 2 mg/kg daily or every other day for maintenance therapy. hepatotoxicity is considered a class characteristic of nsaids, despite the fact that there are many different chemical classes of nsaids, and no consistent mechanism of liver injury. 149 with the exception of aspirin, which is an intrinsic (dose-related) hepatotoxin, the mechanism with other nsaids is believed to be idiosyncratic (either immune or as a consequence of toxic metabolites). 149, 150 toxicity does not appear to be related to prostaglandin inhibition like the renal or gi side effects. 149 preexisting hepatic disease has not been shown to be a risk factor for nsaid-induced liver injury. 150 all nsaids have the potential to cause idiosyncratic hepatotoxicity in dogs, but hepatic reactions appear to be rare. 150 carprofen has specifically been reported as a cause of drug-induced liver injury in dogs. 73 labrador retrievers were overrepresented in the series, but it is not clear whether this is a true breed predisposition. 150 clinical signs (anorexia, lethargy, vomiting, pu/pd) occurred within the first 4 weeks of therapy and icterus was a common finding on physical examination. biochemical evaluation revealed marked increases in liver enzymes (alt activity usually exceeded alp activity) and hyperbilirubinemia. hepatic biopsy findings revealed multifocal to diffuse hepatic necrosis, mild to moderate lymphocytic-plasmacytic inflammation, secondary cholestasis, and variable biliary hyperplasia and bridging fibrosis. 73 concurrent renal toxicity (glucosuria without hyperglycemia, proteinuria, granular casts) also was noted in some dogs. most dogs recovered with discontinuation of carprofen and appropriate supportive care, although some dogs died of alf. general hepatoprotective therapy with same or silybin has been recommended, although the benefits are unproven. nac has been suggested for ancillary treatment when carprofen causes alf. 128 and vomiting. although increases in serum alt activity may be detected, centrilobular hepatic necrosis appears to be uncommon. clinical signs in dogs are more likely when doses exceed 200 mg/kg and can be indicative of methemoglobinemia and/or centrilobular necrosis. 142 laboratory features include methemoglobinemia, anemia, increased serum alt activity, and hyperbilirubinemia. intravenous nac is the treatment of choice for acetaminophen toxicity in dogs and cats. 142 nac increases the synthesis and availability of glutathione, which when conjugated to napqi, decreases toxicity. for maximum effectiveness, nac should be given within 12 hours of acetaminophen exposure; however, there may still be a benefit if given 36 to 80 hours after exposure. nac (10% solution) is diluted 1 : 2 or more with saline and given intravenously through a nonpyrogenic 0.25 µm filter at an initial dose of 140 mg/kg over a 20-to 30-minute period. a maintenance dose of 70 mg/kg is given iv or orally every 6 hours for seven treatments. same also serves as a glutathione source and has been shown to have protective effects against acetaminophen-induced oxidative stress on the erythrocytes in cats and dogs. 143, 145 in an experimental study in cats, silymarin (30 mg/kg po) was as effective as nac for treatment of acetaminophen toxicity when given up to 4 hours after exposure. 140 vitamin c (30 mg/kg iv q6h) may be helpful in the treatment of acetaminophen toxicity because of its antioxidant effects. cimetidine (5 mg/kg iv q8h) is also recommended as adjunctive therapy in the early stages (first 16 hours) because it inhibits hepatic p450 enzymes and decreases napqi formation. the antiarrhythmic drug amiodarone is associated with a reversible hepatotoxicity in dogs. 134, 137 doberman pinschers may be at increased risk. 134 toxicity, which appears to be at least partially dose related (doses of 400 mg/day), was identified in 45% of doberman pinschers treated with amiodarone in one clinical series. 134 clinical signs (anorexia, lethargy, vomiting, diarrhea) and biochemical abnormalities (increased alt and alp activity ± hyperbilirubinemia) developed 6 days to 8 months after initiation of therapy. liver biopsy in one dog revealed multifocal hepatocellular necrosis with mild lipidosis and lymphoplasmacytic inflammation. 137 clinical improvement usually occurs within a few days of stopping the drug, but liver enzyme elevations may not return to normal for 3 months. 134 transient progression of enzyme abnormalities despite discontinuing the drug has also been noted, which may reflect the long half-life of amiodarone causing a delay in systemic elimination. 137 biochemical changes precede clinical signs, so monitoring of liver enzymes at least monthly is recommended. the azole antifungal drugs ketoconazole and itraconazole (and rarely fluconazole) are associated with increased liver enzyme activity and icterus in dogs and cats. 146 hepatotoxicity is more likely with ketoconazole than with itraconazole. cats are more sensitive to the hepatotoxic effects than are dogs, but considerable individual variation occurs. histologic findings are poorly characterized but include bile duct proliferation and infiltration of mononuclear cells. transient mild subclinical elevations of liver enzymes (alt and alp activity) are common, and do not necessarily require a change in therapy. a clinically significant hepatic reaction is suggested by alt activity that exceeds two to three times the upper limit of normal, especially when accompanied by clinical signs of anorexia and vomiting. drug therapy should be stopped for 1 to 2 weeks until appetite and liver enzymes return to normal. a rapid recovery usually occurs, and treatment can be restarted at a lower dose (50% of previous dose decreased appetite, weight loss, pu/pd, vomiting, ascites, and pleural effusion. ascites was due to a combination of hypoalbuminemia and portal hypertension. common biochemical abnormalities included increased liver enzyme activity (alt, ast, alp, ggt) and hypoalbuminemia. other less-consistent findings included hyperbilirubinemia, hypercholesterolemia, and increased serum bile acid concentrations. glucosuria (without hyperglycemia) and renal failure were noted in some dogs, possibly attributable to ccnu renal toxicity. 72 liver biopsy findings were nonspecific (hemosiderinladen kupffer cells, hepatocellular vacuolization, mild to moderate periportal inflammation, and fibrosis) but suggested chronicity. the majority of affected dogs died from progressive chronic liver disease. in a recent study, routine monitoring of alt activity prior to each subsequent dose of ccnu suggested that subclinical elevations of alt activity (greater than five times the upper limit of normal) are common. 155 thirty-two of 109 dogs (29%) had increased alt activity, which developed most commonly after one to three doses of ccnu. 155 increases in alt activity were not associated with cumulative dose. the lower incidence of clinical hepatotoxicity in this study (3 of 109 or 2.8%) versus the kristal study (11 of 179 or 6.1%), was attributed to prompt cessation of ccnu treatment in dogs with significant increases in alt activity. 155 however, it was noted that chronic administration of ccnu could be associated with chronic irreversible hepatopathy, in the absence of a significant alt elevation. the mechanism of hepatotoxicity is suspected to be a result of generation of toxic intermediate metabolites (e.g., isocyanates, diazonium hydroxide), and depletion of glutathione may play a role. 126 preliminary results of a clinical study using denamarin (same and sylibin; nutramax labs, lancaster, sc) for prevention of ccnu hepatotoxicity, suggested that dogs receiving denamarin had less-severe liver enzyme elevations. 156 methimazole, an antithyroid drug, is associated with hepatic injury in cats with hyperthyroidism. clinical findings include anorexia, vomiting, lethargy, jaundice, markedly increased serum liver enzyme activity, and hyperbilirubinemia that usually occurs within the first month of therapy. 126 histologic lesions have not been fully characterized, although biopsy findings in one cat revealed hepatic degeneration and necrosis. clinical signs resolve within a week of discontinuing therapy but biochemical resolution may take up to 45 days. reduced hepatic glutathione concentrations, which have been documented in other species with hyperthyroidism, may predispose to hepatic injury. 126 treatment with same may be beneficial. phenobarbital is associated with chronic hepatic disease and cirrhosis in dogs. 70 most dogs have been treated with phenobarbital for more than a year before the liver disease is apparent. the mechanism of hepatic injury is not known but higher doses, higher blood levels (>40 µg/ml), and long duration appear to be important risk factors. 70 clinical signs in dogs reflect chronic liver disease and include sedation, ataxia, anorexia, weight loss, weakness, ascites, jaundice, coagulopathy, and encephalopathy. phenobarbital-induced hepatic injury should be suspected in any dog with a history of chronic phenobarbital therapy and clinical and biochemical evidence of hepatic dysfunction. routine biochemical screening (every 4 to 6 months) of dogs on long-term phenobarbital therapy is recommended for early detection of hepatic injury. however, mild liver enzyme elevations (especially alp) are commonly seen in dogs treated with phenobarbital, who do not have clinical or histologic evidence of significant liver early recognition of hepatotoxicity (including periodic monitoring of liver enzymes during the first 3 months) and discontinuation of drug therapy provides the best opportunity for full recovery. whether dogs with previous carprofen hepatotoxicity can be safely switched to another nsaid without experiencing a hepatic reaction is unknown. oral diazepam has been incriminated as a cause of acute idiosyncratic fatal hepatic necrosis in cats. 151, 152 intravenous diazepam, and oral oxazepam, clonazepam, and zolazepam also have been implicated. 126, 152 onset of signs occurs within 5 to 13 days of initiating therapy. clinical signs and biochemical evaluation are consistent with acute hepatic necrosis and liver failure. most cats die within 15 days of initial administration of the drug. if treatment of a cat with oral diazepam is unavoidable, liver enzymes should be checked before and within 5 days after starting therapy. if liver enzymes are increased, the drug should be discontinued and symptomatic therapy should be started. ancillary treatment of alf with nac may be beneficial. 9 glucocorticoid therapy in dogs is commonly associated with increased serum alp activity and development of a reversible vacuolar ("steroid") hepatopathy as a result of hepatic glycogen accumulation. these hepatic effects can be seen with virtually any glucocorticoid preparation (including topical ophthalmic and otic preparations) and are influenced by drug preparation (e.g., repositol versus short-acting), dose, duration of therapy, and individual dog susceptibility. in contrast, cats are quite resistant to the hepatic effects of glucocorticoids and only rarely develop these hepatic changes. 153 increased serum alp activity can occur within 3 days after initiating glucocorticoid therapy in dogs and is often striking (up to 64 times normal). glucocorticoids are associated with the induction of a specific corticosteroid-induced isoenzyme of alp, which may account for 60% to 100% of the total alp activity. 154 in contrast, serum alt activity is often normal or only mildly increased. in most dogs, glucocorticoids do not cause significant hepatic dysfunction or clinically relevant hepatic disease and biochemical tests reflecting hepatic function (serum bilirubin, albumin, glucose, blood ammonia concentration, and coagulation tests) are typically normal. serum bile acid concentrations are normal or only mildly increased (<60 mmol/l). hepatic glycogen accumulation causes hepatomegaly (which can be detected on abdominal radiographs), and diffuse or multifocal increases in hepatic echogenicity (detected on ultrasonography). the hepatic effects of glucocorticoids are reversible after drug withdrawal. the length of time required for complete resolution is unpredictable, varying from weeks to months. lomustine ccnu [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] is an oral nitrosourea alkylating agent that is used for chemotherapy of lymphoma, mast cell tumor, histiocytic sarcoma, and brain tumors in dogs. idiosyncratic dose-related hepatotoxicity was described in 11 of 179 (6.1%) dogs given an oral dose of ccnu (50 to 110 mg/m 2 ), with a dosing interval of 3 to 6 weeks. 72 the median time to detection of hepatic disease (from the last dose of ccnu) was 11 weeks and ranged from 2 to 49 weeks. delay in onset was noted, with an inverse relationship between the size of dose, and length of time before abnormal serum alt was detected. 72 a cumulative dose effect was suspected. clinical findings of hepatotoxicity included glutathione precursors (nac, same) and vitamin c in treatment. 21 dogs with hepatopathy are less likely to recover (46%) than are dogs with nonhepatic manifestations of sulfonamide hypersensitivity (89%). 159 tetracycline can predispose to hepatic lipid accumulation because it inhibits protein synthesis and interferes with hepatic secretion of triglyceride-rich lipoproteins. 126 however, it does not appear that these hepatic effects are clinically significant in most dogs and cats, although clinically significant idiosyncratic hepatic injury has been reported. increased liver enzyme activity was a common finding in dogs treated with doxycycline (increased alt activity in 39.4%; increased alp activity in 36.4%), but the clinical significance remains to be determined. 160 aflatoxicosis aflatoxins are metabolites, produced primarily from strains of the saprophytic fungus, aspergillus, which cause toxic hepatitis in dogs and many other species. 138, 161 exposure in dogs may occur through the inadvertent use of aflatoxin-contaminated corn or peanut meal during the commercial production of dog food, or after ingestion of homemade pet foods, moldy garbage, or improperly stored dog food. 161 dogs are relatively susceptible to aflatoxins and the liver is the target organ. clinical cases of aflatoxicosis in cats have not been reported. aflatoxin b1 is most commonly implicated in hepatotoxicity and toxic effects are seen when levels exceed 60 µg/kg of food. 161 aflatoxin b1 is readily absorbed from the gi tract and undergoes hepatic metabolism by cytochrome p450 enzymes, to a toxic intermediate (aflatoxin b1 8,9-epoxide) , which binds to essential molecules within the cell, leading to hepatocyte necrosis and decreased protein synthesis. detoxification of aflatoxin b1 8,9epoxide occurs by conjugation to glutathione. depending on the amount consumed, dogs may present with acute, subacute, or chronic liver disease. high-dose exposure is associated with acute hepatic failure, jaundice, dic, and death. repeated exposure to low doses can lead to chronic liver disease and cirrhosis. in 2005, an outbreak of aflatoxicosis occurred in at least 100 dogs, as a result of eating a commercially available dog food manufactured with aflatoxin-contaminated corn. 138 severity of clinical signs varied significantly among dogs. some dogs died suddenly without preexisting signs of illness. other dogs were presented with signs of anorexia, lethargy, vomiting, jaundice, diarrhea (including melena and hematochezia), abdominal effusion, he, and evidence of a bleeding disorder. common biochemical features included increased liver enzyme activity (especially alt), hyperbilirubinemia, electrolyte disturbances, hypoalbuminemia, hypocholesterolemia, and prolonged clotting times. reduced plasma antithrombin iii and protein c activities and hypocholesterolemia were suggested to be the most sensitive biomarkers of aflatoxin ingestion in dogs with minimal clinical signs, possibly reflecting an early effect of aflatoxin on biosynthesis of certain proteins and cholesterol. 138 in dogs with acute or subacute aflatoxicosis, the liver is enlarged and pale yellow with histologic features of diffuse hepatic vacuolation (lipid accumulation), scattered individual hepatocyte necrosis, biliary hyperplasia, and modest inflammation. collapse of zone 3 hepatocytes around the central vein, associated with perivenular inflammation, may explain clinical features of portal hypertension. with chronic low-level exposure, findings include a small liver with regenerative nodules, acquired pss, and histologic evidence of marked biliary hyperplasia and periportal fibrosis. disease. 157 potential indicators of clinically significant liver injury include increases in alt and alp activity that exceed five times the upper limit of normal; alt activity that exceeds alp activity; any elevation in ast activity; or enzyme elevations accompanied by evidence of hepatic dysfunction (hyperbilirubinemia, hypoalbuminemia, hypocholesterolemia, increased sba). hepatic cirrhosis associated with chronic phenobarbital therapy is characterized grossly by a small, nodular liver and histologically by bridging portal fibrosis, nodular regeneration, biliary hyperplasia, and mild inflammation (see figure 61-19) . 70 these lesions are by no means pathognomonic for phenobarbital-induced hepatic damage; however, in the absence of other known causes of hepatic damage, circumstantial evidence would support drug therapy as a likely cause. chronic phenobarbital therapy also is associated with superficial necrolytic dermatitis (hepatocutaneous syndrome) in dogs. 158 liver biopsy changes were typical of those seen with hepatocutaneous syndrome (marked vacuolar change and parenchymal collapse), which are distinct from the characteristic chronic hepatitis and cirrhosis as described above. 158 phenobarbital should be decreased or discontinued if possible in dogs with biochemical and histologic evidence of hepatic disease. in dogs with phenobarbital-associated toxicosis, clinical, biochemical, and histologic improvement can occur if the drug is discontinued or used at a reduced dosage prior to severe, end-stage liver disease. improvement in clinical signs can be noted within days to weeks of decreasing serum phenobarbital levels. primidone also is associated with chronic liver disease in dogs, likely as a consequence of metabolism of primidone to phenobarbital. phenytoin can cause acute or chronic hepatitis in dogs, as well as jaundice and death. the risk of hepatotoxicity is increased with combination therapy of phenobarbital, primidone, and phenytoin. 126 sulfonamides potentiated sulfonamides (trimethoprim-sulfadiazine, trimethoprimsulfamethoxazole, and ormetoprim-sulfadimethoxine) are associated with the acute idiosyncratic drug-induced liver injury in dogs. 21 trimethoprim-sulfadiazine was implicated in over 20% of hepatic drug reactions in dogs that were reported to the center for veterinary medicine between 1988 and 1990. 71 doberman pinschers are suggested to be at risk for development of polyarthropathy from sulfonamide hypersensitivity, but not necessarily the idiosyncratic hepatic reaction. 159 onset of clinical signs occurs within 5 to 36 days (mean: 12 days) from starting the drug. 159 previous exposure to sulfonamides is not required. doses of potentiated sulfonamides are generally higher in dogs who develop the idiosyncratic hepatic reaction, as compared with other systemic manifestations of sulfonamide hypersensitivity (thrombocytopenia, fever, polyarthropathy, other). 159 biochemical findings include increased liver enzyme activity (alt > alp) and hyperbilirubinemia. liver biopsy usually reveals marked hepatic necrosis; however, cholestasis and marked lymphocytic-plasmacytic inflammation also have been described. 21 the pathogenesis of the idiosyncratic hepatic reaction is unclear. dogs in general may be at increased risk for sulfonamide reactions because they lack genes that express the n-acetylation enzymes, which is a major metabolic pathway of detoxification of sulfonamides in humans. 21 however, this does not explain individual risks among dogs. hepatotoxicity may be a result of p450 oxidation of sulfonamides to reactive metabolites, such as hydroxylamine and a nitroso metabolite, which may be associated with hapten formation, t-cell proliferation, or direct cytotoxicity. 21 impaired detoxification of reactive metabolites via a deficiency in glutathione, cysteine, and ascorbate, may play a role, and theoretically supports the use of hepatocyte uptake of amanitins and has been shown to be protective against experimental a. phalloides liver damage in beagles, when given at a dose of 50 mg/kg iv twice, at 5 and 25 hours after exposure. 141 however, an intravenous form of silymarin is not currently available for clinical use in the united states. experimental studies suggest that penicillin g may also reduce hepatic uptake of amanitins, even several hours after ingestions. 162 iv nac may be beneficial as described for treatment of acetaminophen toxicity. overall mortality rate with amanita mushroom toxicity is high. ingestion of toxin-producing blue-green algae (microcystis aeruginosa) is a rare cause of hepatotoxicity and alf in dogs. 164 algae proliferate in shallow, stagnant water, especially in hot, dry weather. dead or dying algae form a thick blue-green scum on the water's surface, and release the toxic principle, microcystins. toxicity is caused by ingestion of algae-contaminated water. signs occur rapidly (within 1 hour of ingestion) and include vomiting, diarrhea, and lethargy, followed by progressive tachypnea and dyspnea, icterus, and coma. biochemical features reflect hepatocellular injury with increased alt and ast activity (that typically exceed increases in alp activity), and hyperbilirubinemia. however, profound or protracted increases in alt activity may not be detected, because microcystins can interfere with transaminase biosynthesis. 7 hepatic lesions consist of massive hepatic necrosis of the centrilobular to midzonal hepatocytes. treatment is symptomatic and supportive. oxidative injury may play a role, 164 which suggests that glutathione supplementation (nac or same) may be of benefit. the prognosis is guarded. cycads (sago palms) are native to tropical and subtropical regions, and are used as houseplants and in residential landscaping. concentrations of cycasin, the primary toxin in cycads, are highest in the seeds and roots, but present in all parts of the plant. 165 ingestion of as few as one to two seeds can be fatal in dogs. following ingestion, cycasin is metabolized by gi bacteria to its active compound, methylazoxymethanol, which causes gi and hepatic toxicity in dogs. 165 most dogs that ingest cycads develop gi signs, including vomiting, diarrhea, and abdominal pain. neurologic signs (weakness, ataxia, depression, proprioceptive deficits, seizures, coma) are also common, but it is not clear if they are a result of a neurotoxin or he. 165 onset of clinical signs ranges from 15 minutes to 3 days and may last from 24 hours to 9 days. 165 hepatic injury is suggested by findings of progressive depression, icterus, he, and excessive bleeding accompanied by increased liver enzyme activity, hyperbilirubinemia, hypoglycemia, and hypoabuminemia. 166, 167 centrilobular hepatic necrosis is found on liver biopsy. 166 no specific treatment is available. mortality has been reported to vary between 32% and 58%. 165, 167 xylitol xylitol, a 5-carbon sugar alcohol used as a sugar substitute, is associated with hypoglycemia and hepatic necrosis in dogs. 168, 169 xylitol is safe in humans and is commonly used in sugar-free gum and other oral care products, and is available as a granulated powder for baking. xylitol was first introduced into the united states in 2002, and since that time, reports of toxicity to the aspca animal poison control center have increased from two dogs in 2002 to 2512 dogs in 2008. 168 ingestion of more than 0.1 g/kg in dogs is associated with a rapid, severe, increase in blood insulin, which results in signs of hypoglycemia within 30 to 60 minutes of ingestion. when amounts exceed 0.5 g/kg, alf may occur within 9 to aflatoxicosis associated with consumption of a commercially manufactured pet food product should be suspected when there is a geographic or temporal cluster of cases in a household, kennel, or region. the history may reveal recent changes in diet or feeding from a new bag. it should be noted that some dogs may consume contaminated food for weeks to months before signs develop. definitive diagnosis of aflatoxicosis is based on chemical detection of increased levels of aflatoxin (>60 µg/kg) in the food. when aflatoxicosis is suspected, the owner should be advised to retain 1 kg of food in an airtight zippered plastic bag (or four cans of food), for laboratory testing. it is also recommended to save packaging information, including product and date code, to help identify contaminated lots of food. if a sample of food is no longer available, serum or liver samples can be submitted for testing for aflatoxin m1 (aflatoxin metabolite), although usefulness may be limited because of the rapid metabolism and excretion of aflatoxin. detection of aflatoxin m1 in urine is only useful if the dog is still consuming the contaminated diet, as levels fall below detectable levels within 48 hours. there is no specific antidote for aflatoxicosis and treatment consists of symptomatic and supportive management of liver failure. nonspecific hepatoprotective therapy with antioxidants (vitamin e), glutathione replacement (nac, same), milk thistle (silymarin), and l-carnitine have been recommended. 138 the prognosis is guarded if clinical signs of aflatoxicosis are present, with a reported mortality rate of 64% in a series of 72 dogs that consumed aflatoxincontaminated dog food. 138 dogs that survive acute liver injury have the potential to develop chronic liver disease. consequently, monitoring of liver function is recommended in recovering dogs and treatment with thiol donors such as same for 2 months has been empirically recommended. 138 amanita mushrooms amanita phalloides (and other varieties such as amanita verna and amanita bisporigera), are poisonous mushrooms found throughout north america that can cause acute hepatic necrosis in dogs and cats. 162, 163 toxicity is attributed to extremely toxic cyclopeptide toxins called amanitins. ingestion of two a. phalloides mushrooms can be lethal to an adult dog. 163 clinical signs occur within 6 to 24 hours after ingestion and are characterized initially by gi signs such as vomiting, bloody diarrhea, and abdominal pain. the late phase (36 to 84 hours after exposure) is characterized by alf (hemorrhage, marked hypoglycemia, he, and terminal coma) caused by severe massive hepatic necrosis (see figure 61 -20) . toxin-induced renal tubular necrosis may also result in renal failure. 162 biochemical features reflect severe hepatic injury and include increased liver enzyme activity (alt exceeds alp), refractory hypoglycemia, and hyperbilirubinemia. diagnosis is usually made based on positive identification of the suspect mushroom, evidence of its ingestion, and consistent clinical features. mushroom pieces in gastric contents can confirm exposure, but are difficult to identify. accurate mushroom identification requires consultation with an experienced mycologist. the suspect mushrooms should be wrapped in paper towels and stored in a paper (not plastic) bag. 162 definitive confirmation can be established by detecting amanitins in liver or kidney tissue (or serum and urine samples collected during the gi phase) by liquid chromatography-mass spectrometry, through the california animal health and food safety laboratory. 163 gi decontamination procedures are recommended as soon as possible after exposure, as described in the "treatment" section of "drug and toxin-induced liver injury". symptomatic and supportive treatment for alf is indicated, including close monitoring and treatment of marked hypoglycemia. silymarin is believed to reduce lower urinary tract disease ammonium biurate uroliths are formed most of the time in the bladder (and rarely in the renal pelvis) and can cause dysuria, urethral obstruction in males, and seldom uroabdomen as a result of chronic inflammation and subsequent devitalization of the bladder wall. 3 ammonium biurate crystalluria is not pathognomonic for portosystemic shunting, and it may occasionally occur in normal dogs and cats, or be found in certain breeds, such as dalmatians, because of an inborn error of metabolism. prolonged recovery from anesthesia or sedation may occur in seemingly normal dogs and cats that have cpss. the liver plays a crucial role in the detoxification process of toxins and drugs, including anesthetics. patients with cpss or apsc have insufficient functional hepatic mass for detoxification. in addition, dogs with portosystemic shunting have increased endogenous benzodiazepine and gabaergic activities. 4 therefore administration of diazepam or barbiturate to these animals may have prolonged and exaggerated effects (see "pathogenesis" section). recurrent fever together with resultant depression and anorexia can be the only clinical manifestation of cpss in some dogs. 5 portosystemic shunting should be excluded in dogs experiencing recurrent fever. this rare manifestation has only been reported in a few dog. 6 its pathogenesis is not understood. elderly dogs with extrahepatic cpss may present with characteristic signs of hypercortisolism (e.g., pu/pd, polyphagia, thin skin, symmetric alopecia, muscle wasting, pot belly) as primary complaints. these are unusual clinical manifestations of cpss. although small body size is observed in cats with cpss, it infrequently occurs in dogs. many dogs with extrahepatic cpss show no or only nonspecific clinical signs throughout their lives and the shunt is detected as a coincidental finding at the time of necropsy. it is not understood why certain dogs become clinically ill and others do not with the same type of portal vein anomaly. cpss never cause icterus, ascites, or spontaneous hemorrhages. portal vein disorders are much less common in cats than in dogs. most cats are younger than 6 months of age when the first signs appear. 7 cats with cpss are often of smaller stature and have unkempt hair coat. episodic salivation and/or central neurologic signs, for example, compulsive pacing or seizures, as manifestations of he are the most typical presenting complains. 8, 9 portal hypertension clinical signs can develop at any age and arise from the presence of increased hydrostatic portal venous pressure, portosystemic 72 hours. 169 alf is not necessarily preceded by early signs of hypoglycemia. the mechanism of hepatotoxicity is unknown, but has been speculated to be caused by cellular depletion of adenosine triphosphate resulting in hepatocellular necrosis, or production of reactive oxygen species causing oxidative injury. 169 in addition to signs of hypoglycemia, dogs with alf may have vomiting, icterus, and evidence of excess bleeding. biochemical findings include markedly increased alt and ast activity, mild to moderate increased alp activity, hyperbilirubinemia, hypoglycemia, hyperphosphatemia, prolonged pt and aptt, and thrombocytopenia. if ingestion occurred within the last few hours, induction of emesis is recommended (unless showing signs of hypoglycemia). activated charcoal may be of limited value in adsorbing xylitol, but is still recommended if large amounts have been ingested. 168 treatment recommendations include hospitalization for observation and monitoring, dextrose supplementation for control of hypoglycemia, and symptomatic and supportive care for complications of liver failure. hepatoprotective therapy with nac, same, and silymarin may be beneficial. the prognosis is good for recovery in dogs with uncomplicated hypoglycemia and guarded to poor for dogs in liver failure. however, survival after liver failure does not necessarily correlate with amount of xylitol ingested. 169 vascular hepatic diseases include congenital and acquired disorders of the portal vein. congenital anomalies result from (a) hypoplasia or aplasia of the portal vein, (b) macroscopic communications between the portal vein and a systemic vein, or (c) between the portal vein and an artery. acquired diseases result from conditions that increase the hydrostatic pressure in the portal vein (i.e., portal hypertension). macroscopic venous connections between the portal and systemic venous systems result in portosystemic shunting (i.e., blood flows from the portal to the systemic veins) via a congenital portosystemic shunt (cpss) or acquired portosystemic collaterals (apsc). neurologic signs hepatoencephalopathy (he) is a reversible central neurologic manifestation of hepatic insufficiency. 1 cpss causes chronic he. the following grades of chronic he are recognized 2 : grade 1-depression, behavior changes; grade 2-ataxia, compulsive pacing, circling, hypersalivation, head pressing, blindness; grade 3-stupor and seizures; and grade 4-coma. chronic he is characterized by periods of severe (grades 2 to 3) signs (lasting usually several hours to a few days alternating with longer periods (days to weeks) of no or mild (grade 1) symptoms. 2 periods of cortical blindness are accompanied by apparent mydriasis. signs of he may be triggered by ingestion of protein-rich meals. pd means excessive fluid intake (in dogs >100 ml/kg body weight/24 h and in cats >50 ml/kg body weight/24 h). pu may be more difficult to diagnose than pd, as pet owners cannot readily measure urine volume. repeated low specific gravity of morning urine (<1.025 in dogs and <1.030 in cats) is compatible with pu. macroscopic connections exist between major vessel systems or their tributaries. single or multiple portosystemic venous connections allow the portal venous blood to flow directly to the systemic venous system without first flowing through the hepatic sinusoids. this toxin-rich blood will be delivered to all cells of the body through the following route: gut > portal vein > shunt > systemic vein > right heart > lungs > left heart > arteries. a connection through a single, or rarely double, large-bore vein without the presence of portal hypertension is considered to be a cpss. single or multiple connections in the presence of portal hypertension are apsc. whenever a macroscopic venous connection is present between the portal vein (or one of its tributaries) and a systemic vein, the blood will flow from the portal vein to the systemic vein, because the pressure in the portal vein is higher (8 to 10 mm hg) than that in the systemic veins (0-5 mm hg). 8 normal portal venous pressure is approximately 8 to 10 mm hg (10 to 13 cm h 2 o). increased pressure in the portal venous system results in portal hypertension. anatomically, portal hypertension can be classified 17 as (a) prehepatic (i.e., portal vein), (b) intrahepatic or (c) posthepatic (i.e., hepatic veins, thoracic caudal vena cava, or heart). posthepatic (postsinusoidal) portal hypertension. all cardiac diseases that result in right-sided congestive heart failure cause posthepatic portal hypertension. these diseases include the (a) congenital or acquired severe insufficiency of the tricuspid valve (e.g., dysplasia, myxomatous degeneration, annulus dilation caused by dilated or arrhythmogenic cardiomyopathy, pulmonary hypertension of various etiologies), (b) pericardial diseases (pericardial tamponade caused by idiopathic or neoplastic effusion or constrictive pericarditis), (c) various congenital anomalies such as cor triatriatum dexter, atrial septum defect, tricuspid or pulmonic stenosis, (d) intracardiac tumors affecting the right heart, and (e) caval syndrome (caused by dirofilaria immitis heart worms). kinking or compression of the thoracic caudal vena cava (by diaphragmatic hernia or a mass) occurs rarely. compression, stenosis or thrombosis of the hepatic veins (the so-called budd-chiari syndrome) does not occur in dogs and cats. 13,13a in posthepatic portal hypertension the portal and caval pressures increase equally. therefore no apsc develop, and the blood ammonia concentration remains within reference range. the high postsinusoidal hydrostatic pressure causes a large amount of protein-rich hepatic lymph to be filtered through the hepatic capsule into the abdominal cavity causing accumulation of a modified transudate. modified transudate has high protein content because the fenestrated endothelial cells of the hepatic sinusoids keep only 10% to 20% of the plasma proteins in the capillary lumen. marked generalized hepatomegaly and dilated jugular and hepatic veins as well as caudal vena cava are hallmarks of postsinusoidal portal hypertension. intrahepatic portal hypertension. chronic acquired parenchymal liver diseases lead to hepatocyte necrosis and subsequent collagen deposition. the resultant disorganization of the hepatic architecture and contraction of the connective tissue results in obstruction of the intraparenchymal vessels. the underlying disease processes include viral, bacterial, protozoal, immune-mediated, or copperassociated chronic hepatitis; toxic, drug-induced, or idiosyncratic liver damage; lobular dissecting hepatitis; and chronic bile duct shunting via apsc, and the underlying disease that led to portal hypertension. clinical signs of portosystemic shunting are similar, regardless whether it is congenital or acquired in origin (see "clinical manifestations" section). accumulation of a large amount of pure transudate or modified transudate (clear, straw-colored, or slightly turbid blood-tinged fluid) in the abdominal cavity is commonly detected with physical examination and abdominocentesis. the absence of free abdominal fluid does not exclude portal hypertension. depending on the etiology and the anatomic location of the underlying disease that led to the development of portal hypertension various signs may be seen such as jaundice, periodic vomiting, and anorexia. jaundice and hemorrhagic diathesis are absent in congenital vascular disorders. congenital arterioportal fistula causes transudative ascites in puppies between 2 and 6 months of age. 10 primary hypoplasia of the portal vein (phpv), also described as "noncirrhotic portal hypertension," 11 may result in ascites, vague gi signs, and he, or may be entirely subclinical. 12, 13 when the phpv is not severe enough to cause portal hypertension, clinical signs may not be obvious. this mild form of phpv is also known as hepatic microvascular dysplasia 14 and may be detected serendipitously if plasma bile acid concentrations are measured for unrelated reasons. portal hypertension is rare and is not typically associated with ascites in cats. 15 portosystemic shunting because of cpss or apsc tend to cause similar signs, for example, periodic salivation and seizures, as manifestations of chronic he. the most common acquired disease causing intrahepatic portal hypertension is biliary cirrhosis caused by chronic bile duct obstruction. jaundice and acholic feces are typical findings in extrahepatic cholestasis. the liver receives its blood supply from the portal vein (approximately 75%) and the hepatic artery (approximately 25%). with diversion of portal venous blood flow, a compensatory increase of the hepatic arterial flow occurs. arterial vasodilation is mediated by adenosine from energy-depleted hepatocytes. the portal vein transports blood from the spleen and the gi tract to the liver. 16 the smallest portal venous and hepatic arterial branches terminate in the capillary system of the liver, the so-called hepatic sinusoids. a large amount of plasma is filtered through the fenestrated walls of the sinusoids to the space of disse. from here the filtered plasma is taken by lymphatic vessels to the systemic venous system. the remaining sinusoidal blood is then collected by the hepatic veins, which enter the caudal vena cava. normally no the hepatofugal portal flow prevents the splanchnic venous blood from entering the liver and causes the development of alternative pathways and often times accumulation of pure transudate in the abdomen. interestingly, phpv always accompanies congenital arterioportal fistulas both in dogs and cats. 5, 10, 25 when a cpss and arterioportal fistulas are concomitantly present, apsc and ascites do not develop. 25 arterioportal fistula can be extrahepatic in cats. acquired portosystemic collaterals. in healthy mammals, multiple nonfunctional venous connections may exist between the portal and systemic veins. these virtual communications become functional when their lumen becomes sufficiently widened. gradual dilation takes place when sustained increase of the portal pressure takes place without the simultaneous increase of caval pressure. the resultant apsc are multiple, usually thin, tortuous veins with species-specific anatomical locations; however, large-bore veins may also develop. these latter cases should not be misinterpreted as simultaneous cpss and apsc. in the presence of an existing cpss, no apsc would develop even if hepatic cirrhosis develops as there is an already existing portosystemic connection that is able to drain 100% of the portal blood without allowing the development of portal hypertension. 26 splenorenal collaterals are consistently present in almost every dog with apsc. 10, 27 these apsc drain the portal venous blood via the splenic vein through acquired connections to the left gonadal vein. the left gonadal vein enters the left renal vein, which later empties into the caudal vena cava. these splenorenal apsc are thought to prevent the spleen from undergoing congestion, 17 which is why splenomegaly is not a feature of canine pre-and intrahepatic portal hypertensive disorders. 10 shunting of the portal blood is not only detrimental for the brain, but for the liver as well. normal hepatic development and function requires sufficient amount of portal venous perfusion of the hepatic sinusoids. increased arterial perfusion is unable to compensate for portal hypoperfusion. regardless of whether the insufficient portal venous perfusion is caused by cpss or by prehepatic portal hypertension, the result is the same: reduced hepatic mass and function. in both cases, histopathologic evaluation of the liver shows stereotypical reaction: small or invisible portal branches, increased number of arterioles and sometimes bile ductules in the portal tracts, hepatocellular atrophy, and periportal sinusoidal dilation. 13 this secondary portal vein hypoplasia is reversible and is histologically indistinguishable from phpv. because primary and secondary portal vein hypoplasia show identical microscopic features, histopathologic evaluation of liver biopsy specimens is unable to diagnose simultaneous phpv and cpss. glycine and gaba are the most important inhibitory neurotransmitters, whereas glutamate and dopamine are the most abundant excitatory neurotransmitters in the brain. in he, a net increase in inhibitory transmission occurs. 28 this results from upregulation of gaba receptors and downregulation of dopamine receptors. 29 activation of gaba receptors causes opening of chloride channels, which leads to hyperpolarization of the postsynaptic membrane. in the presence of gaba, benzodiazepines increase the frequency, whereas barbiturates increase the duration of the chloride channel opening. 30 the cause of increased gabaergic tone in he is thought to be gut derived, 4 but gaba may also be formed in the neurons from glutamate. 28 endogenous benzodiazepines are proven to be obstruction. the resultant increase in portal pressure may cause development of apsc as well as ascites. modified transudate may accumulate in the abdominal cavity when the disease process predominantly obstructs the postsinusoidal hepatic venules. however, when the disease process affects predominantly the intrahepatic portal vein branches (presinusoidal portal hypertension) pure transudate will accumulate in the abdomen. primary hypoplasia of the portal vein is caused by insufficient development of the intrahepatic portal venous branches, the left portal vein branch, or the whole portal venous system. 12, 18 when the hypoplasia is severe enough to cause intra-or prehepatic portal hypertension, apsc develop. this condition is also known as "noncirrhotic portal hypertension." if hypoplasia is not severe enough to cause portal hypertension, clinical signs will not develop. because no portosystemic shunting is present, the results of rectal ammonia tolerance test (att), portal scintigraphy, and portography are all normal (see "differential diagnosis" section). if either macroscopic or microscopic portosystemic communications is present, hyperammonemia or abnormal att should also be present, but this has never been documented. the only abnormality that could be detected in these dogs is elevation of serum bile acids levels. 14 the most plausible explanation for this is that the hepatic clearance of bile acids by the hypoperfused liver is probably less effective than that of ammonia. the disease can only be diagnosed by histopathologic examination of liver biopsy specimens. this condition also has been described as "hepatic microvascular dysplasia." although the terms noncirrhotic portal hypertension and hepatic microvascular dysplasia have been used to describe this syndrome, both have been replaced by the term phpv. 13 the occurrence of phpv in the cat is very rare and poorly documented. the most common congenital cause of portal hypertension in cats is congenital hepatic fibrosis as a part of polycystic kidney and liver disease complex. 19 this disease may cause clinical signs with or without the presence of macroscopic hepatic or renal cysts. 20 prehepatic portal hypertension. narrowing of the portal vein lumen may be caused by (a) extravascular compression by a tumor, enlarged lymph node, cyst, abscess, or hematoma, (b) idiopathic circumscribed stenosis, 21 or (c) intravascular obstruction by a thrombus or parasites, all of which can lead to portal hypertension. 22 portal vein thrombosis is always secondary either to neoplasia, or to a systemic disorder that causes hypercoagulability such as nephrotic syndrome, immune-mediated hemolytic anemia, hypercortisolism, acute pancreatitis, peritonitis, or sepsis. 23 parasites in the portal vein also may occur, for example, heterobilharzia americana in north america 24 and schistosoma japonicum in east asia. 13 depending on the degree of portal vein occlusion, increased portal pressure with apsc and ascites may develop. as the hydrostatic pressure in the sinusoids does not increase (in contrast to posthepatic portal hypertension), the resultant ascitic fluid will have a low protein content. the narrowed lumen of the portal vein causes reduced portal flow to the liver, resulting in reduction in the size of the liver. portal vein thrombosis itself rarely causes clinical signs and is usually a coincidental finding. when the liver receives little portal venous blood, an insufficient amount of substrate (i.e., ammonia) is available for hepatic urea production. this theoretically results not only in a low plasma urea concentration, but also in a lower renal medullary urea concentration, which impairs renal concentrating ability and causes pu. increased basal plasma concentrations of acth and cortisol as well as increased urinary cortisol-to-creatinine ratios are invariably present in dogs with portosystemic shunting. [43] [44] [45] [46] cortisol interferes with the action of arginine-vasopressin at the renal tubule, causing a nephrogenic-type diabetes insipidus. 47 hypersecretion of acth (and α-melanocyte stimulating hormone [α-msh]) has been shown to arise predominantly from the intermediate lobe of the pituitary. 43, 48 the hormone secretion of this lobe is regulated by tonic dopaminergic inhibition. acth-hypersecretion can be explained by the production of "false" neurotransmitters (e.g., octopamine), whose effect is about one-fiftieth that of dopamine on the dopamine receptors. 35 central diabetes insipidus also contributes to pu in dogs with he. impaired release of arginine-vasopressin from the posterior lobe of the pituitary is caused by a reduced magnitude of response and a highly increased threshold to increased plasma osmolality. 45 release of arginine-vasopressin is inhibited by the gaba inhibitory neurotransmitter system, whose activity is increased in he. 29, 45 pseudohyperaldosteronism cortisol and aldosterone have similar affinities to bind aldosterone receptors. however, cortisol is normally inactivated by 11β-hydroxysteroid dehydrogenase in tissues where aldosterone action is required. 49 high serum bile acids concentrations inhibit this enzyme, and cortisol can bind to aldosterone receptors resulting in increased mineralocorticoid effect. 45 plasma cortisol concentrations are 10-fold those of aldosterone, causing constant and inappropriate pseudohyperaldosteronism. the resultant sodium retention causes secondary water retention and subsequent pu by pressure diuresis. hypokalemia caused by hyperaldosteronism also contributes to pu 50, 51 according to the following mechanism. the presence of aquaporin-2 channels in the renal collecting ducts' cell membranes is necessary for water reabsorption. intracellular signaling pathways through cyclic adenosine monophosphate regulate the insertion of these channels. hypokalemia decreases the sensitivity of cyclic adenosine monophosphate to arginine-vasopressin, which results in decreased insertion of aquaporin-2 channels into the cell membrane. 50 this leads to nephrogenic diabetes insipidus and pu. congenital portal venous anomalies in dogs are typically associated with enlarged kidney volume. increased renal gluconeogenesis as a compensation of insufficient hepatic gluconeogenesis may cause the kidneys to enlarge. 52 in addition, increased systemic circulating growth factor concentrations released from the pancreas may play a role in this increased volume. 53 normally, these growth factors act only in the liver, as they do not reach the systemic circulation in high concentrations. behavior changes and abnormalities in the thirst center due to he may contribute to pd; however this is difficult to prove in individual patients. produced in the intestines of dogs with cpss. 4 their source is not quite clear: they could arise from the diet, intestinal flora, or by endogenous modification of inactive gut precursors. 4 in all forms of portosystemic shunting the concentration of aromatic amino acids are increased in the systemic circulation whereas the concentration of branched-chain amino acids is decreased. high aromatic amino acid concentration results from impaired hepatic clearance and increased production caused by muscular breakdown exacerbated by hyperglucagonemia. 28, 31 the decreased concentration of branched-chain amino acids results from their increased utilization for gluconeogenesis. 28, 32 this amino acid imbalance may result in the development of "false" neurotransmitters (e.g., octopamine) in the brain, which contribute to the development of he. 33, 34 these "false" neurotransmitters have a fraction of dopamine's excitatory effect on dopamine receptors. drugs with dopaminergic effect, such as bromocriptine, may improve signs of he. [35] [36] [37] intestinal toxins such as ammonia and bacteria are normally inactivated by hepatocytes and hepatic macrophages (kupffer cells), respectively, so that toxin-and bacterium-free blood can enter the systemic circulation. in patients with portosystemic shunting the majority of the portal venous blood bypasses the liver, allowing the toxin-and bacterium-rich blood to enter the systemic circulation. ammonia is a neurotoxin and can contribute to the clinical signs of he. 38 other toxins that are believed to play a role in he are endogenous benzodiazepines, gaba, tryptophan, glutamine, serotonin, mercaptans, indoles, and skatoles. 4, 34, 39 hyperammonemia also leads to impaired glial function through increased intracellular glutamine concentration. 38 glutamine is made in the glial cells through incorporation of ammonia into glutamate by glutamine synthetase. 28 chronically increased glutamine concentrations cause swelling of the glial cells resulting in so-called alzheimer ii type degeneration of astroglias. 40, 41 glial dysfunction contributes to the development of he. 40, 42 during hyperammonemia, the reserve capacity of the astrocytic glutamine synthetase is exceeded, and ammonia can enter the neurons. 42 in the neurons ammonia inhibits glutaminase, an enzyme that converts glutamine to glutamate. because glutamate is an excitatory neurotransmitter, its reduced level contributes to the development of he. 28 portosystemic shunting or an atrophic liver alone is insufficient to allow he to develop; he can only develop when they are both simultaneously present. 28 the reserve capacity of the liver ensures that even in advanced chronic parenchymal liver diseases, he does not develop in the absence of portosystemic shunting. alkalosis and hypokalemia can worsen the signs of he. in alkalosis the nh 3 + h + = nh 4 + reaction shifts to the left, causing the more lipophilic ammonia to enter the cells of the cns. during hypokalemia, potassium ions (k + ) move from the cells to the extracellular space in exchange for h + , which later results in extracellular alkalosis and intracellular acidosis. intracellular acidosis causes nh 4 + trapping within the cells. this might explain why the blood concentration of ammonia is not closely related to the severity of the clinical signs of he in individual cases. several mechanisms contribute to the development of pu/pd in portosystemic shunting. high concentrations of sodium and urea in the renal medullary interstitium are essential for the production of concentrated urine. these create a high osmotic gradient between the renal tubular lumen and interstitium, which is necessary for water reabsorption. to result from reduced enzyme activity of the urea cycle. as no clinical signs are present, no treatment is required. the condition resolves spontaneously with age because these pups develop enhanced incorporation of ammonia into glutamine. peritoneal absorption of ammonia-containing urine can cause hyperammonemia, however in such a patient the clinical signs of acute uremia predominate. the presence of urease-producing bacteria (e.g., staphylococci) are necessary to split urinary urea to ammonia. 67 fulminant hepatic failure ingestion of blue algae or certain mushrooms (e.g., a. phalloides) results in peracute insufficiency of hepatic function. these animals develop hepatic coma and die shortly thereafter. hyperammonemia, dic, and icterus are present in most affected patients. arginine is an essential amino acid in the cat. in anorectic cats hyperammonemia may develop along with hepatic lipidosis because of the insufficient amount of hepatic arginine needed for urea synthesis in the urea cycle. 68 thus, in contrast to adult dogs, hyperammonemia in cats can occur without portosystemic shunting. methylmalonic acidemia associated with cobalamin deficiency in dogs, 69a a cat 69 and a suspected "transient hyperammonemic syndrome" in a german shepherd dog 70 have been reported as extraordinarily rare causes of hyperammonemia. if blood sampling and sample processing are performed inappropriately hyperammonia may be erroneously diagnosed. high bile acids concentration may result not only from portosystemic shunting, but also from any primary or secondary hepatic diseases associated with intra-or extrahepatic cholestasis. 71 therefore, the presence of high serum bile acids concentrations is very sensitive, but not a specific indicator of portal venous disorders. 63 the simultaneous presence of hyperammonemia and a large amount of pure or modified transudate in the abdominal cavity indicates the presence of severe pre-or intrahepatic portal hypertension with apsc. biochemical and cytologic analysis of the peritoneal effusion, ultrasonography and central venous pressure measurement may be useful in identifying the source of ascites. 17 suspected intrahepatic causes should be further evaluated by histopathologic examination of liver tissue. in any animal that is presented with central neurologic signs, portosystemic shunting (along with other metabolic encephalopathies, e.g., hypoglycemia or electrolyte imbalance) should be investigated with appropriate blood tests. no single finding is pathognomonic for diagnosing vascular liver disorders. therefore, a combination of history, physical hyperammonemia results in a higher filtered load and urinary concentration of ammonia. high urinary concentration of uric acid results from decreased hepatic conversion of uric acid to allantoin because of reduced hepatic mass and function. the portal vein carries bacteria and endotoxins from the intestines, which are normally phagocytized by the kupffer cells. shunting of portal blood permits these bacteria and/or endotoxins to enter the systemic circulation causing bacteremia and/or endotoxemia, and subsequent fever. 5 systemic antibiotics may result in temporary resolution of clinical signs, however give no definitive cure. if a cpss occurs within hepatic parenchyma it is called intrahepatic. 54, 55 intrahepatic cpss arise either from the left or right portal branches. a left divisional intrahepatic cpss results from the failure of postnatal closure of the embryological ductus venosus. 56, 57 the intrahepatic cpss arising from the right portal branch and all extrahepatic cpss are thought to be developmental anomalies with poorly understood etiology. in dogs the anatomy of the shunting vessel is fairly consistent: intrahepatic cpss may be left, right, or central divisional, whereas extrahepatic shunts drain the portal venous blood via the splenic or the right gastric vein to the caudal vena cava or the azygos vein. 15 the course of the shunting vein in the cat is much more variable. 15, 58 the inherited nature of the disease has been established in several breeds including the irish wolfhound and the yorkshire and cairn terriers. [59] [60] [61] phpv is a congenital anomaly of unknown etiology. although arterioportal fistulas may develop as a result of neoplasm or trauma (e.g., shot wound or liver biopsy), only the congenital form has been reported in dogs and cats. congenital hepatic fibrosis caused by polycystic kidney disease (pkd) is a genetic disease. 19, 62 differential diagnosis high fasting venous blood ammonia concentrations together with high fasting serum bile acid concentrations are very specific and sensitive tests for diagnosing portosystemic shunting. 63 urea cycle enzyme deficiency reduced activity of one or more enzymes of the urea cycle may result in elevated blood ammonia concentration. 10, 64 serum bile acids levels should remain within reference range in patients with urea cycle enzyme deficiencies. normal hepatic scintigraphy and liver histopathology results should confirm the absence of portosystemic shunting. certain metabolites (e.g., citrulline) and enzyme activities (e.g., argininosuccinic acid synthetase) can be measured in urine and liver biopsy specimens, respectively, to establish a definitive diagnosis. the condition is rare in dogs. one suspected feline case has been reported. 65 irish wolfhound puppies healthy irish wolfhounds commonly have moderate hyperammonemia (<120 µmol/l) at the age of 6 to 7 weeks. 66 this is thought chloride (nh 4 cl) solution is administered at a dose of 2 ml/kg fifteen cm into the descending colon using a soft feeding tube. 75 venous blood ammonia is measured before and 20 and 40 minutes thereafter. in the presence of portosystemic shunting the ammonia concentration will increase at least twofold by the 20-and/or 40-minute sampling times. normal att result excludes portosystemic shunting (figure 61-21) . the degree of increment is a semiquantitative measure of the degree of portosystemic shunting. it should be noted that rectal administration of nh 4 cl solution may cause transient irritation of the colonic mucosa during the first 10 minutes. rectal att is a safe procedure, with signs of he rarely occurring. att should not be performed in patients with very high (>150 µmol/l) blood ammonia levels. postprandial measurement of ammonia is thought to decrease the possibility of iatrogenic hyperammonemic he; however the increase of blood ammonia concentration after feeding takes longer and its peak time point is poorly predictable. 76 markedly increased preprandial (i.e., 12-hour fasting) bile acids concentrations are often found with portosystemic shunting as a result of interrupted enterohepatic circulation of the bile acids. 77, 78 high 12-hour fasting plasma bile acid concentration is a sensitive, but nonspecific indicator of portosystemic shunting. specificity can be increased by simultaneous measurement of venous ammonia. as plasma bile acids concentrations are invariably high in icteric animals, the presence of apsc can only be justified or excluded by measuring blood ammonia concentration or performing an att. in case of a normal 12-hour fasting bile acids concentration (<15 µmol/l), measuring an increased postprandial plasma bile acids concentration (>25 µmol/l) 2 hours after a meal increases the sensitivity, but not the specificity of the bile acids test in diagnosing portosystemic shunting. 79 meal-induced gallbladder contraction causes an endogenous bile acid load, which will be absorbed in the ileum and will substantially increase the plasma bile acids concentration when portosystemic shunting is present. an abnormal postprandial increase of bile acids occurs also in cholestatic liver diseases. in patients with portosystemic shunting hypoalbuminemia, hypoproteinemia, hypocholesterolemia, and low plasma urea concentration may be found as a result of their reduced hepatic synthesis. hypoglycemia may be seen as a result of reduced hepatic glyconeogenesis and glycogen storage. low creatinine concentration reflects the increased glomerular filtration rate. 53 none of these biochemical changes is specific for portosystemic shunting; furthermore, many of these findings may be present in healthy pups. the cause of mild increase of plasma alt and alkaline phosphatase activities is not fully understood. microcytosis with or without mild nonregenerative anemia and leukocytosis may accompany portosystemic shunting. relative iron deficiency is thought to cause the microcytosis [80] [81] [82] and bacteremia may induce the leukocytosis. although aptt is often moderately prolonged in dogs with cpss, no spontaneous bleeding tendency or hemorrhage occurs. 83, 84 hemoabdomen is a possible postoperative complication after surgical attenuation of a cpss. acquired parenchymal hepatic diseases examination, laboratory tests, diagnostic imaging results and histopathology of liver biopsy specimens are often required to establish a specific diagnosis. intrahepatic cpss tend to cause clinical signs between 2 months and 1 year of age in large-breed dogs. bernese mountain dogs, irish wolfhounds, hovawarts, and retrievers are predisposed. australian cattle dogs and male dogs more likely have right-sided than leftsided intrahepatic cpss, 72 whereas irish wolfhounds tend to have left-sided intrahepatic cpss. intrahepatic cpss in small-breed dogs are very rare. extrahepatic cpss usually occur in small breeds and can cause clinical signs at any age. 73 the most commonly affected breeds are maltese dogs, miniature schnauzers, dachshund, yorkshire terrier, jack russell terrier, and cairn terrier. no clear sex predisposition is known. the waxing-waning nature of central neurologic signs is suggestive of chronic he. symptoms may improve spontaneously. physical examination often fails to detect abnormalities. occasionally an enlarged left kidney is palpated. the presence of ascites detectable with physical examination should exclude cpss. some authors suggest that copper-colored iris is a typical finding in cats with cpss; however, no reports exist about its positive or negative predictive value. dogs with intra-or prehepatic portal hypertension may have ascites. jaundice is absent in congenital portal hypertensive disorders, but may be present in acquired parenchymal liver diseases. blood ammonia concentration fasting (12-hour) venous hyperammonemia is a very specific and sensitive indicator of portosystemic shunting. as a single test, increased blood ammonia level is usually sufficient to diagnose portosystemic shunting. because ammonia is formed from amino groups of proteins and urea also in the collection tube, the blood sample (in an ethylenediaminetetraacetic acid tube) should be placed directly on ice after sampling and the measurement should be performed within 30 minutes. because contamination of the sample with airborne ammonia may cause false-positive results, samples should be taken using a closed system (needle on a syringe or directly into a vacutainer). hemolysis may artificially increase ammonia concentration because erythrocytes contain two to three times more ammonia than the plasma. measurement of ammonia should be performed in a clean location, where the air is not contaminated with ammonia from congested urine or cigarette smoke. a number of analyzers offer the possibility of ammonia measurement; however some of them are unreliable. 46 measuring arterial ammonia concentration provides no additional value. if venous ammonia concentration is within the reference range or only slightly elevated, rectal att is the cheapest and quickest test to exclude or justify the presence of portosystemic shunting in patients with a suggestive history. most of these "suspicious" animals also have high fasting plasma bile acids levels. 74 a 5% ammonium anomalies, a high-resolution grayscale ultrasound is often sufficient to establish a definitive diagnosis. 10 although certain secondary changes (such as small liver, large kidneys with hyperechoic medulla, and sediment or stones in the urinary bladder) suggest the presence of cpss, diagnosing a cpss requires the visualization of the anomalous vein from its origin to its termination. the urinary bladder should always be evaluated for the presence of urinary calculi. ultrasonography can be used not only during the initial diagnostic workup, but also during the surgical treatment and postoperative followup of cpss. 86 intraoperative grayscale ultrasonography facilitates localizing the course of an intrahepatic cpss, 26 whereas intraoperative color and spectral doppler examination helps to determine the optimal degree of shunt attenuation. 87 scintigraphy free 99m tc-pertechnetate administered into the colon is absorbed into the portal vein and appears in the liver first in animals without portosystemic shunting, or in the heart in patients with portosystemic shunting. 88 isotopically labeled albumin macroaggregates may be directly administered into a splenic vein with ultrasound guidance. 89 the macroaggregates are trapped in the first capillary bed, which is normally the liver. the fraction of portal may lead to spontaneous hemorrhages because of the presence of dic. plain radiographs give very limited additional information (such as small liver, possibly enlarged kidneys, and sometimes visible uroliths) to the history and laboratory results, so they don't have to be part of the routine diagnostic workup of vascular liver diseases. pure ammonium biurate uroliths are radiolucent. abdominal ultrasonography is the first choice of diagnostic imaging modality, once the presence of portosystemic shunting has been established by fasting hyperammonemia or abnormal rectal att (see figure 61 -21). the major advantage is that ultrasonography requires no sedation or anesthesia. its drawback is in the operator dependence. by using a systematic examination protocol one can accurately differentiate cpss from apsc, and intrahepatic from extrahepatic cpss, as well as readily diagnose arterioportal fistulas and prehepatic portal hypertensive disorders. 15, 85, 86 although colorflow doppler highly facilitates evaluation of portal vascular injection of iodinated contrast agent into the portal vein or into one of its tributaries under fluoroscopy used to be the only form of diagnostic imaging available to diagnose anomalous vessels. angiography allows identification of extra-and intrahepatic cpss as well as apsc. 90 the major drawback of selective portography is its invasiveness and the need for general anesthesia. whereas mesenteric portography requires catheterization of a mesenteric vein during laparotomy, 91 splenic portography can be performed by ultrasoundguided percutaneous injection of contrast material into a parenchymal splenic vein. visualizing portal vein segments with hepatofugal flow or shunt segments with hepatopetal flow (figure 61-22) is problematic, as no contrast reaches these parts of the vessels. blood that bypasses the liver will be trapped in the pulmonary capillaries. splenic venous injection of isotopes makes exact calculation of the shunting fraction possible (i.e., activity in lungs/activity in liver + lungs); however, the procedure requires anesthesia. scintigraphy is the gold standard diagnostic imaging method to justify or exclude the presence of portosystemic shunting. however, differentiating congenital from acquired portosystemic shunting or intrahepatic from extrahepatic cpss is not possible. information provided by a scintigram is a "yes" or "no" answer to the question: does the patient have portosystemic shunting? this answer can be reached much more easily and without using radiopharmaceuticals by documenting a single high blood ammonia concentration or by rectal att. images (a, b) . arrows indicate the direction of blood flow. a and b, intraoperative color-flow doppler ultrasound images of the portal vein at the point where the congenital splenocaval shunt originates. note that the diameter of the shunt (sh) is larger than that of the portal vein (pvcaudsh). the portal vein cranial to the origin of the shunt (pvcrsh) becomes narrower than the portal vein caudal to the shunt origin (pvcaudsh) because of hypoperfusion. a and c, because blood always flows toward the lowest resistance, 100% of the portal venous blood flows through the shunt (sh) to the caudal vena cava (cvc). note that the blood from the gastroduodenal vein (gdv) finds lower resistance to flow caudally toward the shunt, than toward the liver. this creates a hepatofugal flow (i.e., flow away from the liver) in the portal vein segment between the entering point of the gdv and the origin of the shunt (pvcrsh). note that the portal vein becomes even narrower cranial to the point where the gdv enters it (pvcrgdv). the diameter of the various portal vein segments varies because of the varying amount of blood that flows through a certain segment. b and d, note that partial occlusion of the shunt increases the resistance in the shunt to such an extent that the blood from the splenic vein (splv) finds lower resistance to flow through the shunting vessel toward the portal vein. this reversed flow in the shunting vessel (*) prevents the portal venous blood from shunting. thus, even though the shunt is only partially closed, it is nonfunctional. also note that the blood in the whole length of the portal vein is forced to flow toward the liver (i.e., hepatopetal flow direction), establishing normal perfusion of the sinusoids. a portion of the splenic venous blood will continue to flow through the attenuated shunt to the cvc, but the splenic blood contains no more toxins than any systemic vein, so no hyperammonemia develops. rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. pine receptor increases the effect of gaba on its gaba areceptor. 4 the cornerstone of therapy in cases of cpss (until definitive surgical therapy) and in all cases of apsc is a high-quality, low-protein diet. 32, 33, 98, 99 commercially available renal prescription diets are ideal and preferable to most hepatic diets, as the protein content is higher in the latter. 28 to decrease colonic transit time and reduce the production of ammonia, oral administration of lactulose is recommended. the dosage of lactulose should be titrated in individual patients to yield soft feces, but not diarrhea. an initial dosage 0.5 ml/kg q12h is recommended. if diarrhea results the dosage should be reduced. lactulose also stimulates the growth of colonic bacteria that can incorporate ammonia into bacterial protein. additional use of antibiotics (e.g., neomycin, metronidazole) has been recommended by some to diminish ammonia-producing colonic flora. antibiotic administration is usually not necessary to control clinical signs of he; moreover, neomycin is thought to antagonize the action of lactulose and may cause the release of endotoxins. 100 furthermore, chronic antibiotic use may contribute to the development of antibiotic resistance. surgical closure of apsc is generally impossible because of their multiple nature, but also contraindicated, as apsc are usually a compensatory mechanism to resolve high portal venous pressures. surgical narrowing (i.e., banding) of the caudal vena cava to reduce shunting by increasing the caval pressure is currently not recommended. in cases of cpsss, attenuation or complete closure of the shunting vein is the choice of treatment. the goals of shunt occlusion are (a) reducing portal flow via the cpss and (b) simultaneously increasing portal venous perfusion of the liver. 86 the shunt should be attenuated as close as possible to the point where it enters the systemic circulation. attenuating intrahepatic cpss is more risky because of the possibility of major bleeding because of excessive dissection of hepatic parenchyma. there are several techniques described, of which none are perfect. the major problem with shunt reduction is that blood from the portal vein will be redirected to newly perfuse the liver and its vasculature. poorly developed portal branches have insufficient capacity to accept even normal amounts of portal venous blood, and in the case of complete shunt occlusion, splanchnic congestion will develop. the goal of surgery is to sufficiently reduce the amount of shunting blood without causing portal hypertension to develop. 86 although complete shunt occlusion would theoretically be ideal, partial shunt attenuation is often sufficient because (a) partial attenuation often results in functional closure 18 (see figure 61 -22), and moreover (b) complete anatomic occlusion may follow in many cases. 101 partial occlusion of extrahepatic cpsss often results in a better outcome because the chance for development of postligation portal hypertension is much smaller compared to complete occlusion. 86 the liver, which may be less than 30% of its normal size, grows very quickly following successful surgery. regeneration may take 2 to 3 weeks to complete in uncomplicated cases. the regenerating liver receives progressively more portal blood flow usually resulting in spontaneous complete closure of the cpss. helical ct, especially the multiscale versions, makes excellent images of the abdominal vasculature (including cpss) relatively quickly after intravenous injection of iodinated contrast agents. 92 three-dimensional reconstruction provides impressive anatomic details of shunting vessels. 93 the drawback is the limited availability of the new-generation scanners and the need for patient anesthesia. although magnetic resonance angiography can provide highquality images of the abdominal vessels, 94 it has not become popular because of its limited availability and high costs. patents require general anesthesia and the examination lasts longer than ct angiography. characteristic mri changes have been described in the brains of dogs and cats with cpss, 95 but these findings are more of research than of clinical interest. histopathologic examination of liver biopsy specimens is essential in identifying the underlying disease process in intrahepatic portal hypertensive disorders. in the routine diagnostic workup of canine cpss liver histopathology gives no additional information. currently, the presence of coexistent phpv cannot be diagnosed preoperatively in dogs with cpss. 18 this is because the histologic findings of liver biopsies are identical in the following conditions: cpss, phpv, cpss with phpv, congenital arterioportal fistula, any prehepatic portal hypertensive disorder (e.g., portal vein thrombosis). 13 however, taking liver biopsies for histopathologic examination is recommended in cats before deciding about surgical closure of a cpss. this is because congenital hepatic fibrosis as part of pkd can be present simultaneously, especially in persian and persian crossbreeds. 20 surgical shunt attenuation is not recommended when congenital hepatic fibrosis and a cpss are simultaneously present in a cat. patients with grades 2 to 4 he may present on an emergency basis. the source of ammonia and other protein breakdown products, which cause the clinical signs of he, is the colon. the purposes of the treatment are (a) to reduce the production and amount of ammonia in the colon by removing its content with warm water enema, and (b) to inhibit the absorption of ammonia by administering lactulose syrup into the emptied colon. lactulose may be given as a retention enema with a soft feeding tube 0.5 to 1.0 ml/kg deep rectally). lactulose is a nonabsorbable disaccharide, which is metabolized by the colonic bacteria into short-chain fatty acids. these fatty acids acidify the intraluminal content causing to form ammonium ion (nh 4 + ) from ammonia (nh 3 ). because of its polarity, nh 4 + will not pass the enterocystic membrane, and is instead trapped in the colonic lumen. 96 it is also essential (c) to correct the acid-base and electrolytic disorders of the patient with iv fluid therapy as alkalosis and hypokalemia can worsen the signs of he. seizures caused by he can be controlled with intravenous propofol. administration of benzodiazepines (e.g., diazepam) and barbiturates should be avoided as they can worsen the clinical signs. 97 binding of a benzodiazepine molecule to its neuronal benzodiazeacute portal hypertension. variable degrees of portal hypertension necessarily develop subsequent to any cpss attenuation. if portal hypertension is severe, circulatory collapse may develop because of sequestration of blood in the splanchnic veins. 116 slight abdominal enlargement as a result of ascites requires no intervention. this usually resolves spontaneously within 1 week of surgery. severe ascites with signs of shock (e.g., depression, tachycardia, hypotension, prolonged capillary refill time, hemorrhagic diarrhea) necessitates emergency surgery and removal of the ligature or constrictor ring from around the shunt. these signs usually develop within 24 hours postoperatively. unfortunately, the prognosis following emergency surgery is poor. portal vein thrombosis. this rare postoperative complication causes sudden onset of shock usually within several days of shunt attenuation. 87, 117 exaggerated shunt occlusion, severe portal hypertension, and stasis of portal venous blood are believed to be the cause of the thrombosis. no survivals have been reported. chronic portal hypertension. when the growth of the liver and development of portal branches is insufficient following partial shunt closure, chronic portal hypertension may induce formation of apsc. this may occur as late as 4 to 8 weeks postoperatively. in such cases, evaluation usually reveals partially patent shunt and increased portosystemic shunting as a result of newly formed apsc. the development of apsc has been documented with all surgical methods. apsc can develop as a result of underdeveloped portal branches or exaggerated shunt attenuation. 18 in most dogs, these apsc remain clinically silent because the hepatic mass has substantially increased following shunt attentuation. 18 all patients should be re-evaluated at 1 month after shunt attenuation by measuring fasting blood ammonia concentration and performing an abdominal ultrasonography. if fasting blood ammonia concentration is within the reference range, rectal att should be performed. if fasting hyperammonemia is present or the att is abnormal, ultrasonography should identify whether shunting occurs via the narrowed cpss, apsc, or both. in the latter cases, lifelong conservative therapy with dietary modifications and lactulose is recommended. a normal att result implies complete shunt occlusion and the pet will generally have a very favorable prognosis. a second surgery to reach further shunt attenuation should only be attempted in patients with persistent clinical signs that have high fasting blood ammonia concentration or abnormal rectal att result 3 months after the first surgery. 18, 86 spontaneous gradual shunt closure would not occur beyond 3 months after surgical ligation. this rare and usually fatal complication of shunt attenuation causes generalized seizures 1 to 3 days postoperatively, almost exclusively in cats and small-breed dogs (often in maltese dogs). 31, 100, 118, 119 its occurrence is unpredictable. the pathogenesis is unknown, but the sudden decrease of endogenous benzodiazepine ligands is thought to play a role (i.e., "benzodiazepine withdrawal syndrome"). 4 because cerebral edema is suspected to be the initial disorder, intravenous mannitol (0.5 to 1.0 g/kg iv, during 20 minutes) may be administered when a patient shows subtle central neurologic signs during the first three postoperative days. once seizures have developed, the prognosis is usually very poor. most patients are euthanized because of uncontrollable seizures or persistent neurologic defects. propofol gradual shunt attenuation is believed to reduce the risk of portal hypertension by allowing the portal venous branches to adapt gradually to the increased flow. 102,103 apsc do develop with gradual shunt attenuation techniques (e.g., cellophane banding and ameroid constrictor ring). cystic calculi should be removed during the same surgical procedure, because resolution of ammonium biurate uroliths with dietary management can only be achieved in 30% of cases. the shunting vessel is identified by midline laparotomy and narrowed or completely occluded by a nonabsorbable ligature. 104 portal hypertension is determined by direct measurement or estimated based on subjective criteria including severe intestinal cyanosis, increased intestinal peristalsis, reduction in arterial blood pressure, and compensatory tachycardia as a result of stasis in the splanchnic circulation. 105 intraoperative doppler ultrasonography will greatly facilitate determination of the optimal degree of shunt attenuation in extrahepatic cpss, helping to prevent severe portal hypertension. 86, 87 cellophane banding instead of a ligature, a 3-mm-thin, three-layer-thick cellophane band is placed around the shunting vessel with or without narrowing of the shunt diameter. 106 gradual shunt occlusion takes place as a result of inflammation induced by the cellophane. 107, 108 ameroid constrictor ring a metal ring filled with a thick layer of casein is placed around the shunting vessel. swelling of the casein occurs as it absorbs fluid. because of the outer metal ring, the casein can only expand centripetally causing gradual occlusion of the shunt over 1 to 3 months. 102, 109, 110 the major drawback of this simple technique is that the rate and magnitude of occlusion is uncontrollable and kinking caused by the weight of the device may cause acute fatal portal hypertension. moreover, because of its relatively large size it can only be easily applied on extrahepatic cpss. 111, 112 laparoscopy laparoscopic shunt narrowing (by clips) is a less-invasive alternative for shunt ligation (see chapter 28) . 113 with this minimally invasive intravascular technique metal spirals with thrombogenic fibers are placed in the shunting vessel. the coils are delivered via a catheter, which is inserted through the jugular vein into the shunt under fluoroscopic guidance in anesthetized animals. 114, 115 the coils cause thrombus formation in the shunt resulting in its partial or complete occlusion. to prevent coil dislodgment from the shunt, an intravascular stent is often placed in the caudal vena cava to cover the point where the shunt enters the caudal vena cava. coiling is an especially attractive method for treating intrahepatic shunts because liver dissection is thereby avoided. in my opinion, the safest and most effective way for attenuation of extrahepatic cpss is doppler ultrasound-guided partial attenuation via surgical ligature. 86, 87 for intrahepatic cpss, coil embolization appears to be an excellent method. ameroid constrictor and cellophane banding would be ideal in extrahepatic cpss, when the portal vein cranial to the shunt origin is severely hypoplastic and the patient is at risk for severe portal hypertension. spironolactone alone does not resolve ascites, furosemide may be added to the therapy. abdominocentesis for removal of abdominal effusion is not recommended because of loss of protein and exacerbation of starling forces permitting further fluid accumulation. dietary and medical management relieves clinical signs only temporarily in most symptomatic young dogs. 99 without surgical shunt attenuation, gradual deterioration of liver function occurs; consequently, conservative therapy alone offers a guarded prognosis in young dogs. in older animals (>6 years) with newly reported signs of cpss, lifelong conservative treatment may be recommended because of the significantly higher complication rates of surgical therapy in these patients. 105 the prognosis depends on (a) whether the cpss is intra-or extrahepatic, (b) the coexistence of phpv, (c) the extent of shunt attenuation, (d) experience of the surgeon, and (e) the age of the dog at the time of diagnosis. intrahepatic cpss generally has poorer prognosis. complete resolution of clinical signs can be expected in approximately 60% to 80% of dogs with extrahepatic cpss in the hands of an experienced surgeon. this same parameter is approximately 50% to 70% for intrahepatic cpss. [102] [103] [104] [105] with extrahepatic cpss, an excellent prognosis can be expected when blood flow is hepatopetal (i.e., toward the liver) in the portal vein segment, which is cranial to the shunt origin. this can be established preoperatively with doppler ultrasound. 86 in cats, regardless of the shunt type and the surgical method, the success rate is approximately 30% to 50% because of the development of postoperative central neurologic signs. 105, 122, 123 the prognosis of portal hypertensive disorders depends on the underlying disease. in the majority of acquired diseases the underlying disorder is chronic and so severe that even stopping the disease process will not cause regression of apsc. primary liver neoplasms are infrequent in the dog and cat, with an estimated prevalence in necropsy studies of 0.6% to 2.6% in the dog and 1.5% to 2.3% in the cat. liver metastases are more frequent than primary hepatic tumors in the dog, and tend to originate from the spleen, pancreas, and gi tract. primary hepatobiliary tumors are more common than metastatic disease in the cat. [1] [2] [3] [4] [5] the etiology of liver cancer in dogs and cats is incompletely understood. potential causes such as aflatoxins, nitrosamines, food additives, parasites, and radioactive compounds have been reported. [6] [7] [8] liver cancer in the dog has many clinical, pathologic, and histologic homologies with liver cancer in humans. [9] [10] [11] [12] in human medicine, chronic diseases of the liver, such as hepatitis b or c infection, as well as cirrhosis, are often associated with (1 to 5 mg/kg, iv) followed by constant rate infusion [cri]) may be used to control seizures. 120 prophylactic treatment with phenobarbital does not reduce the risk of development of postligation neurologic complications, 118 but may be used in long-term seizure management. preventive use of potassium bromide has not been shown to reduce the possibility of postoperative seizures. in every patient with central neurologic signs in the early postoperative period, hypoglycemia and he should be excluded by measuring venous glucose and ammonia concentrations, respectively. blindness and other types of central neurologic signs may develop in cats shortly after surgery. the pathogenesis of these changes is unknown. hemorrhage from liver biopsy sites or shunt dissection in cases of intrahepatic cpss can lead to hypovolemic shock and death in the early postoperative period. dogs with hepatic insufficiency are prone to develop hemorrhagic complications because of decreased concentrations or abnormal synthesis of coagulation factors. 84 postoperative portal hypertension may also contribute to bleeding tendency from hepatic parenchymal dissection. hemoabdomen should be carefully differentiated from severe portal hypertension and septic peritonitis as they all can cause shock and variable degrees of abdominal distention. coagulation parameters (e.g., pt, aptt) should be routinely monitored after surgery and if they are abnormal or if there is evidence of clinical bleeding, fresh-frozen plasma transfusion should be administered (10 to 20 ml/kg, iv). toy-breed dogs are prone to develop hypoglycemia during or shortly after surgery. 100 blood glucose concentrations should be regularly monitored and hypoglycemia should be treated with glucosecontaining infusions. specific treatment should address the underlying parenchymal liver disease based on the histologic results of liver biopsy. he can often be controlled with dietary modification and lactulose. currently, no specific treatment exists for portal venous hypoplasias. renal or hepatic prescription diets and lactulose may alleviate clinical signs of he in case of apsc. diuretic agents may be useful in dogs with ascites. liver lobe resections have been reported in animals with congenital arterioportal fistulas. however, simultaneous presence of phpv in the whole liver prevents postoperative resolution of portal hypertension and the portosystemic shunting via apsc. 10, 25 therefore, the pet owner should be educated that partial hepatectomy may not result in complete recovery of portosystemic shunting, and that lifelong dietary support and/or lactulose will likely be required. severe abdominal effusions should be treated with diuretic agents. 121 the first choice is spironolactone (1 to 2 mg/kg q12h), an aldosterone receptor antagonist as (a) chronic hepatic insufficiency is associated with hyperaldosteronism, (b) concurrent hypercortisolemia is associated with cortisol binding to the mineralocorticoid receptor, and (c) potassium-sparing diuretics prevent development of hypokalemia and alkalosis, both of which would worsen signs of he. if show no clinical signs, and liver cancer is suspected only with increases in serum liver enzyme activities. 3, 5, 7, 23 the most common physical examination findings are cranial abdominal mass (35%), abdominal bloating (30%), and jaundice (18%). jaundice is a less common finding in cases of metastatic cancer. other manifestations include neurologic signs as a result of he; paraneoplastic syndromes such as hypoglycemia and myasthenia gravis; and skin alterations consistent with hepatocutaneous syndrome. 7, 24 no clear breed predisposition is observed with canine liver cancer, although poodles, fox terriers, miniature schnauzers, labrador retrievers, and male dogs are overrepresented in some reports of hepatocellular carcinoma. 1, 12, 19, 25, 26 labrador retrievers and female dogs are overrepresented in reports of bile duct carcinoma. 1, [26] [27] [28] whether male and female cats are equally at risk for bile duct carcinomas is unsettled, 28, 29 but male cats appear to be at greater risk for bile duct adenomas. 30, 31 neuroendocrine tumors are generally observed in younger animals. 1, 32 diagnosis definitive diagnosis of liver cancer can be made only by liver biopsy. asymmetrical enlargements of the liver detected on physical examination, abdominal ultrasonography, or survey radiography should not be assumed to be neoplastic in origin. similarly, laboratory data do not distinguish hepatic neoplasia from other liver pathologies. 33 the clinical approach to an animal with suspected liver cancer should include basic information such as a complete blood cell count, serum biochemistry, coagulation tests, urinalysis, thoracic and abdominal radiographs, abdominal ultrasound ( figures 61-23 and 61-24) , and fine-needle or core biopsy of the liver. a tnm tumor classification system has been used for staging of liver cancer where t represents tumor (t0, no evidence of tumor; hepatocellular tumors; however, there is no established association between hepatic tumors and viral infections in the dog or cat. moreover, canine hepatic cirrhosis does not appear to predispose to hepatocellular carcinoma (hcc). 1 a possible association between hookworm or whipworm infection and liver cancer has been reported, and cats with chronic cholangitis may have an increased predisposition to biliary carcinoma. 7, 13 several liver mitogens and tumor suppressor genes such as epidermal growth factor, tgf-α, vascular endothelial growth factor (vegf), p53, and tgf-β and its receptors (tgf-β-r) have been associated with liver cancers in humans and these may play a similar role in the dog. [14] [15] [16] [17] in human medicine a small percentage of hccs or cholangiocarcinomas originate from hepatic progenitor stem cells. dogs diagnosed with hcc or cholangiocarcinoma do demonstrate activation of hepatic progenitor stem cells in response to liver injury, but hepatic progenitor stem cell expression in liver tumors is relatively low. 18 the hypothesis of cancer development as a multistep process applies to liver tumors in the dog and the cat, as well. 7, 19 precancerous lesions, such as dysplastic nodules, can be identified before the development of overt malignancy in humans. dysplastic nodules are characterized by cell atypia, cellular crowding, trabecular thickness, microacini, and histochemical markers. 20 dysplastic nodules have not been reported in the dog or cat and further studies are needed to understand the chronology of hepatic malignancy in domestic animals. preliminary reports of histochemical markers have been reported in dogs with hyperplastic hepatic lesions and hepatocellular and biliary neoplasms. 11 liver tumors cause damage to the liver by several mechanisms: inflammatory effects, obstruction of the biliary system, obstruction of the vascular compartment or adjacent organs, and spontaneous rupture with hemoabdomen. 21 hepatic tumors are usually resistant to chemotherapy. 7, 19 in one recent study, p-glycoprotein was more highly expressed in hcc than in cirrhosis, which is consistent with the known resistance of hcc to chemotherapy. p-glycoprotein, which is encoded by the multidrug resistance gene (mdr-1), is normally expressed in tissues with excretory function, including the jejunum, kidney, liver, and adrenal gland. 22 primary liver neoplasms are usually classified according to their cellular origin and macroscopic appearance. with respect to cellular origin, these tumors may be hepatobiliary, hematopoietic, sarcomas, or metastases of other tumors (box 61-2). in relation to macroscopic appearance, they can be classified as lobular, multiple nodular, or diffuse (). the combination of histopathologic and morphologic classification has consequences for prognosis and treatment strategy in these animals (tables 61-6 and 61-7) , and the clinician must always address these factors to arrive at correct management decisions. in dogs, malignant tumors are more common than benign lesions. in cats, biliary neoplasms are the most common presentation, particularly intrahepatic benign forms. 6, 7 most animals with liver neoplasia present with nonspecific clinical signs such as anorexia and weight loss. less-frequent clinical signs include vomiting and diarrhea, pd and pu, pale mucosal membranes, and acute weakness because of anemia and hypovolemia coincident with tumor rupture. 7, 19 up to 25% of affected animals diffuse or infiltrating, multiple coalescent nodules in all the lobes, or diffuse disappearance of the liver parenchyma; lobular, nodule or large mass in a single liver lobe; nodular, several nodules throughout the liver parenchyma, or several affected liver lobes. t1, tumor involving one lobe; t2, tumor involving more than one lobe; and t3, tumor invading neighboring structures); n represents regional lymph nodes (rlns) (n0, no evidence of rln involvement; n1, rln involved; n2, distant ln involved); and m represents distant metastasis (m0, no evidence of metastasis; m1, distant metastasis detected). although recommended, this system has not been universally adopted. 34 table 61 -8 illustrates the most typical hematologic and biochemical findings in dogs and cats affected with liver neoplasia. leukocytosis is a result of inflammation and necrosis of large tumors; anemia tends to be moderate and nonregenerative and is thought to be caused by chronic illness, inflammation, or iron deficiency 19 ; thrombocytosis is attributable to a paraneoplastic syndrome characterized by thrombopoietin production, iron deficiency, or anemia. 7 serum liver enzyme elevation is a frequent, but not universal, finding in animals with liver neoplasia. it should be noted, however, that the degree of serum enzyme elevation does not correlate with the degree of liver involvement or severity of disease. in one survey, animals with primary liver tumors tended to have greater elevations in serum alt and alp activities than animals with metastatic disease, while the latter tended to have greater elevations in serum bilirubin and ast. 35 it also has been suggested that an ast-to-alt ratio of less than 1 is more compatible with carcinoma, while a ratio of greater than 1 is more indicative of a sarcoma or carcinoid. 1 other reported biochemical changes include hypoglycemia, hypo-or hyperalbuminemia, and increased serum bile acids. hypoglycemia as a paraneoplastic syndrome associated with hepatocellular carcinoma is attributed to the secretion of insulin-like growth factor ii. 36 unlike dogs, cats usually present with a high incidence of serum creatinine and bun elevations. 28, 29 coagulation factor abnormalities are more commonly associated with hemangiosarcoma, although dic may be evident in end-stage liver cancer or in decompensated patients. coagulation studies should always be performed before undertaking invasive diagnostic procedures. 23 serum α-fetoprotein has been evaluated in the dog, and increases are reported in 75% of animals with hepatocellular carcinoma, and in 55% of those with biliary carcinomas. the use of this biomarker is limited by the fact that it is increased in cases of hepatic lymphoma and other liver pathologies, and only very dramatic elevations in α-fetoprotein may be taken to indicate hepatocellular carcinoma. [37] [38] [39] abdominal radiographs often reveal a mass effect in the cranial abdomen, although this finding will depend upon the size of the neoplasm and the number and size of metastatic tumors. other reported findings include dorsal displacement of the stomach, hepatomegaly, loss of abdominal detail (because of the presence of free abdominal fluid), and, occasionally, biliary tract calcification. thoracic radiography should be considered as part of the staging procedure for animals with metastatic disease. 25, 40 changes in the ultrasound density of the liver may take a variety of forms (box 61-3). most changes are not pathognomonic for a given disease process, and the final diagnosis is established only on the basis of clinical findings, laboratory testing, and results of cytology or histopathology (see . ultrasound is also very useful for evaluating other abdominal structures, and for the staging of cancer. [40] [41] [42] • diffuse or multifocal liver neoplasms tend to present with hepatomegaly, but this depends on the degree of infiltration. liver carcinomas can be diffuse or affect multiple lobes, with variable ultrasound characteristics depending on the presence of necrosis, inflammation, hemorrhage, or cavitation. in these malignant tumors it is common to observe a mixed echogenicity pattern. lymphoma can affect the liver without detectable ultrasound changes, or cause diffuse hypoechogenicity, hyperechogenicity, or mixed echogenicity with or without hypoechoic nodules. consequently, if lymphoma is suspected, even if the liver ultrasound findings appear normal, fine-needle aspiration cytology is recommended. histiocytic neoplasms are more often associated with multiple nodules and hypoechoic masses, although diffuse liver hypoechogenicity has also been described. mast cell infiltration of the liver tends to produce diffuse hyperechogenicity. • nodular patterns • benign nodular hyperplasia is common, particularly in dogs, and accounts for many of the focal liver lesions identified at ultrasound exploration. it has been estimated that 25% to 36% of all nodular masses detected in the liver are nodular hyperplasia. • benign liver adenomas or hepatomas can manifest as a focal mass of variable size and of normally hyperechoic characteristics. • the liver is a frequent location of metastatic spread, fundamentally through the portal system that drains most of the abdominal structures. • primary liver neoplasms such as hepatocellular carcinoma can present as focal or multifocal masses, although less often so than in the case of metastases. focal hypoechoic lesions with a hyperechoic center or core (referred to as target or bull's-eye lesions) are usually associated with metastases, although some benign processes, such as nodular hyperplasia, can generate similar patterns. • biliary obstruction: ultrasound has become an important tool for evaluating biliary obstruction in icteric dogs and cats. primary tumors of the liver, biliary tract, duodenum, or pancreas are capable of causing biliary obstruction. liver cytology has obvious limitations in that it cannot distinguish between liver adenomas and regenerative nodules, and even some hepatocellular carcinoma aspirates may be composed entirely of normal-appearing hepatocytes. in many cases it may prove necessary to resort to ultrasound-guided biopsy, laparoscopy, or exploratory laparotomy. however, cytology may prove useful in determining the presence of lymphoma, mastocytoma, and histiocytic sarcoma, as well as contribute to the initial classification of tumor type. concordance rates between cytology and histopathology findings may be good for some disease processes, but the reported concordance rate varies from 14% to 86%. 44, 45 the treatment to be provided and the prognosis of animals with primary liver cancer depend on the cell of origin, degree of malignancy, and clinical presentation. the clinician should quickly determine if surgery, chemotherapy, radiation therapy, or palliative care is the treatment of choice in individual patients. palliative treatment is the option for animals that are not surgical candidates, for example, tumors with poor response to systemic chemotherapy, and for whom pain management and general liver failure treatment are the best recommendations. the success of newer options such as chemoembolization, metronomic therapy, antiangiogenic drugs, and tyrosine kinase inhibitors in the treatment of these patients has not been clearly established. [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] the macroscopic presentation is clinically very important (see figure 61 -26), as 100% of the diffuse forms have metastasis at the time of diagnosis, versus 37% of the isolated (massive or nodular) clinical presentations. 27 it should be noted, however, that some dogs with massive hcc present without metastasis, and deaths in these cases may be unrelated to hcc. 1, 26, 54 histopathologic subtype and anaplastic characteristics in general influence the prognosis and predictability of metastasis. 1, 26, 55 metastatic spread usually affects the regional lymph nodes, lungs, and peritoneum. 26, 56 high-field mri scanning has an accuracy of 94% in differentiating malignant from benign lesions with a sensitivity and specificity of 100% and 90%, respectively. mri classified malignant hepatic lesions as hcc in all confirmed cases and correctly predicted the histologic grade of five hcc lesions. these results suggested that mri is a useful modality for abdominal imaging in veterinary patients, and that mri accurately differentiates benign from malignant focal hepatic lesions. 43 liver cytology is useful in the initial evaluation of hepatomegaly and usually permits differentiation between primary tumors, metastatic disease, and focal infection (see . however, cytology does not distinguish between benign focal inflammatory disease and progressive chronic liver disease, and it cannot establish the extent and distribution of disease. likewise, a definitive diagnosis of regenerative nodular hyperplasia cannot be established, and the technique is unable to differentiate a benign inflammatory reaction from cell changes associated with other pathologies. contraindications to ultrasound-guided cytology include the following: • coagulation abnormalities-if one or more coagulation test parameters are altered, it is advisable to administer vitamin k 1 via the subcutaneous route 12 hours before cytology. • cavitary masses-the ultrasound detection of a large cavitary lesion in an elderly dog usually contraindicates cytology, a b tumors are more common in cats. 1, 13, 27, 61 three morphologic forms or presentations have been described: lobular, multifocal, and diffuse. in general, only the lobular form should be considered for surgical removal as long as there is no evidence of metastasis. the prognosis for multifocal and diffuse bile duct carcinomas is very poor, surgery is usually not feasible, and most animals die within 6 months of surgery. 29 no effective chemotherapeutic options have been described for these malignancies in dogs or cats. also known as biliary cystadenomas, biliary adenomas, cholangiocellular adenomas, and cholangiomas, these tumors are common findings in aging cats. males appear to be more frequently affected than females (figure 61-28 ). in cats, 50% of these lesions are isolated or lobular and 50% are multifocal. 59 biliary duct adenomas usually do not cause clinical signs unless they grow and compress other structures. 62 despite the benign nature of these tumors, surgical removal is usually recommended because malignant transformation is always possible and because expansion into the porta hepatis may cause life-threatening consequences. 63 liver lobectomy is recommended for cats with single bile duct adenoma or multifocal tumors confined to one or two lobes. in cats, surgical resection of biliary adenomas may provide cure or tumor-free survival of several years. 28, 29, 31, 59, 62, 63 carcinoid tumors neuroendocrine (carcinoid) tumors are infrequent in the dog and cat. in dogs, carcinoids have an aggressive biologic behavior and are usually not amenable to surgical resection as they tend to present as diffuse lesions (see figure 61 -25). 1, 27 carcinoid tumors in dogs have also been described in the gallbladder, and these have been managed successfully with cholecystectomy. 64, 65 carcinoid tumors in cats can be intrahepatic or extrahepatic involving the bile duct and occasionally the gallbladder. 65 the extrahepatic form of carcinoid tumors may cause biliary tract obstruction, icterus, and increases in serum hepatic enzyme activities. biliary tract diversion procedures should be considered for obstructive lesions involving the extrahepatic biliary tract. unlike the circumstance in dogs and humans, female cats are more often affected by these tumors than males (female-to-male ratio of 5 : 1). 66 the prognosis of carcinoid liver tumors in dogs and cats is generally poor, and metastatic disease is present in 90% of the cases at prognostic factors in dogs with massive hcc include need for surgery, liver lobe involvement, serum alt and ast activities, and ratios of alp to ast and alt to ast. 26 liver lobectomy is recommended for cats and dogs with hepatic tumors that have a massive morphologic appearance without metastases. however surgical complications are reported in more than 28% of cases, with a mortality rate of almost 12%. 26 the predilection of massive hcc for left-sided liver lobes has been reported. 27, 54 advanced imaging and intraoperative ultrasonography may provide useful information on the relationship of right-sided and central liver tumors to the caudal vena cava prior to liver lobectomy. 40, 41, 43 even though right-sided liver tumors have a poorer prognosis because of intraoperative death, there is no difference in the survival time after successful surgery. 26 the considerable regenerative capacity of the liver can permit successful resection of up to 80% of hepatic mass if the remaining tissue is functionally normal and critical supportive care is provided. 56 the median survival time for dogs with massive hcc following liver lobectomy is greater than 4 years. without surgery the average life expectancy is 270 days and the prognosis is generally considered poor. 26 tumor recurrence in dogs with massive hcc is rare and reported to be 0% to 13% after lobectomy. 26, 54 the prognosis for dogs with nodular and diffuse hcc is poor. surgical resection is usually not possible because of involvement of multiple liver lobes. no effective systemic chemotherapy or radiation therapy protocols have been described for hcc treatment. hcc is considered chemoresistant in humans although mitoxantrone has been reported to be helpful in some cases. 7, 56, 57 the most likely reason for the poor response to systemic chemotherapy is the expression of p-glycoprotein in hepatocytes. 22 treatment options for nodular and diffuse hcc in humans include liver transplantation and minimally invasive procedures for regional control, such as ablation, chemoembolization, immunotherapy, hormonal therapy, and low-dose metronomic chemotherapy. 56, 58 a recent report recommends therapy with sorafenib, a multikinase inhibitor and antiangiogenic agent. 47, 48 chemoembolization is a procedure commonly used in the treatment of diffuse hepatocellular carcinoma in humans with median survival times of 1 to 2 years compared with 3 to 6 months with systemic chemotherapy. 52, 51 in veterinary medicine, chemoembolization has been reported with moderate success in the palliation of four dogs with hcc. 52, 53 in cats, hepatocellular carcinoma is less frequent, and less data are available. 28, 59 hepatocellular adenomas these tumors are also known as hepatomas and are more common in cats than in dogs. in the dog it is sometimes very difficult to distinguish adenoma from reactive nodular hyperplasia, and biopsy is needed to clarify the diagnosis. the prognosis for adenomas is usually good, but it is advisable to remove focal mass lesions because they can grow and spontaneously rupture with severe bleeding. 19 bile duct carcinoma (adenocarcinoma and cholangiocarcinoma) bile duct carcinoma is the most common liver malignancy in the cat, and the second most common liver malignancy in the dog (see figure 61 -27) . tumor behavior is very aggressive in both species, and metastases are present at the time of diagnosis in 60% to 88% of cases. bile duct carcinomas usually metastasize to the regional lymph nodes, lungs and peritoneum, kidneys, heart, adrenal glands, eye, and bone. 1, 60 bile duct carcinoma can be intrahepatic or extrahepatic, but rarely occurs within the gallbladder. intrahepatic bile duct tumors are more common in dogs, and extrahepatic bile duct must always be considered as a possibility when a hepatic tumor is diagnosed. benign mesenchymal neoplasms, such as fibroma and hemangioma, have been described but are quite rare. 6, 1, 29, 59 lymphoma in dogs the liver can be involved in variable forms of lymphoma, including multicentric, alimentary, and hepatosplenic forms. a study in cats documented that abdominal lymphoma is currently the most common anatomic location and the liver occasionally is the only organ involved. 69, 70 many protocols are recommended for treatment of lymphoma in dogs and cats; most include vincristine, cyclophosphamide, and prednisone, with variable combinations of l-asparaginase, methotrexate, and doxorubicin. careful evaluation of liver function is necessary before starting chemotherapy because many drugs undergo hepatic metabolism and altered hepatic clearance may lead to unpredictable and potentially increased toxicity. 5 surgical resection with liver lobectomy is recommended for cats with primary hepatic myelolipoma and the prognosis is excellent with prolonged survival time and no reports of local recurrence. 7 in dogs with advanced disease, mast cell tumors can metastasize to the liver. primary visceral mast cell tumors are more common in cats than dogs. the spleen is usually the primary site with metastasis to the liver and bone marrow, and the survival time with splenectomy alone can be a year or more. 71 the overall prognosis for disseminated mast cell tumor in the dog is grave. the median survival time reported in one study was 43 days despite therapy with various chemotherapy agents. 72 canine mastocytoma involving the liver can be controlled with cyclophosphamide, vinblastine, and prednisone. 73 recently, tyrosine kinase inhibitors have shown some promise and ccnu has been shown to be active against feline mast cell tumors. 49, 74 hepatic nodular hyperplasia is a common benign lesion observed in the liver of older dogs that can occasionally be observed in some cats. it is characterized by a discrete accumulation of hyperplastic hepatocytes arising as either macroscopic or microscopic hepatic nodules. it reportedly occurs in 70% of dogs older than 6 years and 100% of dogs over 14 years. [75] [76] [77] the wsava standards for clinical and histologic diagnosis of canine and feline liver diseases include hepatic nodular hyperplasia in its classification system of hepatocellular neoplasia so that it may be differentiated from true neoplasia. 78 the etiology of hepatic nodular hyperplasia is unknown. it has been suggested to be a preneoplastic lesion, 76 but this has not yet been reported in the dog. 77 because of hepatocyte microscopic changes, it is suggested that nutritional and metabolic disorders play a role in the pathogenesis of this lesion. 77 hepatic nodular hyperplasia is characterized microscopically by well-differentiated hyperplastic hepatocytes with increased mitotic activity. [75] [76] [77] hyperplastic nodules may be accompanied by concurrent focal intrahepatic cholestasis, mechanical compression on a higado b the time of diagnosis. 7 a better prognosis is observed with extrahepatic carcinoids with a life expectancy of more than a year. 64, 65 liver sarcomas primary liver sarcomas are rare in the dog and cat. hemangiosarcoma is the most frequent primary hepatic sarcoma in cats and leiomyosarcoma the most common in dogs. 7, 19 there also have been reports of hepatic fibrosarcoma, rhabdomyosarcoma, osteosarcoma, liposarcoma, and histiocytic sarcomas in both animal species. 1, 28, 29, 55 these are usually very aggressive tumors, metastasizing in 86% to 100% of cases to the spleen and lungs, or spreading diffusely within the liver. 5 chemotherapy has not been studied in the treatment of primary hepatic sarcomas, although, similar to other solid sarcomas, response rates are likely to be poor. histiocytic sarcomas respond partially to ccnu, with a mean duration of remission of 85 days and a survival of 172 days. 67 continuous low-dose oral chemotherapy may be an effective alternative to conventional high-dose chemotherapy for adjuvant therapy of dogs with hemangiosarcoma. 50 mass resection may offer some palliation in the circumstance of tumor hemorrhage despite irrefutable evidence of metastasis. a cat with a primary extraskeletal hepatic osteosarcoma was treated with surgery and carboplatin and was alive 42 months after diagnosis with no clinical evidence of disease. 68 on the other hand, metastases feline hepatic lipidosis (hl) is a metabolic syndrome found in obese, middle-aged cats that undergo a period of acute anorexia and catabolism. morbidly obese cats are at increased risk and more than 85% of cats with hl suffer from an underlying disorder that contributes to the initial anorectic event. [1] [2] [3] [4] [5] although the underlying pathogenesis of hepatic lipid accumulation in cats has not yet been completely elucidated, several unique biochemical and nutritional features place this obligate carnivore at risk for fat mobilization and fatty infiltration of the liver during periods of anorexia or starvation (box 61-4). 2, [6] [7] [8] [9] [10] [11] there is a general consensus that reduced caloric intake and protein-calorie malnutrition are important predisposing factors. the result is a rapid mobilization of peripheral fat culminating in fatty accumulation in the liver. 1 intracellular processing of fats is an important function of the hepatocyte. during fasting or starvation, fatty acid metabolism becomes deranged in an obligate carnivore as a result of obesity, catabolism, chronic overnutrition, impaired fatty acid oxidation or vldl secretion, and enhanced hepatic fatty acid synthesis ( figure 61 -29). 1,6-11 hl is a disorder of middle-aged to older cats; domestic short-haired cats are more commonly affected. cats with hl often present with a history of acute stress and/or near-complete anorexia of several days duration. [1] [2] [3] icterus is a variable feature of hl. when serum bilirubin concentrations exceed 1.5 mg/dl, clinical icterus can be observed on the pinnae, mucous membranes, sclera, and hard palate in the cat. in general, most cats with hl are obese at the time of presentation, with many cats being 20% to 30% over ideal body weight prior to an episode of hl. other physical features of hl include hepatomegaly, dehydration, vomiting, and weakness. if he develops as a consequence of hl, neurologic abnormalities such as ptyalism, stupor, coma, ataxia, and seizures may be observed. [1] [2] [3] a minimum database, including complete blood cell count, serum chemistry, and urinalysis, almost always reveals severe liver enzyme elevation and other abnormalities such as nonregenerative anemia, stress leukogram, poikilocytosis, and bilirubin crystalluria. 1 the pattern of liver enzyme elevation is typically cholestatic in nature and characterized by marked increases in serum alp activity, followed by smaller increases in serum alt and serum ast activities. serum ggt activity is often normal in affected cats. increased serum bile acids and bilirubin are often observed in cats with hl, and electrolyte abnormalities, such as hypophosphatemia and surrounding hepatic parenchyma, as well as alterations in the microvascular circulation. vacuolar changes are seen frequently, suggesting a reactive or metabolic condition such us hyperadrenocorticism, lipidosis, or hypothyroidism. 77 nodular hyperplasia affects older dogs with a mean age of 11 years without gender or breed predisposition. hepatic nodular hyperplasia does not appear to cause clinical signs or illness. 77 laboratory findings may include mild to marked increases in serum alkaline phosphatase activity and, less commonly, increases in serum alt activity. liver function tests are usually normal with hepatic nodular hyperplasia. 77 hepatic nodular hyperplasia is usually discovered as an incidental finding during a diagnostic workup for other medical problems. nodular hyperplasia is clinically important because it may easily be confused with primary or metastatic hepatic neoplasia during abdominal ultrasound or at surgery. even microscopically, it may be impossible to differentiate hepatic nodular hyperplasia from hepatocellular adenomas, and a large sample (wedge rather than needle biopsy) may be required to confirm hyperplasia from welldifferentiated hccs. [75] [76] [77] routine abdominal radiographs are generally unremarkable and ultrasonographic features are inconsistent because of the varied hepatocellular morphologic characteristics and size of the nodules. 79 multiple nodules varying in size, distributed randomly among the liver lobes, being superficial or deep within the parenchyma are found in most cases. 77 hyperplasic nodules of hepatocytes need to be differentiated from regenerative nodules. hyperplasic nodules develop in livers of normal mass, whereas regenerative nodules arise as a result of compensatory hyperplasia of surviving hepatocytes in a background of hepatic injury, atrophy, and fibrosis. no treatment is usually required. rupture of large nodules may require emergency mass removal and blood transfusion (rare). hepatic nodular hyperplasia has no significance in the morbidity of affected patients. 77 metabolic disorders of the liver are commonly encountered in companion animal practice. this section focuses on the metabolic liver disease induced by concurrent endocrinopathies (hyperthyroidism, hypothyroidism, diabetes mellitus, and hyperadrenocorticism), lipid disturbances (lipoproteinemias, feline hepatic lipidosis, and hyperlipidemias), and metabolic infiltration (amyloidosis). hepatic lipidosis and hyperthyroid hepatopathy are the primary metabolic hepatopathies in cats. in dogs, steroid (or glycogen vacuolar) hepatopathy is the most frequent metabolic liver disorder; diabetic hepatopathy and hyperlipidemic hepatopathies (lipoproteinemias, hypothyroidism) occur less commonly. • essentiality of dietary arginine 6 • low levels of hepatic ornithine 7 • high dietary protein requirements 7 • lack of hepatic enzyme adaptation to low protein 8 • insufficiency of hepatic glutamate reductase 7 • insufficiency of intestinal ornithine transcarbamylase 7 • diversion to orotic acid metabolism 9 • differences in lipoprotein metabolism (hdls) 10, 11 to 90 kcal/kg body weight in most cats. 1, 18 unless he is present, dietary protein should not be restricted (ideal is 35% to 45% protein on a dry matter basis) and even then protein restriction is controversial as protein is needed to support hepatic regeneration. feeding multiple small frequent meals may help to maintain euglycemia and lessen the metabolic impact on the liver. the protein content of the diet should be considered when he is present (see chapter 32). dairy and vegetable-based proteins are higher sources of branchedchain amino acids than meat-derived proteins and may lessen the signs of he. diets high in fiber generally should be avoided because they decrease the nutrient density of the diet. cats with hl occasionally may experience a refeeding syndrome, a condition that results in metabolic and electrolyte disturbances. 12 with the reintroduction of food, insulin secretion promotes intracellular uptake of phosphorus, potassium, and magnesium. hypophosphatemia can result in muscle weakness and hemolytic anemia. gradual reintroduction of food and correction of electrolytes diminishes the risk of refeeding syndrome. glucose intolerance and hyperglycemia are common in cats with hl and can be addressed by decreasing the carbohydrate content of the diet. canned low-carbohydrate, high-protein formulations without added fiber are ideal for the treatment of feline hl as they provide amino acids, limited carbohydrates, and water, and are easily administered through a feeding tube. small amounts of food should be administered via the feeding tube after residual gastric fluid contents have been removed. trickle feeding can be performed by placing liquefied food into an empty fluid bag and allowing gravity to force flow into the feeding tube. alternatively, a large-bore hypokalemia, may be frequently observed. in particular, the presence of hypophosphatemia should alert the clinician to the possibility of refeeding syndrome. 12 presumptive diagnosis of feline hl can be made on the basis of clinical history, physical examination, clinicopathologic features, ultrasound examination, and liver aspirates. 1, [13] [14] [15] ultrasound examination of the liver often reveals hepatic parenchyma that is hyperechoic to that of falciform fat, but a thorough ultrasound evaluation of the gallbladder, pancreas, intestines, kidneys, bladder, and other abdominal structures is essential to rule out other primary disorders, such as acute pancreatic necrosis, which may be the basis of the anorectic event precipitating an episode of hl. definitive diagnosis is best achieved through liver biopsy 16 ; however, anesthesia and biopsy may not be possible in acutely ill patients because of the presence of coagulopathies from vitamin k deficiency. 17 a liver aspirate that reveals more than 80% fatty infiltration of the hepatocytes may be used for presumptive diagnosis of hl. if there is no response to treatment after 3 to 5 days, liver biopsy may be necessary to rule out other underlying hepatobiliary conditions such as cholangitis. a catabolic state develops quickly in the anorexic cat and prompt measures should be taken to place an enteral feeding tube (table 61-9) . nasoesophageal, esophageal, and gastrostomy tubes can be used for this purpose. the caloric needs should be approximately 60 increases in serum ast and alt activities have been reported in approximately 80% of hyperthyroid cats. 22, 23 liver enzyme elevation has been attributed to increased liver metabolic activity compared to blood flow. long-term untreated hyperthyroidism in human beings can ultimately lead to cirrhosis. [24] [25] [26] middle-aged to older cats are typically affected, and there is no breed or sex predilection. because hyperthyroidism is characterized by hypermetabolism, polyphagia, weight loss, pd, and pu are prominent features of the disease. 22, 23 hyperactivity, tachycardia, pupillary dilation, and behavioral changes are also characteristic of the disease and are associated with activation of the sympathetic nervous system. long-standing hyperthyroidism leads to hypertrophic cardiomyopathy, high-output heart failure, and cachexia. long nails, dermatologic conditions, panting, elevated body temperature, and poor grooming or overgrooming are additional clinical signs of feline hyperthyroidism. clinicopathologic features of hyperthyroidism include erythrocytosis and stress leukogram (neutrophilia, lymphocytosis) caused by increased circulating catecholamine concentrations. increased catabolism of muscle tissue in hyperthyroid cats may result in increased bun, but not serum creatinine. most cats will have decreased urine specific gravity, particularly if they are exhibiting pu as a clinical sign. increased metabolic rate results in liver hypermetabolism, therefore serum activities of liver enzymes (alt, ast) are increased in more than 80% of hyperthyroid cats. 22, 23 diagnosis diagnosis of feline hyperthyroidism is achieved by measurement of serum total thyroxine (tt 4 ) concentration. serum thyroxine concentrations are elevated in more than 90% of hyperthyroid cats, making this a very sensitive test of thyroxine-induced hypermetabolism. 22, 23 false-positive test results are rare to nonexistent, suggesting that hyperthyroxinemia is a specific test for feline hyperthyroidism. in a clinically hyperthyroid cat, thyroid hormones still fluctuate on a daily (and hourly) basis with hormone concentration intermittently decreasing into the normal range. 27 to avoid this type of diagnostic error the clinician should repeat blood sampling 1 to 2 weeks after the first test. nonthyroidal disease can have a significant effect on circulating thyroid hormone concentrations. [28] [29] [30] in the case of persisting nonthyroidal illness (e.g., renal disease), the syringe attached to a syringe pump may be useful in delivering the food through the feeding tube. crystalloid fluids supplemented with fortified b vitamins, including thiamine, riboflavin, niacinamide, d-panthenol, pyridoxine, and cyanocobalamin, should be used. 1 nutritional supplements to enhance antioxidant function, such as vitamin e and glutathione precursors (e.g., same) may also be beneficial. amino acid supplements that support hepatic regeneration and metabolism include carnitine and taurine. 1, [18] [19] [20] carnitine functions in the transport of fatty acids into hepatic mitochondria for energy production. taurine is an essential nutrient for cats and is involved in cns, cardiac, and biliary functions. signs of taurine deficiency may be similar to those associated with he. antiemetic therapy is necessary to control vomiting and facilitate feeding of an appropriate type and quantity of diet (see chapters 23 and 35) . injectable antiemetics, such as maropitant (cerenia, pfizer animal health, kalamazoo, mi), a selective nk-1 receptor antagonist, at a dosage of 1 mg/kg sc or iv on a daily basis, is preferred. 21 oral maropitant at the same dosage or oral ondansetron, a 5-ht 3 receptor antagonist at a dosage of 0.1 to 1.0 mg/kg q12-24 h may be used in cats with larger-bore feeding tubes. persistent vomiting should be investigated to identify feeding tube occlusion or other undiagnosed disease. the prognosis depends upon duration of illness, and the time frame of resolution of hepatic enzyme elevation, hyperbilirubinemia, and other biochemical changes. cats that survive an episode of hl have a greater than 50% reduction in liver enzyme and bilirubin concentrations within 10 days of therapy, whereas cats that die usually do so within 7 days of hospitalization. 1 long-term prognosis for recovery is good with the majority of cats having resolution of hl as long as the underlying disease process (e.g., pancreatitis) is identified and treated. hyperthyroidism in cats is caused by adenomatous hyperplasia of the thyroid gland resulting in increased circulating concentrations of thyroxine and triiodothyronine. 22, 23 hyperthyroxinemia increases hepatic metabolism without proportionate increases in hepatic blood flow with the overall consequence of reduced oxygen delivery to hepatocytes. 24 characteristic lesions of superficial necrolytic dermatitis (hard, cracked foot pads and elbows). painful feet caused by footpad lesions are common. clinicopathologic features include mild nonregenerative anemia, microcytosis (with advanced liver dysfunction), increased serum liver enzyme (alp and alt) activities, hypoproteinemia, hypoalbuminemia, and fasting hyperglycemia. serum bile acids are usually increased. serum glucagon is inconsistently elevated, but plasma amino acid concentrations are often less than 50% of normal. 34, 37 diagnosis abdominal ultrasonography may reveal small, normal or increased liver size; however, there usually is a characteristic "swiss cheese" appearance of the hepatic parenchyma as a result of hepatic degeneration, nodularity, and collapse. 36, 38 pancreatic imaging and biopsy are indicated if a glucagonoma is suspected. symptomatic palliative therapies may be beneficial and include high-protein diets with egg white (approximately 2 to 4 egg whites/ day for a 25-kg dog), zinc (2 mg/kg q24h po) niacinamide (250 to 500 mg/dog q24h po), ursodeoxycholic acid (10 to 15 mg/kg/day po), vitamin e (10 iu/kg daily po), same (20 mg/kg/day po 2 hours before feeding), and fatty acid supplementation. some patients will respond to 10% parenteral amino acid solutions (aminosyn, abbott laboratories, chicago) given at a dose of 500 ml over 8 to 12 hours intravenously through a large-bore central venous catheter. if no response is observed following the initial amino acid infusion, therapy should be repeated every 7 to 10 days for a total of four treatments. prognosis is poor for most cases; however, remissions of longer than 2 years have been reported with intensive amino acid and hepatic support therapy. 37 steroid hepatopathy develops following exogenous corticosteroid therapy, or from endogenous hyperadrenocorticism of pituitary or adrenal origin. the dog liver is uniquely susceptible to both glucocorticoid-and sex steroid-induced liver enzyme elevation, glycogen accumulation, and vacuolar degeneration. 39, 40 pathophysiology in healthy dogs, glucocorticoid administration results in significant liver enzyme (alp and alt) elevation in 2 to 3 days. increased alp and ggt activities develop in parallel as the enzymes undergo induction and release from sinusoidal and canalicular membranes. within 7 days of glucocorticoid administration, the glucocorticoidinduced alp isoenzyme increases significantly. glycogen accumulates within the hepatocyte resulting in a vacuolar degeneration typical of the syndrome. 1, 39, 40 steroid hepatopathy occurs primarily in the dog. there is only one reported case of steroid hepatopathy in the cat. 41 a history of corticosteroid administration or signs consistent with endogenous steroid overproduction (cushing syndrome) are usually evident, for example, pd, pu, panting, potbellied appearance, bilaterally symmetric alopecia on the trunk, and polyphagia. in dogs affected with measurement of unbound thyroxine (t 4 ) or free t 4 may be preferable to repeated tt 4 measurements. free t 4 concentrations are a very sensitive test for the diagnosis of hyperthyroidism with 98% of hyperthyroid cats exhibiting elevated serum free t 4 concentrations. the specificity of free t 4 is not as good as its sensitivity; as many as 12% of euthyroid cats with concurrent illness will have high free t 4 concentrations for reasons that remain unclear. 29 as a result, free t 4 should not be used as a screening test, and free t 4 values should be interpreted in light of the tt 4 concentrations. the combination of a high free t 4 with a low tt 4 is indicative of nonthyroidal illness; however a high free t 4 with a high-normal tt 4 is suggestive of hyperthyroidisim. 31 methimazole (tapazole) is the antithyroid drug most often recommended (2.5 to 5 mg q12h). it is available as a transdermal gel or as an oral tablet. methimazole is often used to prepare the patient for surgical thyroidectomy or radioiodine therapy. antithyroid drugs have several side effects. anorexia and vomiting are common side effects of methimazole, whereas rare side effects include self-induced excoriation of the face, thrombocytopenia, bleeding diathesis, agranulocytosis, development of serum antinuclear antibodies, and cholangitis. bleeding, jaundice, and agranulocytosis necessitate immediate withdrawal of the drug. hepatic injury related to antithyroid therapy such as methimazole is well documented in humans and reported in the cat. 22, 32 mild histologic changes are common, but cases of fulminant hepatic failure with central lobular necrosis have been described. 33 prognosis is excellent with definitive therapy of the hyperthyroidism (surgery or radioactive iodine). hepatic reactions to methimazole will necessitate discontinuation of therapy. the etiology is unknown, but hypoaminoacidemia may play a role in the development of diabetic hepatopathy. 34, 35 fatty acid, niacin, and zinc deficiencies also may be involved in the pathogenesis. increased serum glucagon, originally thought to be the cause of diabetic hepatopathy, is found in only one-third of the reported cases. a much stronger association between the skin lesions of superficial necrolytic dermatitis and glucagonoma, hyperglucagonemia, and poorly regulated diabetes mellitus have been observed in both humans and dogs. 34, 35 pathophysiology hepatopathy is thought to occur secondary to the metabolic abnormalities associated with diabetes mellitus, glucagonoma, or nutritional deficiencies. [34] [35] [36] [37] hepatic features include vacuolar hepatocyte degeneration, hepatic parenchymal collapse, and hepatic nodularity. the disorder is seen most frequently in middle-aged male dogs, and has been reported in one cat. [34] [35] [36] [37] acute presentations may include clinical signs such as vomiting, diarrhea, lethargy, weight loss, pd, pu, icterus, and lameness because of dermatopathy of the footpads. in some cases clinical signs are mild or nonexistent. physical examination may reveal poor body condition, lethargy, and lipoproteinemias etiology genetic abnormalities in lipid metabolism lead to diffuse vacuolar hepatopathy and biliary mucoceles. 1 increased circulating cholesterol and triglyceride cause a vacuolar hepatopathy associated with excess lipid accumulation and/or hepatocyte glycogen synthesis and storage. chronic hypercholesterolemia increases biliary cholesterol content and predisposes to cystic hyperplasia, dysmotility of gallbladder smooth muscle, and biliary mucocele. 1 familial hypercholesterolemia and other hyperlipidemias are found in certain breeds of dogs including the miniature schnauzer, shetland sheepdog, briard, west highland white terrier, scottish terrier, cairn terrier, and beagle. mixed-breed dogs may also be affected. clinical signs are usually associated with necrotizing cholecystitis and may include icterus and cranial abdominal pain. more often, dogs are asymptomatic and biliary mucoceles are identified serendipitously during ultrasound evaluation for some other medical problem (such as pancreatitis). clinical pathology findings usually include hypercholesterolemia or hypertriglyceridemia, and elevated liver enzyme activities, particularly alp. necrotizing cholecystitis may be accompanied by leukocytosis, neutrophilia, and hyperbilirubinemia. diagnosis may be made by characteristic ultrasound findings of nongravitational gallbladder sludge, increased gallbladder wall thickening, "kiwi"-shaped mucosal image, and bi-or trilaminar appearance of the gallbladder wall. the hepatic parenchyma may have a pattern of multifocal hyperechogenicity and hypoechoic nodules. 1 the best treatment for biliary mucoceles is surgical removal of the mucocele and/or cholecystectomy and may become an emergency procedure if the clinical signs of necrotizing cholecystitis are severe. medical therapy following surgical removal is usually necessary and includes a fat-restricted diet and lifelong treatment with ursodeoxycholic acid (15 mg/kg po q24h). prognosis is good for patients undergoing successful removal of the mucocele as long as lifelong medical therapy is continued. in dogs and cats, amyloid deposition is usually secondary to sustained systemic inflammatory response, for example, chronic infection, chronic inflammation, immune disorders, and malignancy. 50 amyloidosis is a familial disorder in the chinese shar-pei dog, and in abyssinian, oriental, and siamese cats. [51] [52] [53] [54] hepatic amyloidosis has also been reported secondary to vitamin a toxicity in cats. 55 pathophysiology deposition of amyloid fibrils within and between hepatic sinusoids results in progressive organ dysfunction. light deposits are found in the space of disse and heavier deposits are often found in the atypical hyperadrenocorticism caused by sex steroid overproduction, dermatologic changes (alopecia, poor hair coat) and reproductive manifestations (perianal adenoma in a castrated male or female dog) are often the only signs suggestive of sex steroid imbalance. atypical hyperadrenocorticism with sex steroid excess may present with no clinical signs except increased serum liver enzyme activities. 1 diagnosis of steroid hepatopathy should be based on a history of exogenous steroid administration or endocrine function testing with or without liver biopsy. classically, liver enzyme elevations consist of moderate to marked increases in alp and ggt, and mild to moderate increases in alt and ast. bile acids may also be increased. 40 the low-dose dexamethasone suppression (ldds) test is considered the screening test of choice for endogenous canine hyperadrenocorticism. 42, 43 the ldds test has a high sensitivity at 92% to 95%. only 5% to 8% of dogs with pdh will exhibit suppressed cortisol concentrations at 8 hours. in addition, 30% of dogs with pdh will exhibit suppression at 3 or 4 hours followed by "escape" of suppression at 8 hours. this pattern is considered diagnostic for pdh, making further testing unnecessary. 43 the major disadvantage of the ldds test is the lack of specificity in dogs with nonadrenal illness. 44 the corticotropin (acth) stimulation test is used to diagnose a variety of adrenopathic conditions, including endogenous or iatrogenic hyperadrenocorticism, as well as spontaneous hypoadrenocorticism. 42, 45, 46 as a screening test for the diagnosis of naturally occurring hyperadrenocorticism, the acth response test has a diagnostic sensitivity of approximately 80% to 85% and a higher specificity than the ldds test. 45, 46 in a study by kaplan and peterson, only 15% of dogs with nonadrenal disease exhibited exaggerated response to acth stimulation. 44 i prefer the acth response test over the ldds test as the acth response test is more accurate for the diagnosis of iatrogenic hyperadrenocorticism (if the history is incomplete) and sex steroid imbalance in addition to pdh or adrenal-dependent hyperadrenocorticism. the urine cortisol-to-creatinine ratio (uccr) is highly sensitive in separating normal dogs from those with hyperadrenocorticism; however, the test is not highly specific for hyperadrenocorticism because dogs with moderate to severe nonadrenal illness also exhibit elevated ratios. [47] [48] [49] an elevated uccr should always be confirmed with an ldds test. in the uccr test, urine is collected for 2 days for a baseline uccr. the animal then is given three doses of dexamethasone (0.1 mg/kg, po q6-8 h) and the final uccr is collected 24 hours after the first dose of dexamethasone. failure of the uccr to suppress into the normal range is diagnostic for hyperadrenocorticism. treatment for exogenous hyperadrenocorticism consists of discontinuation of exogenous steroids by slowly weaning the patient to prevent the development of addisonian crisis. treatment for endogenous hyperadrenocorticism can be achieved with chemotherapy (o,p′-ddd, or trilostane) or surgery (hypophysectomy or adrenalectomy). treatment of sex steroid imbalance can be achieved with mitotane or trilostane. prognosis for steroid hepatopathy is good to excellent if diagnosed early and if corticosteroid injury can be abated by discontinuation of steroid therapy or treatment of the underlying disorder. threefold increases in the frequency of hypothyroidism. 61 the pathogenesis of stone formation in hypothyroidism is believed to involve hypercholesterolemia, gallbladder dysmotility, and bilirubin retention. 61 the most common clinical symptoms of hypothyroidism are lethargy, weight gain, depression, hypothermia, and bradycardia. gi signs such as reflux esophagitis, gastric atony, constipation, diarrhea, and hepatopathy with mucocele formation are rare clinical signs of hypothyroidism in dogs. 62 symmetric truncal or tail-head alopecia are a classic findings in hypothyroid animals. 62 hyperkeratosis, hyperpigmentation, secondary pyodermas, and demodicosis are also observed. clinicopathologic findings such as normocytic normochromic anemia, hypertriglyceridemia, and hypercholesterolemia are seen in the majority of hypothyroid animals because of altered lipid metabolism and binding proteins (increased hdls), decreased fecal excretion of cholesterol, and decreased conversion of lipids to bile acids. 63 total serum t 4 concentration and endogenous thyroid-stimulating hormone (tsh) may be used to confirm the diagnosis of hypothyroidism. this combination of tests has been shown to have the highest specificity, sensitivity, and lowest overall cost. if the tt 4 is in the low normal or below normal range and the tsh is high, the animal is suffering from primary hypothyroidism. 64, 65 if the tt 4 and tsh are both low, free t 4 by dialysis should be determined to distinguish euthyroid sick syndrome (normal free t 4 ) from true secondary hypothyroidism (low canine thyroid stimulating hormone [ctsh] resulting from pituitary tsh deficiency). 66 treatment synthetic thyroid hormone supplementation is the treatment of choice for hypothyroidism. levothyroxine sodium therapy is started at a dosage of 0.02 mg/kg given orally twice daily. 66 thyroid function should be monitored every 6 to 8 weeks for the first 6 to 8 months of treatment and then once or twice yearly thereafter. in stable well-controlled animals, the total treatment may be given once daily with excellent clinical results, as long as adequate peak hormone concentrations are achieved. 67 with thyroid hormone replacement therapy in hypothyroid dogs, the prognosis is excellent. inflammatory disease involving the intrahepatic bile ducts is commonly encountered in veterinary practice. cholangitis is recognized more commonly in cats than in dogs, but both species can be affected. the wsava liver standardization group suggests that cholangitis be considered in the following four groups: neutrophilic cholangitis (nc), lymphocytic cholangitis (lc), chronic cholangitis associated with liver fluke infestation, and destructive cholangitis. 1 sinusoidal lumen. amyloid fibrils are readily detected on routine hematoxylin and eosin or diff-quik staining. amyloidosis is confirmed on examination of congo red-stained aspirates or biopsies under polarized light where the extracellular material shows characteristic green birefringence. 50 concurrent amyloid deposition in the kidneys, liver, spleen, and adrenal glands can occur, but clinical manifestations of liver failure are most common. chronic progressive liver failure with clinical signs of anorexia, weight loss, and lethargy, is the typical clinical course in many cases. some animals may instead present with acute collapse following hepatic rupture and intraabdominal hemorrhage. 50 pallor of mucous membranes, hypothermia, and hepatomegaly are the most frequently recognized physical examination findings. typical laboratory findings include regenerative anemia, leukocytosis, thrombocytopenia, marked elevations in serum alt and ast, and marked prolongations in aptt and pt times. radiography is useful in detecting free peritoneal fluid, hepatomegaly, and irregular hepatic borders. ultrasonography reveals a diffuse, heterogeneous echogenicity with highly echogenic ("sparkling") areas and hypoechoic foci. 50 definitive diagnosis requires tissue biopsy and congo red staining. there are no specific treatments for this disorder. colchicine has been recommended because it may block formation of amyloid in the early stages of the disease, but it is of unproven benefit and has been associated with significant side effects. dimethyl sulfoxide has been recommended because it may promote resorption of amyloid. as there are no specific therapies for this disease, treatment is instead largely symptomatic and supportive. with progressive amyloidosis lesions, the prognosis for long-term survival is poor. a familial hyperlipoproteinemia has been reported in cats that is characterized by fasting hyperchylomicronemia, elevated circulating concentrations of vldls, and hypertriglyceridemia. 56, 57 serum cholesterol is only minimally elevated. the underlying biochemical lesion is a reduction in the activity of lipoprotein lipase, and the disorder is transmitted as an autosomal recessive gene. xanthomas accumulate in the soft tissues, including the liver, but clinical signs are more often related to involvement of the peripheral nerves. dietary fat restriction improves clinical signs in some affected animals. 58 etiology decreased circulating thyroid hormone concentration affect hepatic metabolism and cholesterol turnover in the liver. liver function tests are mildly disturbed in almost 50% of patients with hypothyroidism despite normal histologic findings. 59 pathophysiology decreased hepatic metabolism in hypothyroidism is reflected by reduced oxygen consumption. 33, 59, 60 patients with a common bile duct stone and gallbladder stone have, respectively, sevenfold and infection of bile has been commonly identified in cats with ehbdo, 4, 8 clinical examination. previous literature has highlighted differences in clinical presentation between cats with different forms of cholangitis. however, we have recognized few differences between the various forms 4, 9 and suggest that any statistically significant differences cited previously hold little clinical relevance given the large degree of overlap within data ranges. nc can occur in cats of any age, breed, or sex. clinical signs are nonspecific and include anorexia, lethargy, vomiting, and weight loss. the duration of these clinical signs ranges from a few days to a few months and may be shorter in cats with anc than in those with cnc, 3 but this is not a consistent finding. 4, 9 physical examination findings commonly include dehydration and icterus. fever is present in 19% to 37.5% of cases. 4,10 some reports suggest that fever is more commonly associated with anc than cnc, 10 while others recognize no difference. 4, 9 hepatomegaly is seen in fewer than half of the cases. abdominal pain is noted occasionally. 3, 4, 9 diagnosis. definitive diagnosis is made by examination of liver biopsy specimens, with ancillary diagnostics providing supportive information. hematologic findings are variable and may include poikilocytosis, neutrophilia, and left shift, although these abnormalities are present in fewer than one-third of cases. 3, 4, 9, 10 biochemical analysis commonly reveals increased activity of alt, ast, alp, and ggt ranging in severity from mild to severe. however, increased liver enzyme activity may be absent in some cases. serum total bilirubin is increased in most cases. serum cholesterol may become increased in cases with ehbdo. imaging findings are nonspecific for cholangitis, but may provide useful information regarding concurrent disease. abdominal radiographs are rarely helpful. ultrasonographic appearance of the liver in cats with nc can vary greatly, with the most common abnormality being a diffuse change in echogenicity ranging from hypo-to hyperechoic. 11 dilation of intra-and/ or extrahepatic bile ducts, gallbladder distention, increased gallbladder sediment, and thickening of the gallbladder or bile duct walls may be seen. gallbladder distention and bile duct dilation may indicate ehbdo, but these changes may occur in cats with cholangitis lacking obstruction. ultrasonography will also provide information regarding the presence of concurrent disease, such as pancreatitis and inflammatory bowel disease. wedge liver biopsy during laparotomy is the optimal method for obtaining a definitive diagnosis. other biopsy techniques that may be considered include laparoscopic and ultrasound-guided tru-cut needle approaches. tru-cut needle biopsy diagnoses correlate with wedge biopsies in fewer than 50% of cases. 12 diagnostic accuracy of laparoscopic liver biopsies compared with wedge biopsies have not been evaluated. laparotomy and laparoscopy provide the additional benefit of evaluation and sampling of extrahepatic structures. laparotomy should be performed in any cat suspected of having ehbdo. while the optimal sampling strategy is unknown, biopsies should be obtained from multiple liver lobes, as we have recognized wide ranges of severity between different lobes in the same cat. in patients that are not stable enough for liver biopsy, such as those with hypotension, coagulopathy or he, fine-needle aspiration with cytology offers a less-invasive diagnostic approach as it can usually be performed quickly with light sedation. however, liver cytology correlates with biopsy results in only 39% to 60% of cases. 13, 14 cytology is sensitive for identifying the presence of hl, however this is the most common misdiagnosis when using cytology. 14 cytology is cholangitis is a common hepatobiliary disorder of cats, second only to hl. 2 although varying terminology has created some confusion regarding this syndrome, it is clear that feline cholangitis includes a spectrum of disease processes, including forms displaying neutrophilic inflammation and those lacking neutrophilic inflammation. histologically, nc is characterized by the presence of neutrophils in the lumen and/or epithelium of the bile ducts. 1 the disease is recognized to occur in acute and chronic forms. in acute neutrophilic cholangitis (anc) edema and neutrophilic inflammation are seen in the portal areas, with occasional extension of inflammation to the hepatic parenchyma. in chronic neutrophilic cholangitis (cnc) there is a mixed inflammatory infiltrate consisting of neutrophils, lymphocytes, and plasma cells. varying degrees of bile duct hyperplasia and fibrosis will be present depending on the chronicity of disease. etiology. although the true etiology remains unknown, nc is largely suspected to be caused by ascending bacterial infection from the intestine. 1-3 rates of bacterial isolation using traditional methods have varied greatly, from less than 20% to more than 60% in affected cats. 3, 4 recently, fluorescence in-situ hybridization (fish) with a 16s rdna probe that recognizes bacteria in general has been used to identify and localize bacteria in cats with cholangitis. 5 combining traditional culture and fish, bacteria were isolated in three of three (100%) cats with anc and eight of 13 (61%) with cnc. the localization of the bacteria identified using fish supports translocation of enteric bacteria as the cause of infection. although it appears that bacteria play an important role in the etiology of nc in many cases, it is important to note that they are not identified in all affected cats. some authors theorize that nc, and cnc in particular, may have an immune-mediated etiology with persistent inflammation following an initial bacterial infection or other unknown initiating factor. 3, 6 pathophysiology. nc in cats is commonly associated with inflammatory bowel disease and pancreatitis. 3, 4, 7 the pathophysiology underlying the relationship of these diseases is unknown, but rational theories revolve around the unique anatomy of the feline biliary and pancreatic duct systems. in the cat, the common bile duct and pancreatic duct merge prior to entering the duodenum at the major duodenal papilla. 7 cholangitis may develop secondary to reflux of ascending bacteria from the duodenum during vomiting. pancreatitis may result from bacterial reflux into the pancreatic duct, or from pancreatic duct obstruction secondary to cholangitis. 7 in most reported cases, the inflammatory bowel disease associated with cholangitis is moderate or severe, whereas the pancreatitis tends to be mild chronic interstitial disease. 7 nc is also commonly associated with extrahepatic bile duct obstruction (ehbdo). ehbdo has been identified in 40% of cats with anc and 76% of cats with cnc. 4 cholangitis and/or pancreatitis are the most common cause of ehbdo in the cat. 8 in one study, 64% of cats with ehbdo had cholangitis, representing 93% of cats that did not have a neoplastic cause. 8 it is unknown whether cholangitis is the cause or the result of ehbdo. histologic changes consistent with cnc have been seen in the livers of cats with ehbdo secondary to pancreatic carcinoma, cholelithiasis and surgical occlusion of the common bile duct. 8 in contrast, cholangitis has been implicated as the sole cause of ehbdo resulting from proliferation of mucosa within the common bile duct. 8 bacterial choleretic properties that make it a rational choice for treating cholangitis. because of the possibility of immune-mediated mechanisms in the perpetuation of nc, particularly with cnc, corticosteroids may be appropriate in some cases. initial treatment should always involve antibiotics in cats with nc. failure to improve within 2 weeks of antibiotic therapy, or clinical deterioration prior to that time, warrants initiation of corticosteroid therapy. prednisolone at 1 to 2 mg/ kg twice daily is given initially and gradually tapered to the lowest effective dose. antibiotics should be continued concurrently with corticosteroids for a minimum of 4 weeks. the duration of corticosteroid therapy varies between individual patients. many cases can be gradually tapered off of corticosteroids over 4 to 6 months, while others require lifelong therapy. surgical intervention is required in cats with ehbdo; however, the optimal surgical procedure is unknown. biliary diversion (cholecystocholedochostomy, choledochoduodenostomy, or cholecystojejunostomy) and choledochal stenting are the most common procedures. surgery in cats with ehbdo is associated with significant perioperative morbidity. in many cases, profound hypotension develops intraoperatively after 45 to 60 minutes as a result of decreased vascular responsiveness and decreased myocardial contractility and is often refractory to interventions such as fluid or vasopressor therapy. 8, 18, 19 whichever surgical procedure is chosen, it is clear that anesthesia time should be minimized and long-term medical management will be necessary. biliary diversion is associated with short-term mortality rates of 36% to 57% 8, 18 and is associated with long-term complications. 8, 19 in a small case series describing choledochal stenting in cats with pancreatitis and cholangitis, five of seven experienced long-term survival (≥7 months), but reobstruction occurred in two of seven and chronic vomiting and recurrent cholangitis were reported. 19 prognosis. the prognosis for cats with nc is typically good. 3, 4, 10 survival to discharge was reported in 72% of all cats with cholangitis in one study. 4 median survival time of 29.3 months has been reported in cats with nc, with no difference between anc and cnc. 10 prognostic factors have not been identified. given the high rate of perioperative morbidity and mortality, it seems likely that cats with ehbdo have a worse prognosis than those without ehbdo. thirty percent to 40% of cats with ehbdo secondary to inflammatory disease die within a week of surgery. 8, 18 however, in those that survive to discharge, long-term survival has been reported. 18 the wsava liver standardization group describes lc as a common, slowly progressive, chronic disease of cats characterized histologically by infiltration of small lymphocytes (and occasionally plasma cells or eosinophils) restricted to the portal areas associated with varying degrees of fibrosis and bile duct hyperplasia. 1 they remark that inflammation centered on the bile ducts may be present, but is not a hallmark of the disease. it is also stated that welldifferentiated lymphoma may be difficult to differentiate from lc. based on the existing literature regarding lc in cats, the description from the wsava group includes several clinically and histopathologically different subsets that may or may not revolve around a common pathogenesis. recognition of these different subsets within the umbrella of lc may have therapeutic and prognostic ramifications. several investigators describe a group of cats with lc where inflammation is confined to portal regions and there is a lack of targeting of bile ductules or biliary epithelium. 2, 20, 21 this has been insensitive for identifying cholangitis in cats, diagnosing fewer than 30% of cases. 14 cytologic examination of bile may prove more sensitive for the diagnosis of nc in cats. in five of seven cats with cnc evaluated at this institution, bile cytology revealed neutrophilic inflammation, presence of bacteria, or both. techniques have been described for safely obtaining bile via ultrasound-guided percutaneous cholecystocentesis in lightly sedated cats. 15 samples for aerobic and anaerobic bacterial cultures should be obtained in any cat suspected of having cholangitis. gallbladder bile is preferred to liver tissue as the culture source. in a group of 58 cats suspected of having hepatobiliary disease, bile cultures isolated pathogens in 36% compared with only 14% of liver cultures. 16 in the same study, 22 dogs and cats had both liver and bile cultured and none had a positive liver culture in the absence of a positive bile culture. 16 in a group of cats with cholangitis, bile cultures were more likely to isolate pathogens (75% vs. 33%) and less likely to yield contaminants (4% vs. 29%) than liver cultures. 4 in a small study comparing bile versus liver cultures in 22 cats with various hepatobiliary diseases, bile culture was positive in five (four had cnc) while liver culture was positive in only two. in the two cats with positive liver cultures, the same organism was isolated from bile. 17 it is important to recognize that many cats with nc (and other hepatobiliary diseases that mimic it clinically) are not stable enough to tolerate diagnostic testing. in such patients, the risk of aggressive diagnostics may outweigh the benefits of obtaining a definitive diagnosis. in these cases, the diagnosis may be suspected based on clinical response to supportive care, including broad-spectrum antibiotic therapy. treatment. optimal treatment protocols for cats with nc are unknown and the recommendations herein are based solely on anecdotal clinical experience. antibiotics are the mainstay of treatment. drug selection is ideally based on results of bacterial culture and susceptibility testing. in cases where cultures are not performed, or while results are pending, broad-spectrum coverage should be provided. the most commonly isolated pathogens are aerobic and anaerobic bacteria of enteric origin, 16 including e. coli, enterococcus spp., and clostridium spp., among others. 3, 4, 16 effective empiric antibiotic combinations would include a penicillin, a fluoroquinolone, and metronidazole. the optimal duration of antibiotic therapy is unknown, but we recommend a 4-to 6-week course for initial treatment. supportive care and treatment of specific sequelae of liver disease should be included as indicated. nutritional support is required in many cats and is best accomplished by use of enteral feeding tubes. we recommend placement of esophageal feeding tubes in cats with cholangitis if they are anorexic and stable enough for general anesthesia. in unstable patients, nasoesophageal feeding tubes offer a less-invasive method of providing short-term support. several medications and nutritional supplements (including ursodeoxycholic acid [udca], same, milk thistle, vitamin e, vitamin c, carnitine, taurine, and phosphatidylcholine) have been suggested for treating cats with cholangitis. while most of these compounds have theoretical benefits, a clinical benefit has not been proven. to optimize client compliance and avoid adverse drug reactions, i prefer to minimize the number of medications given to feline patients. because most cats with anc respond well to antibiotic therapy, i rarely include other medications in our treatment protocol. however, in cats with anc that do not quickly respond to antibiotics and in many cats with cnc, i like to use udca. among its theoretical benefits, udca has immunomodulatory and distinction between lc and well-differentiated (small cell) lymphoma can be a challenge even for experienced pathologists. preliminary data using immunohistochemistry and pcr for t-cell receptor clonality has not proven useful in differentiating between the two conditions. surprisingly, cats with both lc and lymphoma had monoclonal t-cell receptors, oligoclonal t-cell receptors, and polyclonal t-cell receptors. 20 using light microscopy, the following features were unique to lc and not present in cats with lymphoma: ductopenia, bile duct targeting by lymphocytes, and the presence of lipogranulomas within portal regions (representing a residual marker of cell death). 20 until more studies are done evaluating molecular techniques, these features may prove useful in differentiating the two conditions. interestingly, bile duct hyperplasia and fibrosis were present in cats with lc and those with lymphoma. this may suggest that an inflammatory state precedes the development of lymphoma, 20 which has been reported anecdotally. treatment. the therapeutic approach to cats with lc should be similar to that described for cats with nc in regards to supportive care and symptomatic treatment of the sequelae of liver disease. because bacteria have been isolated from some cats with lc, i recommend treatment with broad-spectrum antibiotics while awaiting results of bacterial cultures. in contrast to nc, long-term treatment of culture negative cats with antibiotics is not warranted. immunomodulation and immune suppression are the major components of treatment based on a presumed immune-mediated etiology. cats that are culture negative, or that have failed to respond to antibiotics within a few days, should be treated with prednisolone at 1 to 2 mg/kg twice daily. responders should be tapered gradually over 4 to 6 months to the lowest effective dose. other drugs that are useful for immunomodulation include metronidazole and udca. cats that fail to respond completely to corticosteroids and/ or other immunomodulators, or who relapse while being treated, may require additional immunosuppressive drugs. although these drugs have not been well evaluated in cats with lc, chlorambucil and methotrexate are suggested by some authors. 3 cats with small cell lymphoma often respond to combination therapy with prednisolone and chlorambucil, but they may require a multidrug weekly sequential chemotherapy protocol. prognosis. cats with lc have a variable prognosis, 3,6 likely a result of being diagnosed at different stages of a chronic disease process. survival of greater than 5 years has been reported, and many cats that die appear to succumb to disease unrelated to the liver. 22 other cases have been reported that fail to respond to treatment and die more acutely, 6 though this is uncommon in my experience. this is a disease that likely requires lifelong management and monitoring with relapse of illness possible as medication doses taper. trematode parasites of the families dicrocoeliidae and opisthorchiidae may inhabit the gallbladder and bile ducts of cats and rarely dogs. 26 there are multiple species with worldwide distribution. the most commonly identified species include the dicrocoelid platynosomum concinnum and the opisthorchid amphimerus pseudofelineus. [27] [28] [29] p. concinnum is mainly found in tropical and subtropical areas, including the southeastern united states. 26 a. pseudofelineus has a wider area of distribution throughout north and south america. 26 the life cycle is similar for both dicrocoelids and opisthorchids. 26 parasite eggs are ingested by a land snail (subulina octona or eulota [bradybaena] similaris), develop into cercariae and enter a second referred to as lymphocytic portal hepatitis. 2, 21 the connection between this histopathologic finding and clinical disease in cats is unknown, as it may represent a common change associated with aging. it was identified in 82% of cats older than 10 years of age and 96% of cats older than 15 years of age from a necropsy population that did not have primary liver disease. 21 it is also possible that this lesion represents a response to inflammation at a distant site, as it is similar to the lesion of nonspecific reactive hepatitis associated with chronic extrahepatic disease. 1 although clinical signs have been described in cats with lymphocytic portal hepatitis, 10 the common occurrence of concurrent disease in these cats makes it difficult to know if the clinical signs are attributable to the lesions in the liver. in another subset of cats, lc is marked by inflammation targeting bile ductules and infiltrating biliary epithelium, leading to progressive ductopenia. 20, 22, 23 these cases seem more likely to have clinical disease attributable to their liver pathology, although side-by-side comparisons of cases with and without bile duct targeting have not been performed. cats with this form of lc in the united states have a similar clinical picture to cats with nc. 9 in the united kingdom, this lesion has been associated with ascites, icterus, and hyperglobulinemia in young cats and termed progressive lymphocytic cholangitis. 22, 23 etiology. although the etiology of lc is unknown, theories suggest that it is an immune-mediated or infectious phenomenon. genetic factors may also play a role, as persian cats are overrepresented in the united kindgom. 22, 23 immunohistochemistry in affected cats has provided evidence for an immune-mediated pathogenesis, although the inciting antigen is unknown. 20, 23 bacteria have been identified in the liver or bile of fewer than 20% of cats with lc. [3] [4] [5] 17, 20 although helicobacter pylori has been isolated from the liver and bile of cats with cholangitis, the evidence for this organism playing an important role in feline cholangitis is not compelling at this time. 24, 25 pathophysiology. as with nc, concurrent inflammatory bowel disease and pancreatitis appear to be common in cats with lc, 3, 4 although some authors report it to be uncommon. 23 the theory that reflux of duodenal bacteria into the biliary and pancreatic ducts incites inflammation may hold true for cats with lc, although a common immune mechanism must be considered. clinical examination. the clinical picture of cats with lc varies widely and has significant overlap with other forms of hepatobiliary disease in cats, including nc. 4, 9 although some studies describe a predominance of older cats, 3 others describe more younger cats. 22, 23 nonspecific clinical signs, including anorexia, lethargy, vomiting, and weight loss, may be chronic and intermittent. 3, 6, 22 physical examination findings may include icterus, hepatomegaly, or ascites, but none are consistent findings. signs of he (dullness, ptyalism, seizure) may develop in severely affected cats. diagnosis. definitive diagnosis is made by liver biopsy. as discussed for nc, ancillary diagnostics will provide information to support hepatobiliary disease, but are not specific for lc. hematology results may be unremarkable, even though marked lymphocytosis has been described in some cases. 3 activity of serum liver enzymes is increased in many, but not all cases and varies in severity. hyperglobulinemia has been described. 3, 6, 22 abdominal radiographic and ultrasonographic findings are nonspecific, but may aid in the recognition of concurrent disease. amoxicillin-clavulanate, milbemycin oxime, and amitraz prior to the onset of clinical signs. 31, 32 the proposed toxic etiology is based on these case histories and histopathologic similarity to humans with idiosyncratic drug toxicity. other toxic insults and viral infection, such as canine distemper, can also result in destructive cholangitis. 1 affected dogs typically present for signs referable to cholestasis, including anorexia, icterus, vomiting, and acholic feces. 31, 32 activities of serum liver enzymes and total bilirubin are moderately to markedly increased. abdominal ultrasound may be unremarkable, showing only mild dilation of intrahepatic bile ducts. 32 definitive diagnosis is made by liver biopsy. optimal treatment options are unknown, but should undoubtedly involve discontinuation of any medications that preceded illness. corticosteroids for immune suppressive and antiinflammatory effects and udca for immunomodulatory and choleretic effects would be rational treatments options, but their efficacy has not been documented. the prognosis appears to be poor, as six of the eight reported cases were euthanized within 6 weeks and the remaining two had only shortterm followup (<6 months). 31, 32 nc, as described in the cat, has been rarely reported in dogs. [33] [34] [35] [36] [37] bacteria have been isolated from the majority of cases reported, including e. coli, klebsiella spp., proteus mirabilis, streptococcus spp., and clostridium spp. [34] [35] [36] [37] although the bacterial species would support ascending infection from the intestine, the literature is too sparse to make conclusions about the pathophysiology of this disease in dogs. bacterial infection could also spread hematogenously or via translocation from the portal circulation. the clinical presentation and diagnostic findings are similar to those reported for cats with nc. affected dogs present with lethargy, anorexia, vomiting, and icterus usually of acute onset. fever is reported in approximately half of the cases. [33] [34] [35] [36] [37] neutrophilia with or without a left shift is common. activity of serum liver enzymes is typically increased and most dogs have mild to moderate elevation of serum total bilirubin. ultrasonography is nonspecific, with the liver varying from normal to hyperechoic with some heterogeneity. 34, 36 thickening and hyperechogenicity of the gallbladder wall is common. 36 definitive diagnosis is made by liver biopsy with changes similar to those reported in cats with nc. most dogs have some degree of mixed inflammatory infiltrate, similar to cnc. [33] [34] [35] [36] [37] this infiltrate extends into the hepatic parenchyma in the majority of cases. aerobic and anaerobic culture of bile or hepatic tissue should be performed, though bile has been the source of bacterial isolation in most cases. treatment involves antibiotic therapy guided by culture and susceptibility results. duration of treatment should be prolonged, as antibiotic courses of 8 to 12 weeks or greater have been required to completely eliminate bile infections. clinical improvement precedes bacterial eradication. 36 the prognosis appears to be good in most cases, although dogs with concurrent disease may have a worse prognosis. liver cysts arising from the intrahepatic bile ducts are rarely encountered in veterinary practice. although cysts may be acquired secondary to trauma, neoplasia, inflammation, or biliary obstruction, 1,38 the vast majority of cases described in the literature are congenital in origin. congenital cystic liver diseases result in dilation of various segments of the intrahepatic bile ducts, and they are associated with varying degrees of hepatic fibrosis and cysts in other organs (most commonly the kidneys). little is known about inheritance patterns of cystic disease in dogs and cats. the various morphologic patterns of cystic disease likely represent abnormalities of bile duct intermediate host. 26, 27, 29 the dicrocoelids tend to use an arthropod, while opisthorchids utilize fish. typically cats acquire infection by ingesting the second intermediate host. in the case of p. concinnum, the sporocysts leaving the snail intermediate host may be eaten by a paratenic terrestrial isopod host (pill, sow, or dung bugs). 26, 29 cats are infected by ingesting this form in a variety of lizard or amphibian intermediate hosts. cercariae migrate from the cat intestine to the gallbladder and bile ducts, where they develop into adults. eight weeks or more after infection eggs are passed in the feces to complete the life cycle. 27 clinical signs are proportional to parasite burden. cats with light infections are often asymptomatic. 27, 28 clinically ill cats may present with nonspecific signs such as anorexia, lethargy, vomiting, or diarrhea. severely affected cats present with signs of ehbdo such as icterus and acholic feces. [27] [28] [29] [30] preliminary diagnostics are nonspecific for fluke infestation. eosinophilia proportional to the parasite burden may be present. 29 serum liver enzyme activity may be mildly to moderately increased, although it is normal in many cases. [28] [29] [30] abdominal ultrasound often reveals evidence of ehbdo. [28] [29] [30] definitive diagnosis by identification of flukes or fluke eggs in the feces is difficult as small numbers of eggs are shed daily, the eggs have varying morphology at different stages of development, and the eggs are quite small. 27 fecal concentration-sedimentation using the formalin-ether technique is the most reliable method of identifying eggs in stool. 27, 29 eggs may also be identified in cytologic preparations of bile. 28, 30 eggs or adults may be seen in liver biopsy specimens, but they are inconsistently identified. 1, 27, 28, 30 histopathologic changes seen in the liver are characterized by dilation of larger intrahepatic bile ducts associated with papillary projections and marked periductal and portal fibrosis. mild to moderate inflammation may be present within the ducts (neutrophils and macrophages) and in the portal areas (neutrophils, lymphocytes, plasma cells). eosinophils may be present in limited numbers. 1 rarely, chronic cholangitis associated with liver flukes can result in the development of cholangiocarcinoma. 1, 29 optimal treatment protocols have not been established, but praziquantel at 10 to 20 mg/kg daily for 3 days appears to be the most effective. [26] [27] [28] [29] [30] doses as high as 40 mg/kg daily have been used successfully, 28 but this dose has also been fatal in cats. 26 sporadic resumption of egg shedding following praziquantel has been reported, suggesting that it does not completely eliminate infection. 26, 27 for this reason, continued treatment at 12-week intervals has been recommended. 29 symptomatic and supportive therapy should be tailored to the individual patient. cats with ehbdo require surgical decompression. glucocorticoids and udca may have some benefit in controlling inflammation and providing choleresis. although infected cats may remain asymptomatic, patients with ehbdo appear to have a grave prognosis. long-term survival has only been reported in rare cases. 28, 30 cholangitis is rarely reported in dogs. reports of cholangitis in dogs include two distinct entities: destructive cholangitis and nc. destructive cholangitis is characterized histopathologically by a loss of bile ducts (ductopenia) within the smaller portal areas, associated with cholestasis, portal inflammation consisting primarily of macrophages, neutrophils, and occasionally eosinophils, and progressive portal fibrosis. 1, 31, 32 this is a rarely reported lesion of unknown etiology. it has been postulated that the lesion represents an idiosyncratic drug toxicity. however, of the eight cases reported in the literature only three had a prior drug history: two having received potentiated sulfonamides and the other having received adult polycystic disease is most commonly recognized as polycystic kidney disease in persian cats. 44, 45 it has also been reported in cats of other breeds and in dogs. 39, 43 this is similar to autosomal dominant polycystic kidney disease in humans. inheritance in persian cats is autosomal dominant. 45 liver cysts are thought to represent a late defect in the development of peripheral intrahepatic bile ducts. the liver may contain multiple cysts ranging from less than 1 mm to greater than 12 cm in diameter. these cysts typically contain clear, colorless fluid. discrete fibrotic areas containing small, irregularly formed bile ducts, referred to as von meyenburg complexes, may be present. 1 in the kidneys, multiple cysts may form in any segment of the kidney but may involve only a small percentage of the nephron population. this is in contrast to the diffuse cysts seen with congenital dilation of the large and segmental bile ducts and juvenile polycystic disease. 1, 40 hepatic cysts are present in 10% to 40% of cats with polycystic kidney disease, while hepatic fibrosis is recognized in up to 48%. 44, 45 the hepatic cysts are usually incidental findings and the animals are not clinically ill unless they develop renal failure secondary to cysts in the kidneys, which happens in adulthood. biliary atresia is an extremely rare congenital disorder, having been reported in only one dog and one cat. 46, 47 in both cases, the common bile duct was not patent because of atresia. in the dog, the occluded segment of bile duct was histologically comprised of fibrous tissue with minimal inflammation. 47 the etiology is unknown, but this lesion likely represents an embryologic nonfusion of the cranial (hepatic) and caudal (cystic) anlages of the bile ducts during development. 1 other possible explanations include ischemic, toxic, traumatic, or infectious insults occurring pre-or postnatally. 47 affected animals have presented at 4 to 6 months of age with clinical signs of depression, anorexia, vomiting, or lameness associated with rickets because of inadequate vitamin d absorption. 46, 47 affected animals show icterus, hepatomegaly and acholic feces. serum biochemistry abnormalities are consistent with ehbdo. definitive diagnosis is made at exploratory laparotomy. surgical biliary diversion is a viable treatment option depending on the location of atresia, but it was unsuccessful in the one case reported. 47 a guarded prognosis should be given as for any animal undergoing a biliary diversion procedure (see discussion under "neutrophilic cholangitis"). cholestasis is impaired bile flow resulting in the accumulation of bile components in the blood. 1 intrahepatic cholestasis occurs secondary to a variety of primary or secondary hepatobiliary diseases. 1, 48, 49 increased activity of serum liver enzymes, particularly alp and ggt, is common with intrahepatic cholestasis but is not specific for the condition. clinically patients may appear jaundiced, but the predominant clinical sign will be related to the underlying disease process. cholestasis is marked by the presence of bile plugs in canaliculi, phagocytosed bile in kupffer cells, and bile granules within hepatocytes. these changes are easily recognized in cytologic and frozen preparations, but are less apparent in paraffin-embedded specimens, particularly in cats. 1 when cholestasis is identified, ehbdo should be ruled out. this should be easily accomplished by abdominal ultrasonography as animals with intrahepatic cholestasis lack the dilation of intra-and extrahepatic bile ducts that is typical of ehbdo. 48 however, exploratory laparotomy should be considered in highly suspicious cases for confirmation. development at different stages of their formation. the wsava liver standardization group suggests that cystic disorders be classified into one of the following groups: congenital dilation of the large and segmental bile ducts; juvenile polycystic disease/congenital hepatic fibrosis; and adult polycystic disease. 1 congenital dilation of the large intrahepatic bile ducts (i.e., the hepatic ducts and segmental ducts) has been described in dogs. [39] [40] [41] the lesion is similar to that of caroli disease in humans and it is thought to represent an early defect in the formation of the intrahepatic bile ducts. 1, 40 the disease is marked by extreme, diffuse, grossly evident dilation of the extrahepatic portion of the large intrahepatic bile ducts containing pale-yellow viscous fluid. the gallbladder and common bile duct are normal, as these have a separate embryologic origin from the intrahepatic bile ducts. the liver is normal to mildly increased in size, with diffuse cysts of varying sizes throughout. histologically there are areas of marked bridging portal fibrosis containing multiple dilated bile ducts. the lobular architecture of the liver is normal. although concurrent ascending cholangitis commonly occurs in humans, this is rarely reported in dogs. [39] [40] [41] in addition to the hepatic lesions, affected dogs have fusiform, radially arranged renal cysts with moderate to marked fibrosis throughout the renal cortex and medulla. [39] [40] [41] affected dogs are presented early in life, ranging from 13 weeks to 3.5 years. [39] [40] [41] clinical signs include vomiting, weight loss/failure to thrive, decreased appetite, lethargy, ascites, and rarely icterus and neurologic signs. the clinical signs are typically chronic in nature. gi signs, neurologic signs, and ascites are likely a result of portal hypertension caused by pressure of the cysts on the portal vein. hepatomegaly may be noted on physical examination. activity of serum liver enzymes is typically normal to mildly increased, although marked increases in activity of alt and alp have been reported. 40 renal azotemia is present in some patients. ultrasonographically, cystic dilations of the intrahepatic ducts (most with associated calcification) are easily recognized, even though the renal cysts are not always apparent. definitive diagnosis is made on the basis of the gross and histologic findings described above. rational treatment options and prognosis are unknown, as only one dog in the literature has been treated. this dog was doing well on a low-protein diet at 5 months of followup. 40 most of the affected dogs have been fairly stable, and supportive care may be warranted despite the appearance of severe cystic disease. juvenile polycystic disease/congenital hepatic fibrosis has been described in litters of cairn terriers, west highland white terriers, and cats. [42] [43] [44] this form is analogous to autosomal recessive polycystic kidney disease in humans, and the inheritance appears to be autosomal recessive in the few families of veterinary patients that have been described. 42, 43 the liver cysts are thought to represent an intermediate defect in the development of the intrahepatic bile ducts. 1, 40 the liver involvement is primarily microscopic including fibrotic portal areas containing abnormally structured, dilated small bile ducts. the result is a grossly enlarged and firm liver. 40 renal cysts are present and are identical to those described for dogs with dilation of the large and segmental bile ducts. 40 affected animals are usually presented at less than 8 weeks of age for abdominal distention because of renomegaly and hepatomegaly. 42, 43 however, one 12-yearold cat with similar lesions has been reported. 44 most affected animals are ill or have died at the time of presentation. 42, 43 liver enzyme activity has been increased in the few animals in which it was evaluated. 43 abdominal ultrasound may identify cysts and definitive diagnosis is made based on histologic findings as described. treatment has not been attempted, and the prognosis appears to be grave. hepatic progenitor cells. 50 they may be identified at intrahepatic or extrahepatic locations. they appear to have a more aggressive course than cholangiocellular carcinomas, with the majority being present in multiple lobes and greater than 90% having metastasized at the time of diagnosis. 52, 54, 55 the major pathogenetic mechanisms of canine and feline extrahepatic biliary tract disease are obstruction, inflammation, and exudation. the major causes of extrahepatic biliary tract obstruction (ehbo) are pancreatitis, gallbladder mucoceles (in dogs), cholelithiasis, parasitic infections (in cats), and tumors. ehbo is more common in dogs than cats. biliary tract inflammation (e.g., cholecystitis, cholangitis) is primarily caused by bacterial infection, but can be nonseptic or parasitic, especially in cats. gallstones may be associated with biliary tract disease (e.g., infection, obstruction), but the majority of them appear to be clinically silent. stones in the gallbladder are termed choleliths while stones in the biliary tract are termed choledocholiths. biliary tract exudation or leakage can be caused by traumatic (primarily of the bile ducts) or spontaneous rupture (primarily of the gallbladder). the latter is caused by necrotizing cholecystitis, which can be caused by sepsis (e.g., infectious cholecystitis), pressure necrosis (e.g., mucocele), or infarction. biliary tract tumors are rare and of uncertain cause. the common bile duct passes through the lesser omentum and the pancreatic parenchyma before entering the mesenteric wall of the duodenum. in the dog, it empties near the opening of the minor pancreatic duct at the major duodenal papilla, whereas in the cat it joins with the major pancreatic duct before emptying into the duodenum. inflammation and edema with pancreatitis may be sufficient to cause compression and obstruction of the bile duct. this is probably the most common cause of canine ehbo. 1 there is, however, no consistent relationship between clinical severity of the pancreatitis and likelihood of ehbo, possibly because pancreatitis can affect different regions of the pancreas. pancreatitis is a rare cause of ehbo in the cat. chapter 60 discusses the breeds at increased risk for pancreatitis and causes. gallbladder mucoceles occur primarily in dogs. in such cases, the gallbladder is filled with inspissated, semisolid mucus that may extend into the bile ducts causing obstruction. mucoceles can exceed the storage capacity of the gallbladder thereby causing pressure necrosis on the wall of the gallbladder. they can also spontaneously rupture (often at the fundus) 2,3 causing bile peritonitis. the cause of gallbladder mucocele is unknown, but might include dysfunction/hyperplasia of mucus-secreting cells in the gallbladder mucosa. mucoceles may become secondarily infected. 2 gallstones are often clinically silent and observed incidentally only at the time of abdominal imaging. they can be associated with cholecytitis, 4 but they rarely cause ehbo because they must be small enough to enter the cystic bile duct but large enough to lodge there. intrahepatic cholestasis is associated with extrahepatic bacterial infection in dogs. 49 this syndrome is well characterized in humans and may occur in other species such as the cat. it represents an important differential diagnosis for hyperbilirubinemia in animals without primary liver disease, as over 40-fold increases in total bilirubin have been reported. the physiology behind this mechanism is incompletely understood, but it is thought to result from reduction of bile salt-dependent and -independent bile flow caused by bacterial toxins and/or inflammatory mediators. 49 tumors of biliary origin in dogs and cats include cholangiocellular adenoma, cholangiocellular carcinoma, and carcinoid. 50 they are uncommon, representing less than 1% of all canine and feline neoplasms. [51] [52] [53] [54] the tumors of epithelial origin, cholangiocellular adenoma and carcinoma, are the most common, comprising 40% of all hepatic neoplasms in dogs [51] [52] [53] and 56% to 80% of all hepatic neoplasms in cats. [54] [55] [56] tumors showing characteristics of both hepatocellular and cholangiocellular carcinoma have been reported rarely. 50, 52 in dogs, 70% to 100% of biliary epithelial tumors are malignant, 51,52 while in cats 35% to 43% are malignant. [54] [55] [56] cholangiocellular adenomas commonly contain cystic components, especially in cats, and have been referred to as biliary or hepatobiliary cystadenomas in this species. 57,58 cholangiocellular tumors arise predominantly from intrahepatic bile ducts in both species. extrahepatic location is more common in cats than in dogs and is always associated with malignancy. [51] [52] [53] [54] 56 in both species, cholangiocellular carcinomas are more likely to present as multiple or diffuse tumors than are adenomas. 51, 52, 54 carcinomas are highly metastatic (70% to 90% rate) with local lymph nodes, peritoneum, and lung being the most common sites of metastasis. 51, 52, 54 the etiology of biliary neoplasia is unknown. affected dogs and cats are typically middle-aged to older, although animals with malignancy may present at a younger age than those with benign disease. 52, 56 clinical signs are usually vague (such as anorexia and lethargy) and somewhat chronic. malignant tumors are more likely to cause clinical signs, as many benign tumors are incidental findings not associated with illness. 51, [56] [57] [58] hepatomegaly or the presence of a cranial abdominal mass may be identified on physical examination. increased activity of serum liver enzymes is more commonly associated with malignancy, often being absent with benign tumors. 51, 56, 58 even though abdominal radiographs will often identify the presence of hepatomegaly or an hepatic mass, ultrasonography is the preferred imaging method for identification of biliary neoplasms. this modality allows for determination of the cystic nature of the tumors 58 and for the evaluation of metastatic potential. fine-needle aspiration and cytology are of limited utility for diagnosis of biliary epithelial tumors. carcinoma was correctly identified via liver mass aspiration in only 20% of cases in one study. 14 however, cytology is recommended as it likely exhibits high specificity, especially for metastatic lesions. surgical excision and biopsy is the optimal diagnostic and therapeutic technique for tumors confined to one or two liver lobes. surgical excision appears to be curative in cats with benign tumors, 56,57 but malignant tumors carry a poor prognosis in both dogs and cats with many cases not surviving to discharge and survival greater than 6 months not reported in any case. 56, 59 adjunctive chemotherapeutic protocols have not been reported. carcinoids, also referred to as neuroendocrine tumors, are far less prevalent in dogs and cats than the tumors of biliary epithelial origin. 52, 54, 55 these tumors are thought to develop from neuroendocrine cells in the epithelium of bile ducts or gallbladder or from anorexia and vomiting as more prominent symptoms. fever is uncommon, icterus inconsistent, and leukocytosis is often insignificant, even with marked bacterial infections of the biliary tract. gallstones are generally asymptomatic. they can be associated with cholecystitis or ehbo. bilious abdomen can be a clinically mild condition, or it can be associated with life-threatening signs. septic bilious abdomen causes extremely severe peritonitis with systemic inflammatory response syndrome (e.g., anorexia, vomiting, abdominal pain, poor perfusion, fever, and death). these patients may be in the initial hyperdynamic state (e.g., red mucus membranes, bounding pulse, fever, or hypothermia) or, if initially undiagnosed, can be in the late hypodynamic state (e.g., pale mucus membranes, weak pulse, and hypothermia). intraperitoneal bile seems to make septic peritonitis more severe. in contrast, some animals with sterile bilious abdomen (e.g., as a consequence of automobile trauma) are essentially normal except for ascites and icterus. plain radiography is occasionally diagnostic of biliary tract disease. some gallstones are radiopaque (figure 61-30 ). 4 finding air in the gallbladder or in the wall of the gallbladder (i.e., emphysematous cholecystitis; figure 61 -31) is diagnostic of infection with a gas-producing bacterium. "porcelain" gallbladder (i.e., a radiopaque gallbladder because of intramural mineralization of the gallbladder) is associated with carcinoma. 15 ultrasound is generally accepted as the most important and sensitive method for diagnosing extrahepatic biliary tract diseases. 7 many patients that are not suspicious for biliary tract disease are fortuitously diagnosed when ultrasound or radiographs are requested for various reasons. 14 in difficult cases (e.g., partial ehbo versus complete ehbo), nuclear scintigraphy techniques can be used 16 ; however, this seems to be rarely required. diagnosis of ehbo generally relies upon the use of ultrasound. dilation of the bile ducts (normal canine bile ducts are ≤3 mm; normal feline bile ducts are ≤2 to 2.5 mm) is primarily caused by ehbo, and is generally seen by 3 days postobstruction. if one is unsure whether the ducts are dilated, repeating the examination in 3 days should be helpful. enlargement of the gallbladder is not diagnostic of ehbo because anorexia and starvation of any cause may do the same thing. however, failing to find a dilated gallbladder parasites occasionally cause obstruction. platynosomum fastosum (i.e., p. concinnum) is a fluke that inhabits the gallbladder and/or bile ducts of cats that are infected by eating lizards or toads. 5 natural infections are found primarily in florida, hawaii, and the caribbean. it may be asymptomatic or may cause obstruction or fibrosis. they are rare causes of cholecystitis. tumors may cause obstruction, and may be one of the more common causes in cats. 6, 7 cholecystitis is most commonly caused by bacterial infection, ostensibly from bacterial migration up the bile duct. various grampositive, gram-negative, aerobic, and anaerobic bacteria (e.g., clostridium, staphylococcus spp., enterococcus spp., streptococcus spp., klebsiella spp., e. coli, helicobacter spp.) have been reported. [8] [9] [10] [11] infections caused by gas-producing bacteria can produce emphysematous cholecystitis. because of a shared biliary and pancreatic ductal system in the cat, hepatobiliary disease is a well-established risk factor for pancreatitis in the cat. aseptic inflammation of the gallbladder (necrotizing cholecystitis) has been reported, and infarction is one such cause. 11, 12 exudation leakage of bile into the abdomen can be a result of mechanical forces (e.g., automobile trauma) that cause a shearing effect resulting in transaction of the common bile duct or one of the other bile ducts. mechanical rupture has also been reported following gunshot trauma. necrosis of the gallbladder raises the risk for rupture of the gallbladder and occasionally the bile ducts. canine and feline gallstones are typically composed of cholesterol, bilirubin, or may be mixed (as opposed to human gallstones which are usually caused by cholesterol). 4 feline gallstones are typically calcium carbonate or mixed stones. 13 biliary tract diseases can cause severe clinical signs (e.g., anorexia, depression, vomiting, icterus, abdominal pain) or they may be relatively asymptomatic. most symptomatic patients have serum biochemical abnormalities (e.g., increased serum alt, alp, and serum bilirubin). hypercholesterolemia is common in patients with ehbo. hyperbilirubinemia by itself does little besides cause icterus; therefore, patients with extremely high serum bilirubin concentrations can be relatively asymptomatic. renal failure has been attributed to excessively high serum bilirubin concentrations, but it is unclear that bilirubin is the cause or that this is common. shetland sheepdogs 14 appear to have an increased risk for biliary tract disease, and cocker spaniels might also (see chapter 62) . besides icterus, clinical signs in patients with ehbo are primarily a result of the cause of the obstruction, not the obstruction itself. canine pancreatitis in particular may cause severe clinical signs (e.g., anorexia, vomiting, abdominal pain-see "complications of liver disease" section). however, not all patients with pancreatitisinduced ehbo have severe pancreatitis. causes of ehbo that are insidious (e.g., mucoceles, tumors, and stones) are often unsuspected until the patient becomes icteric. biliary tract inflammation such as septic cholecystitis is welldocumented in dogs and cats. shetland sheepdogs appear to be at a greater risk of inflammatory biliary tract disease than most other breeds. 14 clinical signs vary, but most animals are clinically ill with cholecystitis is often diagnosed by ultrasound-guided percutaneous aspiration of gallbladder bile for cytology and culture. this is a relatively sensitive and specific procedure for diagnosing biliary tract infection. 2, 18 the finding of a dilated bile duct coincident with a normal-size gallbladder suggests cholecystitis, prior ehbo, or a rare congenital problem such as caroli disease. 19 it is important to note that patients with bacterial cholecystitis may have no ultrasonographic or gross abnormalities. ultrasound may be suggestive, but is relatively insensitive for cholecystitis. 8, 20 consequently, it is probably best to routinely aspirate bile for cytology and culture in patients with hepatobiliary disease. gallstones are relatively easy to diagnose, some can be found by radiographs, but almost all can be found by ultrasonography ( figure 61 -33). with ehbo, the underlying cause is always the primary concern. ehbo in and of itself is not the primary consideration when deciding upon therapy. pancreatitis, for example, is primarily a medical disease (see chapter 60) . surgery is rarely appropriate in the management of pancreatitis even when it is causing ehbo. if deemed necessary, ehbo caused by pancreatitis can be relieved by percutaneous aspiration 21 or placement of a biliary tract stent. 22 if absolutely necessary, a cholecystoduodenostomy may be performed, but this surgery should be avoided if possible. these procedures are seldom necessary because almost all patients with ehbo caused by pancreatitis will experience resolution of the obstruction with medical therapy. gallstones should be removed only if they are causing obstruction or cholecystitis. it is usually better to perform a cholecystectomy 4,13 as opposed to a cholecystotomy; the former has a lower morbidity and mortality rate in people and presumably in dogs and cats as well. biliary tract tumors can seldom be cured surgically. if a patient has ehbo caused by something that cannot be treated medically (e.g., tumor, pancreatic stricture, traumatically torn bile duct), then a biliary bypass procedure can be performed. cholecystoduodenostomy can relieve the obstruction, although the surgery requires special surgical skills. this surgery can predispose the patient to recurrent, ascending cholecystitis or other complications 10,23 and should only be performed in patients that in a patient with clearly dilated bile ducts suggests biliary tract inflammation or prior ehbo. any dog with ehbo should be suspected of having acute pancreatitis until proven otherwise. chapter 60 details the diagnosis of pancreatitis. if acute pancreatitis is eliminated in a patient with ehbo, then one should look for gallstones and tumors, first by imaging and then by exploratory surgery or laparoscopy if imaging fails to provide a diagnosis. mucoceles are readily diagnosed by ultrasound, although there is some debate about what constitutes a mucocele. "classic" mucoceles are described as producing a "kiwi fruit" appearance without gravitydependent bile movement. "sludge" in the gallbladder is a common finding (and it has gravity-dependent movement), but is not clinically significant. 17 the gallbladder of clinically normal dogs may appear abnormal on ultrasound examination (figure 61-32) , while patients with significant biliary tract disease may appear essentially normal. whether or not patients with ultrasonographic abnormalities of the gallbladder will become symptomatic cannot be reliably predicted. the gallbladder is filled with hyperechoic material (arrows) that was not gravity dependent. the dog was asymptomatic for biliary tract disease and was still in good health without therapy for the gallbladder 10 months after this image was taken. . the stones were found fortuitously during an abdominal ultrasound; there was no evidence that they were causing any clinical signs. gb that has a risk of dehiscence. in general, one should either remove the gallbladder, aspirate gallbladder bile, gently express the gallbladder, or leave it undisturbed. if the leakage is associated with septic peritonitis, then that is a true emergency and requires immediate, aggressive medical and surgical therapy. gallstones may be monitored if the patient is asymptomatic. tumors can rarely be resected; they are generally inoperable when found. rare examples exist of patients with biliary tumors being cured surgically. biliary flukes can be treated with praziquantel (20 to 40 mg/kg sq for 3 days). chapter 60 discusses pancreatitis in greater detail. biliary mucoceles that have not ruptured often have a good prognosis; however, one report found a 32% mortality rate in patients without a rupture versus 68% mortality in patients with bile peritonitis. 14 the report authors recommended a more preemptive approach (i.e., versus waiting until the patent is symptomatic). another group found no difference in mortality between rupture and nonruptured mucoceles (i.e., 21%). 3 gallstones generally are innocuous, and even when they are causing signs have a good prognosis as long as rupture has not occurred. bacterial cholecystitis has a good prognosis as long as the gallbladder is intact and not at risk for rupture. septic bilious peritonitis has a very guarded to poor prognosis, depending upon the severity of the peritonitis. gallstones are usually asymptomatic, but can occasionally cause a problem. they are usually easily removed and resolved. biliary tumors are usually a poor prognostic finding 6, 7 because of late diagnosis. flukes can be treated, but without early recognition, extensive tissue injury may or may not resolve after therapy. the liver serves many important functions including metabolism (carbohydrate, protein, lipid, nucleic acid, xenobiotics, porphyrins, vitamins, minerals, glutathione, endogenous hormones), coagulation factor synthesis, biliary secretion, and immune surveillance (see chapter 1). it is not surprising, therefore, that animals with liver disease experience a broad range of complications reflecting perturbations in one or more of these functions. the hepatic portal venous system accounts for up to 75% of the total hepatic circulation and serves to transport nutrients from the gi tract to hepatic sinusoidal capillaries (and hepatocytes) before coalescing once again into central veins, hepatic veins, and caudal vena cava. in health, the multiple branching of the main portal vein, venules, and capillaries reduces the overall resistance to blood flow (circuits in parallel reduce resistance), and therefore the pressure required to perfuse these capillaries is maintained at a low level of less than 5 to 6 mm hg. 1,2 disease processes that obstruct flow through the intrahepatic branches of the portal vein or sinusoids elevate this pressure and result in significant portal hypertension. hepatocyte swelling impedes portal flow in acute pathophysiologic states, and fibrosis further impedes this flow in absolutely require it. generally speaking, ehbo caused by pancreatitis that is not resolving as quickly as desired is not necessarily a good indication for this procedure; patience and medical therapy generally resolve the problem. in fact, pancreatitis can be a complication of surgery in this region. 24 many animals with hepatic disease are concurrently treated with antioxidants and other hepatoprotectants. although most of these drugs will not hurt patients with biliary tract disease (in fact, they may be beneficial if the disease is extending into the hepatic parenchyma, such as cholangitis/cholangiohepatitis), care must be taken before administering udca. udca is a choleretic agent that stimulates bile flow. this could be disadvantageous in a patient with complete ehbo. mucoceles usually need to be removed surgically. 2, 3, 14 medical management may be attempted (e.g., fat-restricted diet plus udca), but there is a substantially increased mortality rate for patients that experience gallbladder rupture (figure 61-34) ; consequently, surgery is probably the safest course. most patients are not at risk of immediate rupture. as long as ultrasonography does not suggest impending rupture, one should make sure the patient is an optimal anesthetic risk. bacterial cholecystitis, uncomplicated by ehbo or stone, should generally first be treated with antibiotics, usually for 4 to 6 weeks. however, some patients with bacterial cholecystitis cannot be cured with antibiotic therapy, ostensibly because the infection has localized in the gallbladder mucosa. these patients consistently relapse after discontinuation of antibiotics, and therefore a cure may be achieved only with antibiotics plus cholecystectomy. when performing this surgery, great care should be taken to avoid causing a stricture or obstruction of the common bile duct. postcholecystectomy bile duct obstruction may prove fatal. it is also very important to be avoid traumatizing the pancreas to avoid severe pancreatitis. 24 biliary tract leakage is treated based upon the underlying cause. if the gallbladder has ruptured, cholecystectomy is usually most appropriate. if the bile duct has ruptured, it is very difficult to successfully anastomose the two ends. in that instance, one generally must perform a cholecystoduodenostomy and ligate both ends of the torn bile duct. it is important to avoid biopsying the gallbladder as clinical signs associated with he in dogs and cats include depression, behavioral changes, circling/head-pressing, ataxia, apparent blindness, abnormal swallowing or salivation, stupor, seizures, and coma (table 61-10) . salivation is much more common in cats than dogs with he. brain dysfunction in he was historically considered to be a neurotransmitter dysfunction but current evidence suggests lowgrade cerebral edema caused by astrocyte swelling is the predominant pathologic change. 8 there is consensus that ammonia is the key toxin in he but blood ammonia concentrations do not always correlate with severity of clinical signs. this is because a large number of other factors interact with the effects of ammonia to precipitate he. astrocytes play a central role because they express glutamine synthetase and detoxify the ammonia that reaches the cns. intraastrocyte accumulation of osmotically active glutamine in he results in astrocyte swelling and thus low-grade cerebral edema. 8 this is largely reversible if the precipitating factors are treated, but edema can become severe and result in irreversible cns changes in severe and acute he. precipitating factors include inflammatory cytokines, benzodiazepine-type sedatives, and disturbances in amino acid metabolism and dopaminergic neurotransmission. [8] [9] [10] the source of ammonia is primarily absorption from the gut, although other sources also exist as a result of interorgan metabolism. gut-derived ammonia was traditionally assumed to be a by-product of intestinal bacterial metabolism in the colon. this remains an important source in some conditions such as melena. however, recent studies in other species suggest that small intestinal enterocyte metabolism of glutamine as their main energy source is the most important source of postprandial ammonia absorption in the portal vein. 9, 10 this is also likely the case in most dogs on normal diets as it is very unusual for undigested protein to reach the colon, although the source of gut-derived ammonia has never been investigated in dogs. in normal dogs, ammonia is transported to the liver via the main portal vein, and further metabolized chronic pathophysiologic states. sustained portal hypertension is associated with many, but not all, of the complications of liver disease. portal hypertension is an important, potentially life-threatening complication of liver disease in the dog. it is rare or poorly documented in the cat. portal hypertension develops most often in dogs with chronic liver disease and cirrhosis. it is occasionally recognized as a congenital lesion in young animals with arteriovenous fistulas, 3 or as a hypoplastic disorder of the intrahepatic portal vein branches resulting in a condition referred to as noncirrhotic portal hypertension. 4, 5 prehepatic portal hypertension can develop secondary to portal vein thrombosis or congenital hypoplasia of the extrahepatic portal vein, but these are less common. sustained portal hypertension may progress to splanchnic congestion, gi ulceration, ascites, and encephalopathy. portal venous hypertension produces vascular stasis and venous congestion and increases the risk of gi ulceration, particularly in conjunction with other risk factors such as anorexia and steroidal and nonsteroidal antiinflammatory drug usage. portal hypertensionrelated ulceration in the dog is typically duodenal although bleeding esophageal varices, similar to those reported in humans, are occasionally observed. 3 glucocorticoids should be used only with great caution in dogs with portal hypertension. reduced systemic blood pressure is another consequence of portal hypertension and splanchnic venous congestion. changes in systemic blood pressure activate the renin-angiotensin-aldosterone system (as described in chapter 8) and renal sodium retention, and thus increase total circulating fluid volume. the increase in circulating fluid volume (the "overfill" hypothesis) is believed to be the triggering event for the development of ascites in animals with portal hypertension. 6 this is why aldosterone antagonists are the initial treatment of choice in ascites caused by portal hypertension (for more details see chapter 8). ascites is a negative prognostic indicator in dogs with chronic hepatitis, 7 although individual animals with chronic hepatitis and ascites can be managed and maintained for many months. with sustained portal hypertension, multiple acquired psss develop and serve as a conduit for portal blood flow directly into the systemic circulation. shunts serve to dissipate some of the increased portal pressure thus reducing the risk of adverse complications such as venous congestion, gi hemorrhage, and ulceration. they do, however, raise the risk of yet another complication of liver disease-hepatoencephalopathy. he is a syndrome of potentially reversible brain dysfunction resulting from impaired liver function. it results from either severe hepatocyte dysfunction or more commonly the presence of portosystemic collateral circulation, either congenital or acquired, where a variable combination of shunting of portal blood and hepatocyte dysfunction contributes to the clinical signs. it can be acute or chronic in presentation. acute he is most often a result of acute fulminating liver failure (see "consequences of hepatocyte and biliary tract injury" section) and carries a poor prognosis. more chronic he is usually a result of congenital or acquired psss. 2 platelet counts, d-dimers, fibrinogen, or protein c) in 24 (57%) of 42 dogs affected with liver disease. 18 coagulation abnormalities are also common in cats with liver disease and one study found abnormalities in 18 (82%) of 22 cats with liver disease. 19 multiple mechanisms of coagulopathy are possible in liver disease patients. in alf cases such as xylitol toxicity in dogs, 20 hl in cats, 21 and cirrhosis in dogs, 18 loss of normal hepatocyte function results in severe coagulation factor deficiency. vitamin k deficiency has also been implicated in coagulopathy particularly in cats in which cholestasis impedes bile salt secretion, emulsification, and micellarization of fat and fat-soluble vitamins and fat-soluble vitamin absorption. 21 concurrent inflammatory bowel disease and pancreatitis exacerbate this condition in many cats with cholangitis. 21 finally, platelet abnormalities (cytopenia and cytopathy) may contribute to coagulopathy in dogs with liver disease. 18, 22 the liver has the unique ability to regulate its growth and mass. hepatocyte loss caused by viral, bacterial, or chemical injury, or partial hepatectomy triggers hepatocyte replication. 23, 24 liver injury not only stimulates hepatocyte turnover but may also stimulate biliary proliferation and activation and proliferation of hscs. these changes usually occur together in an orchestrated wound-healing response. in the case of hepatocyte loss, normally quiescent hepatocytes replicate to restore the liver functional capacity and mass. these are the main cells that regenerate liver mass. however, in severe injury or where hepatocyte turnover is inhibited by senescence, a progenitor cell reserve may also replicate and regenerate liver mass. 24 although hepatocytes are capable of replication, they have very slow turnover in a normal liver and there are negative consequences of long-term increased stimulation and turnover in chronic liver disease. it has been shown that cycling hepatocytes suffer irreversible erosion of telomeres, which leads to senescence. 25 functional capacity is a relative rather than absolute parameter. the set point for growth regulation is the ratio between liver mass and body mass rather than liver mass per se. the optimization of the ratio indicates that the liver reaches a state in which it performs the amount of metabolic work needed to meet the functional requirements of the body. 24 gene expression in the regenerating liver is a multistep process with at least two critical steps: the transition of quiescent hepatocytes into the cell cycle ("priming"), and the progression beyond the restriction point in the g 1 phase of the cell cycle. hepatocytes must first be primed before they can fully respond to growth factors. as many as 70 different genes participate in the early response to hepatectomy, but tnf and il-6 appear to be the major cytokines involved in the priming of hepatocytes. 20 the proliferative effect of tnf on hepatocytes is further influenced by reactive oxygen species, nitric oxide, and glutathione content, and at least four transcription factors (nuclear factor kappa b, stat3, ap-1, and c/ebpβ) play major roles in the initiation of early liver regeneration. 23, 24 progression through the cell cycle beyond the initiation phase requires growth factors, primarily hepatocyte growth factor and tgf-α. the subsequent expression of cell-cycle genes, particularly cyclin d 1 , establishes the stage at which replication becomes growth factorindependent and autonomous. at this point, the hepatocyte is irreversibly committed to replicate and the cell-cycle replication machinery takes over. the regenerative capacity of the residual hepatocytes may restore liver mass and function after as much as 65% to 70% hepatectomy. 23, 24 progenitor cells are only activated in severe liver injury. they appear to reside in a "niche" that is a particular regulatory environment. 24 a recent immunohistochemical to urea by hepatocytes in the krebs-henseleit cycle. with portosystemic shunting or severe hepatocyte dysfunction, ammonia accumulates in the brain (and other tissues) where it is taken up by astrocytes and results in edema as described previously. dogs with he also show disturbances in cns aromatic amino acid metabolism. 11, 12 the aromatic amino acids (tyrosine, tryptophan, and phenylalanine) accumulate in, the cns in portasystemic shunting. in the brain, β-phenylalanine and tyrosine are metabolized to phenylethanolamine and octopamine, both of which can act as false neurotransmitters. however, dietary supplementation with branched-chain amino acids (e.g., leucine, isoleucine, valine) does not convincingly improve he in either dogs or humans. 9, 12 however, some dietary protein sources appear to be better than others in dogs with he. dogs on soya protein diets show a lower plasma ammonia concentration than those fed meat protein. 13 dogs with he have also traditionally been fed a protein-restricted diet. however, protein restriction is no longer advocated in humans with he 9 and it may be the digestibility and type of protein rather than a reduced amount that are most important in dogs. more studies are needed to investigate this. several other metabolic alterations exacerbate clinical signs associated with he, including acid-base disorders, electrolyte abnormalities, particularly hypokalemia, hypoglycemia, hypoxemia, and arginine deficiency (cats). an important trigger in humans and rodents is inflammation: recent studies confirm that inflammatory cytokines are synergistic with ammonia in precipitating he and that controlling inflammation in other organs is an important part of managing the patient with he. 14, 15 there is anecdotal evidence that this is also true in dogs. the liver has significant structural and functional reserve capacity to support ongoing metabolic needs during mild to moderate forms of liver injury. moreover, the liver has the ability to regenerate liver volume and cell mass during the recovery phase of most forms of liver injury. signs of liver failure develop earlier with acute forms of liver injury than with chronic, progressive liver disease. zones 1, 2, and 3 hepatocytes of the hepatic acinus have differing functions. zone 1 (periportal) hepatocytes, for example, have a high capacity to cycle ammonia through the urea cycle thereby reducing the toxicity of ammonia. zone 3 hepatocytes (nearer the hepatic vein) have a lesser capacity for ammonia and instead convert it to glutamine. 16, 17 in health, zonation permits flexibility in hepatic function such that in metabolic acidosis, for example, the liver can rapidly divert ammonia toward glutamine production, which is necessary for h+ ion excretion in the kidney. in severe acute liver injury, this becomes an important "tradeoff" because acute selective destruction of periportal (zone 1) hepatocytes more readily results in signs of encephalopathy because of the reduced ability of zone 3 hepatocytes to detoxify ammonia. in chronic liver disease, if hepatocytes undergo piecemeal necrosis at different rates in different zones, the remaining hepatocytes can assume some of those functions, so that clinical signs of deficiency are not seen until later in chronic disease processes. coagulopathy is a complication of both acute and chronic liver disease in dogs and cats. a recent study reported one or more coagulation abnormalities (prolongation of coagulation times, changes in the cause of chronic hepatitis is usually unknown. 30 there is increasing evidence that fibrosis and even some forms of cirrhosis in humans and rodent models are reversible if the underlying cause is removed. 28, 31 the challenges are removing the cause and also defining the point at which cirrhosis moves from a reversible to irreversible state. increased fibrous septal thickness, smaller nodule size, and reduced cellularity together with increased collagen cross-bridging have all been associated with an irreversible cirrhotic state in rodents and humans. 28 it is unknown whether liver fibrosis or cirrhosis in dogs is reversible clinically. cases of chronic hepatitis in dogs very rarely have sequential liver biopsies over a long period of time to assess progression of disease and noninvasive markers of fibrosis remain to be validated. serum hyaluronic acid is increased in dogs with cirrhosis 32 as is tgf-β 33 but the usefulness of these markers in following progression in clinical cases has not been assessed. identifying a reliable noninvasive marker of fibrosis for sequential studies in humans and dogs remains a challenge. 34 study suggests that canine and human liver progenitor cells are functionally very similar. 26 further characterization of the molecular events regulating hepatocyte replication and liver regeneration should improve outcome in animals affected with severe liver disease. the normal ecm of the liver provides cells with positional information and a mechanical scaffold for adhesion and migration. the ecm consists of collagens, glycoproteins, proteoglycans, glycosaminoglycans and molecules that are bound specifically by the ecm, such as certain growth factors, cytokines, matrix metalloproteinases, and processing enzymes such as tissue transglutaminase and procollagen propeptidases. a normal liver contains a very small amount of fibrous tissue as a percentage of its total mass. 27, 28 acute or chronic liver injury causes a dynamic wound-healing response with both production and removal of fibrosis. 29 hscs are the major source of the collagens that comprise fibrosis and cirrhosis, as well as of the tissue inhibitors of metalloproteinases (timps) that inhibit collagen degradation. it is the balance between collagen production by hscs and its degradation by matrix metalloproteinases that determines the severity and reversibility of the fibrotic response. following acute or chronic liver damage, hscs are stimulated to multiply and to undergo a complete phenotypic transformation from quiescent vitamin a-storing cells to contractile myofibroblasts which synthesize large amounts of ecm. 25, 29 important stimuli for hsc transformation and multiplication in liver injury include oxidative stress; chemokines including platelet-derived growth factor; vegf and tgf-β; adipokines; and parts of the innate immune system including toll-like receptor ligands. 29 the role of adipokines in stimulating fibrosis is increasingly being recognized in humans, where nonalcoholic fatty liver disease can lead to fibrosis and cirrhosis. they are produced by hscs themselves, as well as fat cells, and increased leptin and reduced adiponectin drive fibrosis. 29 their importance in dogs is unknown and it is also unknown whether vacuolar or fatty liver diseases progress to fibrosis in dogs, but these are important questions to answer in the future given the widespread occurrence of obesity in dogs. the contractile function of activated hscs contributes significantly to the development of portal hypertension, 28 and increases in angiogenic chemokines such as vegf and platelet-derived growth factor not only contribute to fibrogenesis by hscs, but also to the development of portal hypertension, so that the two pathologic processes are inextricably linked. 29 activated hscs have greatly increased production of timps, particularly timp1 and timp2, which prevent the action of matrix metalloproteinases in the ecm. the degree of fibrosis and reversibility then depend on the balance between perpetuation of hsc proliferation and secretion and resolution of hsc by either apoptosis or senescence of hscs or, indeed, their reversion to an inactive state. many factors contribute to hsc apoptosis, senescence, or reversion, including reduction in timps and nuclear factor kappa b and increased fas and p53. 29 however, in spite of all this understanding of the molecular mechanisms of fibrosis, a truly effective treatment for hepatic fibrosis in either humans or dogs has yet to be found. 29 it is important to remember that a normal fibrotic response (scar) is important in walling off pathogens and tissue injury and inhibiting this response without removing the inciting cause (e.g., a viral cause) could lead to spread of the pathology. 28 future treatment strategies for fibrosis should therefore incorporate treatment of the underlying cause of disease. this is clearly a problem in dogs where organizational principles of the liver bile formation and enterohepatic circulation functional and morphological relationships between the feline main pancreatic and bile duct sphincters the extrahepatic biliary tract in common domestic and laboratory animals hormonal regulation of bile secretion physiology of cholangiocytes hepatic stellate cell (vitamin a-storing cell) and its relative -past, 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comparisons with man comparative analysis of hepatocellular carcinoma in men and dogs inactivation of tgfbeta signaling in hepatocytes results in an increased proliferative response after partial hepatectomy mdm2 and p53 polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis b virus infection epidermal growth factor-induced hepatocellular carcinoma: gene expression profiles in precursor lesions, early stage and solitary tumours identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma withrow and macewen's small animal clinical oncology status epilepticus after ligation of portosystemic shunts progressive remission of portosystemic shunting in 23 dogs after partial closure of congenital portosystemic shunts gradual occlusion of extrahepatic portosystemic shunts in dogs and cats using the ameroid constrictor methods of gradual vascular occlusion and their applications in treatment of 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partial ligation of, or ameroid ring constrictor placement on, the left hepatic vein in twenty-eight dogs laparoscopic portosystemic shunt attenuation in two dogs transvenous coil embolization of portosystemic shunt in dogs transjugular coil embolisation of an intrahepatic shunt in a cat complications associated with the diagnostic, medical, and surgical management of portosystemic shunts portal vein thrombosis as a complication of portosystemic shunt ligation in two dogs neurologic dysfunction in dogs following attenuation of congenital extrahepatic portosystemic shunts neurologic dysfunction in three dogs and one cat following attenuation of intrahepatic portosystemic shunts use of propofol to manage seizure activity after surgical treatment of portosystemic shunts portal hypertension part ii: clinical assessment and treatment diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats magnetic resonance imaging of focal splenic and hepatic lesions in the 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discrimination tests in dogs with hyperadrenocorticism effects of nonadrenal disease on the results of diagnostic tests for hyperadrenocorticism in dogs. (abstract) plasma cortisol response to exogenous acth in 22 dogs with hyperadrenocorticism caused by adrenocortical neoplasia evaluation of the hypothalamic pituitary-adrenal axis in clinically stressed dogs urinary cortisol in the assessment of pituitary-adrenal function: utility of 24-hour and spot determinations urine cortisol:creatinine ratio as a screening test for hyperadrenocorticism in the dog evaluation of a urine cortisol:creatinine ratio as a screening test for hyperadrenocorticism in dogs spontaneous hepatic rupture in six cats with systemic amyloidosis severe cholestatic liver disease secondary to liver fluke (platynosomum concinnum) infection in three cats destructive cholangiolitis in seven dogs suspected druginduced destructive cholangitis in a young dog primary cholangiohepatitis in a dog cholangiohepatitis in a dog gallbladder aspirate from a dog bacterial cholangitis/ cholangiohepatitis with or without concurrent cholecystitis in four dogs concurrent bacterial cholecystitis and cholangitis in a diabetic spitz dog clinical-pathologic conference congenital cystic disease of the liver in seven dogs congenital dilatation of the bile ducts (caroli's disease) in young dogs congenital dilatation of the large and segmental intrahepatic bile ducts (caroli's disease) in two golden retriever littermates polycystic disease of the kidney and liver in the cairn terrier polycystic kidney and liver disease in two related west highland white terrier litters polycystic kidney and liver disease in cats autosomal dominant polycystic kidney disease in persian and persian-cross cats congenital biliary atresia in a cat: a case report 48. van den ingh ts, rothuizen j, van den brom we: extrahepatic cholestasis in the dog and the differentiation of extrahepatic and intrahepatic cholestasis cholestasis associated with extrahepatic bacterial infection in five dogs morphological classification of neoplastic disorders of the canine and feline liver canine hepatic neoplasms: a clinicopathologic study the pathology of liver tumors in the dog canine biliary carcinoma: epidemiological comparisons with man a morphologic and immunohistochemical study of hepatic neoplasms in cats nonhematopoietic hepatic neoplasms in cats: 21 cases (1983-1988) relationship between inflammatory hepatic disease, inflammatory bowel disease, pancreatitis and nephritis in cats pathogenesis and outcome of extrahepatic biliary obstruction in cats a survey of feline inflammatory hepatobiliary disease using the wsava classification clinical features of inflammatory liver disease in cats: 41 cases (1983-1993) correlations between ultrasonographic findings and specific hepatic diseases in cats: 72 cases (1985-1997) diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats comparison of liver cytology and biopsy diagnoses in dogs and cats: 56 cases accuracy of ultrasound-guided fine needle aspiration of the liver and cytologic findings in dogs and cats: 97 cases percutaneous ultrasound-guided cholecystocentesis in healthy cats bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease: 1998-2003 prospective evaluation for bacterial infection in hepatic tissue and bile of cats with diffuse hepatobiliary disease cholecystoenterostomy for treatment of extrahepatic biliary tract obstruction in cats: 22 cases treatment of pancreatitis-associated extrahepatic biliary tract obstruction by choledochal stenting in 7 cats advances in characterization of feline nonsuppurative cholangitis/cholangiohepatitis syndrome characterization of portal lymphocytic infiltrates in feline liver progressive lymphocytic cholangitis in the cat immunohistochemical characterization of the lesions of feline progressive lymphocytic cholangitis/cholangiohepatitis detection of helicobacter pylori in bile of cats association of helicobacter with cholangiohepatitis in cats feline clinical parasitology liver flukes (platynosomum concinnum) in cats cholangiohepatitis and choledochectasia associated with amphimerus pseudofelineus in a cat platynosomum concinnum infection in cats congenital dilatation of the bile ducts (caroli's disease) in young dogs ultrasonographic evaluation of gallbladder wall thickness in cats therapeutic percutaneous ultrasound-guided cholecystocentesis in three dogs with extrahepatic biliary obstruction and pancreatitis choledochal tube stenting for decompression of the extrahepatic portion of the biliary tract in dogs: 13 cases extrahepatic biliary tract surgery in the cat: a case series and review surgical management of gallbladder mucoceles in dogs: 22 cases (1999-2003) chronic bile duct ligation in the dog: hemodynamic characterization of a portal hypertensive model important clinical syndromes associated with liver disease esophageal varices due to a probable arteriovenous communication in a dog portal hypertension associated with primary hypoplasia of the hepatic portal vein in dogs idiopathic noncirrhotic portal hypertension in dogs: 33 cases (1982-1998) fluid retention in cirrhosis: pathophysiology and management ascites is a negative prognostic indicator in chronic hepatitis in dogs pathogenetic mechanisms of hepatic encephalopathy dispelling myths in the treatment of hepatic encephalopathy gut ammonia production and its modulation cerebrospinal fluid glutamine, tryptophan, and tryptophan metabolite concentrations in dogs with portosystemic shunts effects of a branched-chain amino acid-enriched diet on chronic hepatic encephalopathy in dogs soy protein isolate versus meat-based low-protein diet for dogs with congenital portosystemic shunts ammonia and the neutrophil in the pathogenesis of hepatic encephalopathy in cirrhosis changing face of hepatic encephalopathy: role of inflammation and oxidative stress regulation of hepatic ammonia metabolism: the intercellular glutamine cycle nonlymphomatous hepatobiliary masses in cats: 41 cases (1972-1991) biliary cystadenomas of cats ultrasonographic evaluation of biliary cystadenomas in cats partial hepatectomy in two dogs extrahepatic biliary tract obstruction: a retrospective study of 45 cases (1983-1993) ultrasonographic appearance and clinical findings in 14 dogs with gallbladder mucocele gallbladder mucocele in dogs: 30 cases cholelithiasis in dogs: 29 cases (1980-1990) severe cholestatic liver disease secondary to liver fluke (platynosomum concinnum) infection in three cats pathogenesis and outcome of extrahepatic biliary obstruction in cats cholecystoenterostomy for treatment of extrahepatic biliary tract obstruction in cats: 22 cases bacterial culture results from liver, gallbladder or bile in 248 dogs and cats evaluated for hepatobiliary disease bacterial cholangitis/ cholangiohepatitis with or without concurrent cholecystitis in four dogs association of helicobacter with cholangiohepatitis in cats long-term survival and risk factors associated with biliary surgery in dogs: 34 cases surgical treatment of 23 dogs with necrotizing cholecystitis canine gallbladder infarction: 12 cases surgical treatment of cholelithiasis in cats: a study of nine cases gallbladder disease in shetland sheepdogs: 38 cases porcelain gallbladder associated with primary biliary adenocarcinoma in a dog correlation between hepatobiliary scintigraphy and surgery or postmortem examination findings in dogs and cats with extrahepatic biliary obstruction, partial obstruction, or patency or the biliary system: 18 cases prevalence of gallbladder sludge in dogs as assessed by ultrasonography percutaneous ultrasound-guided cholecystocentesis in healthy cats cross-species immunohistochemical investigation of the activation of the liver progenitor cell niche in different types of liver disease is liver fibrosis reversible now there are many (stages) where before there was one: in search of a pathophysiological classification of cirrhosis evolving challenges in hepatic fibrosis chronic hepatitis in dogs: a review of current understanding of the aetiology, progression, and treatment reversibility of liver fibrosis blood hyaluronic acid as a marker for canine cirrhosis transforming growth factor beta-1 signalling in canine hepatic diseases: new models for human fibrotic liver pathologies therapeutic targets in liver fibrosis structural-functional organization of hepatic glutamine and ammonium metabolism coagulation disorders in dogs with hepatic disease coagulation abnormalities in 22 cats with naturally occurring liver disease acute hepatic failure and coagulopathy associated with xylitol ingestion in eight dogs proteins invoked by vitamin k absence and clotting times in clinically ill cats correlation between coagulation profile findings and bleeding complications after ultrasoundguided biopsies: 434 cases (1993-1996) liver regeneration new concepts in liver regeneration defining therapeutic targets for liver fibrosis: exploiting the biology of inflammation and repair key: cord-000083-3p81yr4n authors: nan title: poster exhibition date: 2009-01-31 journal: hepatol int doi: 10.1007/s12072-009-9123-4 sha: doc_id: 83 cord_uid: 3p81yr4n nan background: biliary atresia (ba) is one of the most common causes of neonatal cholestasis and the most frequent hepatic cause of death in early childhood. the incidence rate of ba is higher in asian countries, occurring in approximately 1 of 8,000 (asian countries ) to 1 of 18,000 (european countries) live births. early identification and prompt intervention is very important. to im prove the early diagnosis, we used proteomic technology to screen serum biomarker for ba. methods: two-dimensional electrophoresis (2-de) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (maldi-tof-ms) were employed to screen serum biomarkers specific to ba sera from idiopathic neonatal hepatitis. after pretreatment including albumin and immunoglobulin (igg ) depletion, sera were subjected to 2-de and there after image analysis. the differentially expressed protein spots were identified by maldi-tof-ms. result: from optimized 2-de gel images, thirty-four spots were differentially expressed and identified by maldi-tof-ms to be eight proteins. overall, kininogen 1 variant was under expressed and alpha-1-b-glycoprotein, leucine-rich alpha-2-glycoprotein 1, 'sp40,40', a1bg protein, vitamin d-binding protein/group specific component , apolipoproteina-, aqgv 3103 were over expressed in ba group com pared to idiopathic neonatal hepatitis. conclusion: 2-de based serum proteome analysis can be useful in detecting protein expression alteration and new discovered biomarkers might be an aid in the diagnosis of ba, though further validation is needed. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 ims, bhu, varanasi, india background: a variety of autoreactive antibodies are detected in patients with chronic liver disease. this prospective, nonrandomized study was undertaken to evaluate the nature & prevalence of various autoantibodies in patients with chronic liver disease of diverse etiologies. methods: study population included 53 patients (75% males), who met defined criteria for chronic liver disease. detailed clinical, laboratory and sonographic evaluation was done. sera were tested for asma, anti-lkm type1, ama, apa, ana, by standard methods. p<0.05 was considered significant. results: among various etiologies for chronic liver disease, hepatitis b was most common (28%), followed by alcohol (19%), autoimmune hepatitis in 15%, hepatitis c (6%) and miscellaneous (2%). 30% of patients were labeled as cryptogenic after detailed investigations. ana (>1/80) was positive in 100% of definite aih, 33% of hcv related cld but at titer of >1/40, 66.6% of hcv related cld & 60% of probable aih were found positive. asma (>1/40) was positive in 6% of hbv related cld, 10% of alcohol related cld, 33% of definite aih, 40% of probable aih, 33% of hcv related cld but asma in titer of >1/80 was positive only in 33% oh definite aih. apa was detected in 12.5% of cryptogenic cld, 13.3% of hbv related cld & 20% of alcohol & probable aih related cld each. ama was detected in 1% of cryptogenic, hbv, aih (definite) & hcv related cld each, and 2% of alcohol related cld & 100% of pbc. conclusions: apart from aih there is high prevalence of ana & sma in hcv related cld while other antibodies has low prevalence in non-aih related clds. this study also suggests that prevalence of various autoantibodies should be borne in mind while considering the diagnosis of cld especially of mixed etiology. conclusions: oa infusion did not lower ammonia levels or improve survival. results: the mortality (50.0%) of patients in lamivudine group with meld score from 30 to 40 was lower than that (86.1%) of control group ( 2 =23.319, p=0.000). univariate analysis showed that mortality was significantly related to age (p=0.005), meld score (p=0.009), treatment method (p=0.000), pretreatment hbv dna load (p=0.000), the decline of hbv dna load during therapy (p=0.006) and encephalopathy (p=0.007). in multivariate analysis, in patients with meld scores 30-40, treatment method (p=0.004), pretreatment hbv dna load (p=0.009), decline of hbv dna load during therapy (p=0.014) and encephalopathy (p=0.019) were independent predictors of mortality; for meld scores above 40, only meld score (p=0.015) was independent predictive. conclusions: lamivudine treatment significantly decreases the 3 month's mortality of patients with meld score 30-40, and a low viral load pre-treatment and quick decline of hbv dna load are good predictors for the survival of lamivudine treatment. background/aims: early identification of patients with fulminant hepatic failure (fhf) who need a liver transplantation is very important. to construct a prediction model for early diagnosis and prognosis of fhf, we studied dynamics of metabolic profiles using a d-galactosamine/lipopolysaccharide (galn/lps)-treated mouse model. methods: balb/c mice were used to construct fhf model and sacrificed for blood collection at 4, 5, and 6 hour after treatment, respectively. levels of plasma metabolites were quantified using gas chromatography/time-of-flight mass spectrometry and data were processed using partial least squares discriminant analysis (pls-da). results: distinct clustering differences were observed 5 and 6 h after treatment between survival and dead groups. at 5 h, plasma levels of some metabolites differed significantly between survival, dead and control groups. ketogenesis and the tca cycle were inhibited in both survival and dead groups, but in dead group, the urea cycle was also inhibited and glycolysis was elevated. pls-da indicated that principal component weighting was greatest for plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate. the y-predicted scatter plot in pls model assigned samples to survival or dead groups using an apriori cutoff of 0.10 with 100% sensitivity and specificity. similar results were observed in 11 fhf patients with different outcomes. pe012 association between polymorphisms in the interleukin-10 gene promoter and hepatitis b-related acute liver failure conclusions: the pls model based on metabonomics analysis can be used to predict outcomes well, and plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate may constitute a set of markers for early diagnosis and prognosis of fhf. il-10 promoter are associated with the susceptibility to hepatitis b-related alf in the chinese population. il-10 a-592c may be a regulatory polymorphism that affects gene regulation. hepatocyte cell death in aclf: mechanism and significance -an immunohistochemical study. p. sakhuja 1 , a. rastogi 2 , s. s hissar 1 , a. singh 1 , a. kumar 2 , r. gondal 1 , s.k. sarin 1 1 gb pant hospital, 2 institute of liver and biliary sciences, new delhi, india background: acute on chronic liver failure (aclf) is defined as acute hepatic insult complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. caspases play an essential role in apoptosis. cox-2 is an inducible immediate early gene responsible for the release of prostaglandins during inflammatory response. we studied the immunohistochemical expression of cox-2 and caspase-1 in liver tissue to assess their role in pathophysiology and in predicting outcome of aclf. method: a retrospective analysis of 50 liver biopsies with clinical diagnosis of aclf was undertaken. patients were divided into two groups a and d based on clinical outcome (alive/died respectively). immunohistochemical analysis for cox-2 and caspase was performed on 39 and 36 cases respectively and scored from 0-8 as per intensity and distribution. score 6-8 indicated high intensity with focal to diffuse distribution, and was considered significant. results: etiology of acute liver failure was viral or alcoholic. increased expression of caspase was observed in 10/21 cases in group d and none of the cases in group a(n=15) (p=0.001). increased expression of cox-2 was observed in 4/21 cases in group d and none of the cases in group a (n=18) (p=0.052). conclusion: increased immunoreactivity of caspase in liver biopsies of patients of aclf may indicate worse prognosis and its important role in the pathophysiology of aclf. immunostaining for caspase is useful for assessment of prognosis and possibility of anti-apoptotic and anti-fibrotic therapies in future. conclusion: hhgf expression vector (pcmv-hhgf) has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hhgf in vivo. j.h. kim 1 , k.w. kim 1 1 asan medical center, seoul, korea background: splenic artery embolization (sae) is performed to increase hepatic arterial flow or to decrease portal venous flow in recipients of liver transplantation (lt). thus, the purpose of this study was to estimate sae effect on the basis of changes in caliber of related vessels and splenic volume on pre-sae and serial post-sae ct scans in lt recipients. methods: between 2003 and 2007, among 73 lt recipients who underwent sae and serial follow-up ct, 43 with no compounding factor that may obscure sae effect were included in this study. they underwent ct before and after (1week, 1month, and 1year) sae. a radiologist retrospectively measured diameters of ca, cha, sa, sv and splenic volume on serial ct scans. their diameters and splenic volume on each ct were compared with those on the prior and pre-sae ct. the difference was compared using repeated-measures anova tests. results: cas decreased between 1week and 1month after sae (p<.05), but were stable before 1week and after 1 month. chas increased within 1week (p<.05) but decreased between 1week and 1month (p<.05) and remained stable after 1month. compared with pre-sae ct, chas were larger for 1month after sae. sas continuously decreased for 1year (p<.05). svs decreased for 1 month (p<.05) and remained stable after 1 month. compared with pre-sae ct, sas and svs were smaller from 1week after sae and on. splenic volume continuously decreased for 1year except a period between 1week and 1month. conclusion: the increase of hepatic arterial flow persists for 1month after sae, but returns to baseline thereafter. the decrease of portal flow may lasts for at least 1year after sae. poster exhibition -cholangioca and other liver neoplasm poster session, hall 5b background: now, rfa has becoming an important practice of hcc therapy. in this study, we evaluated whether rfa therapy for metastatic liver tumor has a beneficial effect on patients' survival. methods: forty six patients were treated by rfa for metastatic liver tumor from july 2001 through february 2008 in our hospital, of the 46, 33 patients were metastasis either form colon or stomach cancer. these 33 patients were analyzed in this investigation. cumulative survival rate from initial rfa therapy was calculated by kaplan-meier method. predictive factors for survival were identified using cox proportional hazard regression model. results: the mean age of the 33 patients were 64. 6 (range, 40-79) . the mean size of the tumor is 28mm (range,8-70mm) and the numbers of tumor foci are 2.7 nodules range,1 18. the survival rates of patients treated by rfa were 49.5% at 3 years and 31.8% at 5 years in colon cancer, 15.6% at 3 years and 15.6% at 5 years in gastric cancer. in this series of 33 patients, primary cancer: colon (p=0.002 odds ratio 0.132 95%ci 0.037-0.473), younger patients ( 64) (p=0.041 odds ratio 0.312 95%ci 0.102-0.955) and multiagent chemotherapy (p=0.012 odds ratio 0.223 95% ci 0.069-0.723) were significantly correlated with better survival. conclusion: the survival of patients treated by rfa for metastatic colon cancers had better survival than those of gastric cancers. in addition, good indication of rfa is for metastatic colon cancers, younger patients and has to be treated by multiagent chemotherapies. utility of contrast enhanced ultrasonography with sonazoid in radiofrequency ablation (rfa) for liver metastasis e. goto 1 , s. shiina 1 , r. tateishi 1 , r. masuzaki 1 , k. enooku 1 , t. sato 1 , j. imamura 1 , t. goto 1 , y. sugioka 2 , h. ikeda 1,2 , h. yoshida 1 , m. omata 1 1 department of gastroenterology, university of tokyo, 2 department of clinical laboratory, tokyo, japan background & aims: contrast enhanced ultrasonography (ceus) with sonazoid is effective for liver metastasis because enhance defect in kupffer imaging is well delineated. the aim of this study is to investigate the detection ability of ceus and the utility of sonazoid in rfa for metastasis liver tumors. material & methods: from january 2007 to december 2007, a total of 346 liver metastatic nodules in 87 patients (62 colon cancer, 13 breast cancer, 3 gastric cancer, 3 islet cell tumor, and 6 others) admitted to receive rfa were studied. the detection ability of liver metastasis was compared between ceus and conventional us using enhanced ct as reference standard. the mean numbers of treatment session of rfa were compared between patient treated with ceus assistance and historical controls matched for size and number of tumors. results: the detection rate was 78.6% with conventional us and 96.4% with ceus (p=0.0004). 83 nodules in 25 patients were not detected by conventional us and detected after injection of sonazoid. in addition, 12 nodules in 2 patients were detected not by ct but only by ceus. the mean number of session was 1.5±0.5 as compared to 2.0±1.0 in the historical controls (p<0.001). conclusions: ceus with sonazoid is useful for detection of liver metastasis. sonazoid is an excellent supportive agent in rfa of liver metastasis. background/aims: carcinogenesis of intrahepatic cholangiocarcinoma (icc)-associated liver fluke infection accumulated genetic and epigenetic alterations. cholangiocarcinoma cell line (kku-m213) is adenosquamous carcinoma which rare variants and not commonly found in icc. however, interactions of liver fluke-associated icc proceed to genetic alterations in adenosquamous carcinoma that have been not elucidated. objectives: to analyze the whole genome-wide genetic alterations in kku-m213 using microarray comparative genomic hybridization. methods: dna of kku-m213 and matched-sex reference were differentially labeled with fluorescence dries (cy3 and cy5) and mixed together with cot-1 dna. the mixture was hybridized on array with spotting 2,464 human bacterial artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13.1, 4q21.1, 4q21.2, 5p tel, and 5p15.3, whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3. conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13. 1, 4q21.1, 4q21.2, 5p tel, and 5p15.3 , whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3 . conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. acknowledgements: this work was supported by faculty of medicine, kku, thailand (grant no. i51117 background/aims: we studied the clinical efficacy of arterial chemoinfusion therapy through an implanted port system for patients with intrahepatic cholangiocarcinoma (icc). thirty patients with unresectable icc or intrahepatic recurrence of icc after surgery were studied. comparison was made between patients who received arterial chemoinfusion therapy through an implanted port system with adriacin and lecithin-added lipiodol emulsion in 5 patients and 5-fluorouracil (5-fu) in 7 patients. eighteen patients were treated without port system. results: disease was stable in 5 patients with adriacin and lecithin-added lipiodol emulsion and in 3 patients with 5-fu. disease was progressed in 4 patients with 5-fu. the mean survival period was 20.8 months in patients with adriacin and lecithin-added lipiodol emulsion, 9.3 months in patients with 5-fu, and 10.5 months in patients without port system (p=0.02, p=0.04). conclusion: arterial chemoinfusion therapy through an implanted port system is useful for patients with intrahepatic recurrence of icc after surgery. pe038 s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh. india background: a number of dietary factors have been involved in the pathogenesis of cholelithiasis. cholesterol overfeeding is the primary means of inducing supersaturated bile and cholesterol gallstones in animal models.aim of the study was to investigate the rate of epithelial cell death and proliferation in gallbladder during gallstones formation. methods: balb/c mice was divided into two groups control in this group animals were fed normal chow diet, high fat diet group in this group (2% cholesterol,0.5% sodium cholate, 5% butter fat and 15% coconut oil) mixed with chow diet was fed to the mice for 4 weeks. cell apoptosis and proliferation was assayed in gallbladder epithelial cells. histological analysis of gallbladder sections were done with hematoxylin and eosin staning. results: mice fed high fat diet had apoptotic as well as necrotic epithelial cells. rate of proliferation was enhanced after 24 and 48 hrs in mice fed high fat diet group as compared to the control group. the histopathological section of control gallbladder has normal morphology whereas gallbladder wall thickness was markedly increased; epithelial cells appeared more elongated in mice fed high fat diet. conclusion: results obtain show that high fat diet markedly induced biliary epithelial cell proliferation and biliary epithelial cell apoptosis. it has been determined that when there is an injurious stimulus that leads to apoptosis, it is later followed by reparative proliferation and when there is no injurious stimulus, apoptosis occurs late in the course as part of remodeling. background: obstructive jaundice can be caused by malignancy. the treatment can be drainage by biliary stenting. in advanced malignant jaundice, the stent placement is often difficult. objective: to evaluate the success rate of malignant obstructive jaundice evaluation of ercp and success rate of stent placement. methods: retrospective study based on data of ercp from october 2004 until july 2008. results: we evaluated 139 patients who has done ercp examination, 131 (94,2 %) patients have clinical diagnosis of obstructive jaundice. there were 73 (55,7%) male and 58 (44,3%) female, age range 20 -84 (median age was 51). there were no malignancy in 66 (50,4 %) patients; malignancy in 48 (36,6%) patients and 17 (13%) patients need further evaluation.. from 114 patients, 57 (50 %) patients attempted to have stent placement, 50 (43,9 %) patients do not and 7 (6,1 %) patients have no data. we done descriptive study on 57 patients attempted to have stent placement, 32 (56,1 %) patients succeed in stent placement whereas 25 (43,9 %) failed. malignancy was showed to be a factor of stent failure (malignancy: 23 fail and 10 success (30,3 %) vs non malignancy: 2 fail and 22 success (91,7%)). conclusion: ercp can identify the cause of obstructive jaundice in 87 % patients. the success rate of stent placement was 56,1 %. the success rate of biliary stenting in malignant obstructive jaundice was 30,3 % whereas in non-malignant cases was 91,7 %. papillary carcinoma was the most frequent cause of malignant obstructive jaundice. background: in hydatid disease of the liver cystobiliary fisula (cbf) constitutes an entity characterized by the occurrence of a life-threatening cholangitis with increased morbidity. aim: to study the different diagnostic and therapeutic aspects of cystobiliary fistula in hydatid disease of the liver. patients and methods: fourteen patients with complicated cysts were divided into 2 groups; group a: nine patients presented with cholangitis, and group b: five patients had history of jaundice. in all patients, the diagnosis of cbf was confirmed by erc (endoscopic retrograde cholangiography). preoperative endoscopic sphincterotomy (es) was done in group a with retrieval of hydatid daughter cysts. seven patients (subgroup a1) were subsequently submitted to surgery entailing endocystectomy in 5 and hepatic resection in two. the remaining 2 patients in group a (subgroup a2), were managed by endoscopic therapy only. patients of group b (n=5), were not submitted to preoperative es and were subsequently managed by hepatic resection in one patient and endocystectomy in four. results: there was no mortality in the studied group. postoperative bile leak occurred in four cases in group b. in contrast, none of the patients who were submitted to preoperative es (subgroup a1) had bile leak. all patients received albendazole treatment. conclusion: erc is important in confirming the diagnosis of cbf. also, therapeutic erc has a place in the treatment algorithm of cbf as it was found to be a safe and a reliable therapeutic alternative especially in high risk patients for surgery. v. singh 1 , g. singh 1 , g.r. verma 1 , v. gupta 1 , s. ghosh 1 , r. gupta 1 , r. kapoor 1 , n. sharma 1 , a. bhalla 1 , s.k. mahi 1 1 background: endoscopic palliation in malignant hilar biliary obstruction requires ercp. however, contrast injection leads to cholangitis. recently, contrast-free metal stenting with or without mrcp has shown encouraging results. however, mrcp and metal stents are costly. there have been no reports on the use of air cholangiography in these patients. methods: we prospectively studied the role of air cholangiogaphy assisted unilateral plastic stenting in these patients. results: ten patients with unresectable malignant hilar biliary obstruction were studied. air cholangiography detected type ii obstruction in 8 and type i in 2 patients which is similar to mrcp. all patients underwent unilateral plastic stenting. a successful endoscopic drainange was achieved in 100% patients. cholanngitis occurred in none and there was no 30-day mortality. no major complications were observed. conclusion: air cohlangiography assisted plastic stenting in these patients is a safe and effective method of palliation. however, it requires a larger study. introduction: a description of igg4-related sclerosing cholangitis (igg4-sc) without pancreatic lesion has recently been reported. in addition to imaging, diagnosis relies on findings of elevated serum igg4 and immunodetection of invading igg4-positive cells. here we report a case of igg4-sc with only slight common bile duct abnormalities and normal pancreatic findings. case study: a 65-year-old man suffering from cephalalgia, general malaise and muscle ache was admitted to our hospital. his blood examinations on admission revealed eosinophilia, mild anemia, liver dysfunction and an igg level of 2820 mg/dl (igg4 374 mg/dl). although ercp did not reveal typical stenosis or irregularities of the bile duct wall, visualization of peripheral bile ducts was slightly impaired. echography revealed thickening of the intrahepatic bile duct and gallbladder walls as well as adenopathy. due to a gradual increase in pleural effusion and a progression of anemia, oxygenation was begun on the seventh day of illness. based on the combination of eosinophilia, elevated serum igg4 levels, image findings and a negative result for helminth, igg4-sc was suspected. liver biopsy was performed on the ninth day of illness and steroid therapy was initiated, after which symptoms and laboratory findings improved. the igg4-positive plasmocytic infiltrate present around the portal region at the time of biopsy disappeared within eight months of treatment. summary: this case displayed two unusual features that are not generally observed with igg4-sc: complications due to hemolytic anemia, and destruction of the peripheral bile duct with little damage to the common bile duct. introduction: various systemic diseases have been reported to be associated with igg4. although steroids are effective in the treatment of igg4-related diseases, there are some reports on relapses with their treatment, and cases are often difficult to differentiate from malignant diseases. we encountered a case of autoimmune pancreatitis with sclerosing cholangitis (aip-sc), in whom ca19-9 was elevated with episodes of exacerbation and an elevated serum igg4 concentration. igg4 staining was also useful for the diagnosis. case study: an 81-year-old woman noticed tumors beneath the bilateral jaw and was found to have an elevated level of ca19-9 (304) seven years previously. her left submandibular gland was removed and diagnosed as sclerosing sialadenitis. four years previously, she was diagnosed as having diabetes mellitus complicated by a recurrence of ca19-9 (419) elevation and liver dysfunction. cholangiocarcinoma was suspected based on ercp, but was not confirmed by histologic findings of bile duct biopsy. elevated igg4 and other test results established the diagnosis of aip-sc, so steroid therapy was initiated, after which symptoms and laboratory findings improved. this recurrence of ca19-9 elevation (634) was diagnosed as a relapse of aip-sc based on an increased igg4 level and histologic findings. summary: some papers have reported that igg4-positive cells are found in liver tissue in this disease, but such cells were not detected in the liver specimens in our case. this might be because intra-liver sites may have differed in the degree of morbidity, and long-term steroid therapy might have suppressed inflammation in the liver tissue. s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh, india background: cholelithiasis, a gallstone disease is major cause of morbidity affecting millions of people throughout the world. aim of the present study was to investigate the predisposing factors that lead to the formation of gallstones. methods: the study was carried out on gallstones, bile and serum of patients. gallstones and bile were divided into three groups' cholesterol, pigmented and mixed gallstones. blood of the patients was divided into two groups with gallstones and without gallstones patients. trace elements and various biochemical estimations were carried out. clinical history of the gallstones patients was recorded from the hospital records. results: trace elements analysis in bile and gallstones showed that calcium is the main element in all the three types of stones. iron was the main element in mixed gallstones. in pigmented gallstones magnesium and zinc were the major trace elements. liver function tests and lipid peroxidation levels in sera were significantly increase whereas, antioxidant enzymes concentrations in sera were significantly decreased in patients with gallstones. clinical history of the gallstones revealed the cases had jaundice, diabetes mellitus and estrogen replacement therapy respectively. conclusion: results suggest that trace elements in gallstones and bile as well as clinical history of patients with chronic cholelithiasis could be the underlying factor in the pathogenesis of gallstones. the concentration of products derived from the free radicals reactions increases with degree of inflammation. such a condition increases risk of bile saturation which would further contribute to the progress of gallstones formation. background and aims: diseases of the biliary tree and gallbladder are being described with increasing frequency among patients with the acquired immunodeficiency syndrome (aids).therefore there is a need to do a research about the risk factors of gallbladder diseases in hiv/aids patients. so it can be useful to clinicians to predict the possibility of a patient having gallbladder disease and consider the options of further plans. the aim of this study was to find the prevalence and varieties of gallbladder diseases in hiv/aids patients. methods: a cross sectional study was performed in patients with hiv/aids who visited ciptomangunkusumo hospital, jakarta. the risk factors (route of transmision,cd4,arv,hepatitis) and clinical presentations were studied.ultrasonography examinations were performed to detect gallbladder annormalities. results: 68 patients with hiv/aids match the study criteria. there were gallbladder abnormalities in 22 (32.4%) subjects, which 19 (27.9%) had acalculous cholecystitis and 3 (4.4%) had cholecystitis with cholelithiasis. on bivariate analysis, there was a significant association between abdominal pain, jaundice and the use of arv to gallbladder abnormalities (p = 0.000; 0.000; 0.004; 0.012). however, there was no association between age, sex, transmision route of hiv, hepatitis and cd4 to gallbladder abnormalities. conclusion: hiv/aids patients are susceptible to opportunistic gallbladder infection. acalculous cholecystitis is the most frequently encountered gallbladder abnormalities of hiv/aids patients in this study. poster exhibition -hbv poster session, hall 5b long-term stopping therapy t.b. trung 1 , p.h. phiet 1 1 university medical center, hochiminh city, vietnam background: among the approved nucleos(t)ide analogues therapies for chronic hepatitis b, lamivudine was used widely, sometime inappropriate in practice due to high safe and low price but lamivudine is associated with the highest rate of drug resistance. objectives: the aim of the study was to determine the ymdd variants after long-term stopping treatment in lamivudine-resistant patients using more sensitive technique. methods: 16 blood samples from lamivudine resistant patients were collected after long-term stopping therapy. the ymdd variants are detected using technique pcr restriction fragment length polymorphism (pcr-rflp) at hcmc university medical center results: after stopping lamivudine treatment 25 months (6-72 months) ymdd mutants were detected in 14 (87,5%) of 16 patients. among them 13 (92.9%) had the most important m204v/i mutant, 1(7 1%) had accompanying l180m mutant. it means that once drug resistant mutants have been selected, they are archived for the long time even if treatment is stopped. many of patients have the features characterized for the patients in immune tolerance phase (young age, hbeag positive, normal alt). the treatment of this group is not strongly recommended due to low efficacy and high risk of drug resistance. conclusion: the most important m204v/i mutant was still detected with significant portion of the virus population after long-term stopping therapy in lamivudine resistant patients. the options of retreatment for this patients when necessary are limited due to cross-resistance. the management of chronic hepatitis b should be followed strickly the recommendations of specialized association to avoid this problem. background/aim: whether liver stiffness measurement (lsm) using transient elastography is reliable to assess liver fibrosis in the settings of severe acute exacerbation of chronic hepatitis b (chb) is uncertain. methods: we prospectively recruited consecutive patients with severe acute exacerbation of chb (alanine aminotransferase or alt >10x upper limit of normal). the relationship of alt levels and lsm were serially assessed and liver biopsy was performed after alt normalization. results: eleven patients (10 male, median age 43 years) were followed up for 25 weeks; 9 patients received anti-viral therapy. overall, lsm was positively correlated with alt levels (r=0.67, p<0.001). at initial presentation, the median serum alt and lsm was 1136 (581-2210) iu/l and 26.3 (11. 1-33. 3) kpa. a progressive reduction in lsm was observed during subsequent visits in parallel with the reduction of alt levels. even after the normalization of alt at week 12, lsm of 9 patients continued to drop at week 25. at the last visit, the median alt was 27 (11-52) iu/l and lsm was 7.7 (4.7-10.8) kpa. among the 5 patients who had liver biopsy performed at week 25, 4 patients had f2 fibrosis (lsm 5.7-8.1 kpa) and 1 patient had f3 fibrosis (lsm 8.6 kpa). conclusions: lsm using transient elastography may misdiagnose liver cirrhosis in patients suffering from severe acute exacerbation of chronic hepatitis b. lsm should be assessed after normalization of alt levels in order to accurately assess the degree of fibrosis. h.c. lai 1 , s.w. lai 1 , k.f. liao 1 , c.s. liu 1 , t. lin 1 , c.c. lin 1 1 china medical university hospital, taichung, taiwan background: in 2007, chronic liver disease was the seventh leading cause of death in taiwan. hepatitis b and hepatitis c are two major causes of chronic liver disease in taiwan. the purpose was to investigate the seroepidemiology of hepatitis b surface antigen (hbsag) and hepatitis c virus (hcv) antibody in taiwan. method: this was a hospital-based cross-sectional study. we analyzed viral hepatitis data from 2695 subjects who received health checkups at one medical center in taichung from 2003 to 2004. all subjects were divided into three age groups, including 20-39, 40-64 and 65. this study emphasized the prevalence of hbsag and hcv antibody by gender and age. the statistical analysis was performed by t test and 2 . result: there were 1526 men (56.6%) and 1169 women (43.4%). the mean age was 49.2 (standard deviation 12.2, range 20-84). the overall prevalence of hbsag was 14.7%, with statistically significant difference(ssd) between gender (17.4% for men vs 11.2% for women, p <0.001). the prevalence of hbsag was decreased with age in men, with ssd (p <0.001), and also decreased in women, without ssd (p =0.08). the overall prevalence of hcv antibody was 5.2%, without ssd between gender (4.8% for men vs 5.7% for women, p =0.272). the prevalence of hcv antibody was increased with age both in men and in women, with ssd (p <0.001). conclusion: we hope this study can provide the epidemiological data for further studies of hepatitis b and hepatitis c virus infection in taiwan. s.m. wu 1 , x. zhou 2 1 wuhan medical treatment center, 2 center for gene diagnosis, zhongnan hospital, wuhan university, china e-selectin is revealed to facilitate leukocyte adhension to the endothelium and migration into inflamed tissue in inflammatory diseases. chronic hepatitis b virus infection is regarded as a chronic inflammatory process. to examine the possible involvement of e-selectin in the etiology of chronic hbv infection, we analyzed two polymorphisms of e-selectin and determined the plasma souble e-selectin levels in patients with chronic hbv infection and controls. the frequency of c allele of the a561c polymorphism was significantly increased in patients with lc campared with controls. no significant positive association was observed between the g98t polymorphism and chronic hbv infection. but in patients with lc, divided according to the child-pugh classification, the frequency of t allele was of significant difference between child'class a and class b plus c. plasma levels of soluble e-selectin were significantly increased in patients with chronic hepatitis and liver cirrhosiscompared with controls. in the liver cirrhosis group, levels of se-selectin were significantly decreased from child' class a to class c. in each group, patients with c allele of the a561c polymorphism showed higher soluble e-selectin levels than those with a allele. this is the first report describing the association between e-selectin polymorphisms and hbv-related hepatic fibrosis. our data showed the a561c polymorphism of e-selectin gene is associated with disease progression in patients with hbv infection and controls the expression of plasma soluble levels, the g98t polymorphism may be related to fibrotic severity in patients with liver cirhosis. background: chronic hepatitis b (chb) patients with high serum hbv-dna and normal serum alanine aminotransferase (alt) levels might be considered for treatment if histopathological findings show fibrosis stage 2 or more. however, to our knowledge there is no recommendation with regard to the therapeutic agents for this group of patients. objective: this study was aimed to evaluate the efficacy of nucleoside analogues (entecavir or telbivudine) in treating chronic hepatitis b patients with high serum hbv-dna and normal serum alt levels. patients and method: this was an open-label study in chb patients with high level serum hbv-dna levels between january 2007 and october 2008. patients were included if they showed normal serum alanine aminotransferase (alt) level at two measurements within a 3-month interval and had fibrosis stage > 2 on liver biopsy specimens. patients were treated with entecavir 0.5 mg/day or telbivudine 600 mg/day. the primary endpoint was the reduction or undetectable of serum hbv-dna at 24 week and 48 week of treatment, while the secondary endpoint was hepatitis b e antigen (hbeag) seroconversion. results: during a 2-year period, 37 chb patients with high level serum hbv-dna with normal alt two times with 3 months interval underwent a liver biopsy. twenty-eight (75.7%) of 37 pts showed fibrosis stage 2 on histological findings (metavir score). twelve of these 28 patients received nucleoside analogues, 7 (58.3%) of them were men. patients' median age was 42 (range: 24-52) years. there were 5 patients with stage-2, 6 patients with stage-3 and 1 patient with stage-4 fibrosis. eleven (91.7%) patients had genotype b virus. at baseline, the mean serum alt level was 32 + 11.8 u/l and mean hbv-dna level was 2.48 x 10 6 iu/ml, ranging from 1.23 x 10 3 to 2.4 x 10 7 iu/ml. six patients received entecavir and the other six received telbivudine therapy. undetectable hbv-dna was achieved by 9 (75.0%) patients at week-24 and 2 (16.7%) patients at week-48 of treatment. one patient who had the highest hbv-dna level had viral load reduction to 1.6 x 10 4 iu/ml at week-48 of treatment. two out of 5 patients with positive hbeag achieved hbeag seroconversion at week-48 of treatment. conclusion: this preliminary study has shown that nucleoside analogues might be considered in the treatment for chronic hepatitis b patients with high serum hbv-dna and normal serum aminotransferases levels. j. chen 1 , x.j.. wu 1 , y. wang 1 , g.q. wang 1 1 department of infectious diseases, peking university first hospital, beijing, china background: the dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are new members of cd28/b7 family, as co-stimulatory molecules expressing on t cells and antigen present cells (apcs). their engaging can downregulate the t cells function, including proliferation, cytokines secretion and cytotoxicity. in periphery blood, pd-1 was upreguated on virus specific-t cells, leading to the impairment of t cells. blocking the pd-1/pd-l can improve the function of t cells. methods and patients: 21 patients with chronic hepatitis b (chb) were treated by pegylated ifn -2b (pegintron from schering-plough, once a week, 0.5 or 1 g/kg/weight). the periphery blood were taken at 0 weeks, 4 weeks, 8 weeks, and 12 weeks. periphery blood mononuclear cells (pbmc) were isolated from fresh heparinized blood by ficoll-hypaque (density: 1.077g/l) density gradient centrifugation. then the cells were incubated with apc-conjugated anti-pd-1 antibodies. the pd-1 expression on lymphocytes was detected by flow cytometry (fcm). results: the pd-1 expression on lymphocytes at 0 weeks was 14.47±5.8%, at 4 weeks was 9.68±3.75%, at 8 weeks was 6.95±2.39%, at 12 weeks was 6.08±1.31% (p<0.05). conclusion: treatment with ifn -2b can downregulate the pd-1 expression on lymphocytes and may partially restore the function of t cells. to investigate the effects of nucleoside analogs therapy in hepatitis b related acute-on-chronic liver failure, we treated 55 hbv related acute-on-chronic liver failure patients with entecavir. as control, the remaining 74 were not treated with nucleoside analogues. results show the survival rate of entecavir therapy group has no significantly difference with none-treated group (p>0.05). although entecavir greatly reduced hbv replication during different therapy times (p<0.001), the meld score and liver function (alt, albumin, bilirubin, prothrombin time) had no significant changes (p>0.05). further more, we analyzed the meld score and liver function in different hbv-dna level patients .no significantly difference was observed (p>0.05). there is no significant correlation between hbv-dna level and meld score in different therapy times (p>0.05).the hbv-dna level between patients with over 3 months and less than 3 months survival patients showed no significant difference either (p>0.05). however, meld score and some parameters of liver function (albumin, bilirubin, prothrombin time) showed significant difference (p<0.05). these results suggest hbv-dna loading may not be a direct factor to increased liver injury and suppression of hbv replication may not reduce the severity of liver failure in hbv related acute-on-chronic hepatitis. s. firdoos 1 , u. adeeb 1 , a. mehmood 1 , m. gill 1 1 islamabad specialists clinic, islamabd, pakistan background: before the availability of etv, it was common to use adv for treatment of chronic hepatitis b patients. primary nonresponse and suboptimal response is a common problem with adv treatment. methods: we wanted to study the outcomes of entacavir therapy in this subset of patients. study was conducted between april 2007 to april 2008. we enrolled 30 chb patients who had non response to 12-24 weeks of 10 mg adv therapy. non response and suboptimal response was defined as non dimunition of at least one log of hbvdna from baseline after 12 weeks of therapy and persistence of 3 log10 after 24 weeks of therapy respectively.they were switched to 1mg entacavir before breakfast daily for at least 12 months.they had serial alt cbc and hbvdna measured every 12 weeks. results: out of 30 patients 20 male and 10 were female. only 8 patients were hbeag(+).mean hbvdna level prior to adv exposure was 6.5 log copies/ml.mean duration of exposure to adv was 26 weeks.5 patients lost to f/u.we did intention to treat analysis. 15 out 30 (50%) patient has, undetectable level of hbvdna after 12 weeks of therapy labelled as group 1.6 out of 30 (20%) had hbvdna level reduced by a mean of 3 log 10 copies/ml labelled as group 2.on week 24 treatment analysis all 15 patients from group 1 was hbvdna undetectable, 2 additional patients from group 2 had undetectable hbvdna. conclusion: entacavir therapy results in rapid suppression of hbvdna levels in majority of patients with primary nonresponse or partial non response to adv therapy. background: except for serum alt level, baseline factors predictive of therapeutic response to lamivudine in patients with hbeag-positive chronic hepatitis b remain largely unknown. we thus studied the influence of pre-therapy viral factors on end-of-treatment responses to lamivudine therapy. methods: a total of 116 treatment-naïve hbeag carriers who had pre-therapy serum alt level> 5xuln and received lamivudine for 18 months reimbursed by the national health insurance were prospectively enrolled. hbeag seroclearance and combined hbeag seroclearance, alt normalization as well as undetectable hbv dna at the end of therapy were defined as primary and secondary endpoint, respectively. the pre-therapy viral factors including viral load, genotype, precore stop codon (pc)/ basal core promoter (bcp) status, and pre-s deletion were determined to correlate with therapeutic endpoints. results: the frequency of patients with detectable pc mutation (g1896a), bcp mutation (a1762t/g1764a), and pre-s deletion at baseline was 22.4%, 21.6%, and 12.1%, respectively. after completing 18-month lamivudine therapy, overall hbeag seroclearance rate was 56.0%. patients with hbeag seroclearance had a higher prevalence of baseline pc mutation than those without (30.8% vs, 11.8%, p= .015). by multivariate analysis, the odds ratio of patients with pc mutation to develop hbeag seroclearance was 3.33 (p= .024). in addition, the presence of pc mutation also correlated with the combined response. conclusions: for hbeag-positive chronic hepatitis b patients with serum alt> 5xuln, pc mutation could predict a higher hbeag seroclearance rate at the end of 18-month lamivudine therapy. the efficacy of adefovir dipivoxil against all patterns of lamivudine resistant hepatitis b d.j. kim 1 , y.d. park 1 , y.g. kwon 2 , h.g seo 1 1 daegu fatima hospital, 2 kunngpook national university hospital, daegu, korea background: our aim was to evaluate the efficacy of adefovir dipivoxil (adv) and determine patient-dependent or laboratoroy variables that are predictive of hbeag loss and ivr for hepatitis b patients resistant to lamiduvine. also we evaluated the activity of adv against all patterns of lamivudine-resistant hbv. method: 179 hbv-infected patients with lamivudine resitance received adv for 6 months. quantitative hbv dna, hbeag/anti hbeag, alt was checked every 3-6months. the hbv polymerase of 161 patients were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. result: there is no significant difference in all patterens of hbv mutation about hbv dna reduction at 24w, 48w, 72w. there is no significant difference in all patterens of hbv mutation about alt normalization at 24w, 48w, 72w. conclusion: adefovir dipivoxil demonstrated similar potent anti-hbv efficacy regardless of the different patterns of lamivudine-resistant hbv mutations. g. novelli 1 , m. rossi 1 , v. morabito 1 , f. pugliese 1 , p. berloco 1 1 la sapienza university, rome, italy background: hepatitis b (hbv)-related end-stage liver disease is one of the most common indication for liver transplantation (lt). a number of patients dying while on the waiting list or removed because of being too ill is progressively increasing. we valued the possibility to improve the model end-stage liver disease (meld) of patients awaiting liver transplantation using a albumin dialysis: molecular adsorbent recirculating system (mars). methods: we treated 34 patients (19 male and 15 female) with a mean age 49.5. inclusion criteria: serum bilirubine > 15mg/dl, meld 25, inr > 2.1, encephalopathy grade ii. all patients were treated with mars mean 9±2.5 hr cycles and mean 9 treatments (range 3-15). all patients received standard medical treatment in addition to mars dialysis. the patient survival was valued at six months. results: we obtained a significant change of cytokines levels as interlukine 6 (p< 0.02) and tumor necrosis factor alfa (p<0.01) in association with an improvement of kidney, hepatic and hemodynamic parameters. at the end of mars treatments we observed a significant reduction of meld score (p<0.003). the results of meld show a rebound effect between the end of treatment and the follow up at six months without returning at starting values (p<0.005). twenty patients lived and 14 dead for clinical complications. conclusion: the improved meld score with mars gave patients on lt waiting list more time of survival, thus allowing them more opportunity for liver transplantation. entecavir for treatment of lamivudine-refractory patients chronic hepatitis b h.t. dat 1 , p.t.t. thuy 1 1 medic medical centre, ho chi minh, vietnam lamivudine treatment is associated with frequent development of resistant hepatitis b virus. this incidence especially is higher in longer time of treatment and loss of treatment benefit. entercavir is a new antiviral agent shown its high efficacy even in cases of mutations with lamivudine resistance. in this study, we evaluate the efficacy, the safety of entercavir in treatment of lamivudine-refractory patients chronic hepatitis b. sixty chronic hepatitis b patients with evidence of lamivudine resistance were randomly divided into two groups in proportion of 3:1. group i (n=45) used entecavir 1mg/day, group ii (n=15) used lamivudine 100mg/day. treatment time was 48 weeks. histology, alt, hbvdna were evaluated in the end of the treatment. age, sex, alt, hbvdna, genotype, hbeag were analyzed to evaluate their influences to the treatment. the results have showed hbvdna<2000 copies/ml in entecavir group 37.78% vs. 0% lamivudine group (p<0.01). hbvdna negative in entecavir group was 17.77% and incidence of seroconversion of hbeag was 8.82%. alt was normal in entecavir group 77.77% vs. 26.66% in lamivudine group (p<0.001).histologic improvement in entecavir group was 37.77% vs.6.66% in lamivudine group (p<0.05). patients with hbeag negative, genotype b, low viral load were shown better results. entecavir was shown to be efficacious in treatment for chronic hepatitis b patients experienced with lamivudine resistance. entercavir is safe, with almost no side effects. factors such as hbeag negative, genotype b, low viral load seems to be better in response to treatment. recurrence or mutation of entecavir resistance should be studied further in future. j.m. kim 1 , s.k. hwang 1 , b.h. choe 1 1 department of pediatrics, kyungpook national university hospital, daegu, korea backgrounds: by analyzing the characteristics of children with chronic hepatitis b who have lost hbsag by long-term lamivudine treatment, the selection of target patients could be relevantly predictable in the treatment of chronic hepatitis b in children. methods: a total of 75 hbeag positive children (< 18 y-o) were recruited who have visited kyungpook national university hospital from mar. 30, 1999 to may 8, 2008 . they were treated with lamivudine for at least 6 months. hbeag seroconversion occurred during lamivudine treatment in 49 out of 75 children. they were divided into hbsag clearance and non-clearance group. parameters influencing treatment results were analyzed according to hbsag loss. result: thirteen out of the 49 (26.5%) patients with hbeag seroconversion were classified as hbsag clearance group, while 36 (73.5%) as non-clearance group after lamivudine treatment. twenty five of 49 patients with hbeag seroconversion were under 6 years old, in 10 (10/25, 40%) of whom hbsag loss occurred as well. twenty four of 49 patients were over 6 years old, in 3 (3/24, 12.5%) hbsag loss occurred, that showed significantly difference (p-value= 0.029, or: 4.667, ci: 1.094-19.902) compared to younger group. age was significantly lower in hbsag clearance group (5.1±4.3 years) than non-clearance group (8.2±5.0 years) (p=0.043), but no difference was observed in other parameters. anti-hbs appeared in 12 patients. conclusion: in the treatment of hbeag positive chronic hepatitis b with lamivudine, age was significantly lower in hbsag clearance group than non-clearance group. background: dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are members of cd28/b7 family, was reported to transfer inhibitory signal, leading to the dysfunction of t cell. background: hepatitis b viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. mutations of hbsag allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus in reduced to undetectable levels. methods: immunohistochemical analysis of tissue samples from 56 patients with chronic hepatitis b (chb), 12 acute hepatitis b (ahb) patients and 10 health controls was performed. results: pd-1 was positively expressed on lymphocytes infiltrating the portal area.pd-l1 expression was the same as pd-1,also expressed in interlobular.pd-l2 expressed on kupffer cells and dendritic cells.pd-1-,pd-l1-,and pd-l2-positive cells express index of chb patients were much more than that of health controls and ahb patients(p 0.05).between groups in chb,the expression rate increase with the disease progression (p 0.05). methods: 58 chronic hepatitis b patients with both positive for hbsag and hbsab were studied.serological markers of hbv were detected by elisa and microparticle enzyme immunoassay. hbv dna levels were determined by fluorescent quantitative pcr, s gene fragments were directly sequenced, liver function was analyzed by automatic biochemistry analyzer au400. correlation test was conducted to evaluate their dependablity. conclusion: overexpression of pd-1 and pd-l within liver might be involved in inhibiting the immune response and be a mechanism of chronicity in hbv infection. results: the level of hbsag and hbsab was 254.4±68.3 s/n and 39.4±38.1 miu, respectively. hbv dna was detectable in 46 patients. fifty-one mutations of s gene were detected in 38 patients, and the relating amino acid substitution was at the sites of 36, 39, 47, 63, 77, 89, 90, 115, 126, 129, 139 and 154. eight (15.7%) out of 51 mutations were located at the "a" determinant region in 14 patients, while no mutation was found at the sites of 124, 137 and 147. however, the mutation did not affect hbv replication. hbv dna was positive correlated with hbeag. conclusions: change in hbsag antigenicity due to s gene resulted in concurrent hbsag and hbsab. the existence of hbsab did not affect hbv replication. the damage of liver failure in those patients was slight. background: hbv infection is common in bangladesh. we often encounter young patients incidentally detected with hbeag negative chronic hepatitis b (chb) in our clinical practice. however the characteristics of these patients is yet to be studied in this country. the aim of this study was to study the characteristics of young bangladeshis incidentally detected with hbeag negative chb. methods: we did percutaneous liver biopsies of 36 chb patients aged between 8 to 20 years. they were all hbeag negative with persistently normal or raised serum alt values. we did pre-core mutation (pcm) study in 4 patients who were randomly selected. results: 56% patients had significant necro-inflammation (hai-ni >3), while significant fibrosis (hai-f >2) was seen in 17.6%. serum alt (cut off 42 u/l) was raised in 38.2%, while high hbv dna load (>10 5 copies/ml) was observed only in 26.5%. pcm was negative in all 4. conclusion: although chb patients between 10-20 years of age are supposed to be in immune clearance phase, which is characterized by low hbv dna and hbeag positivity, the study shows that hbeag negative chb is an entity that can also be seen in this age group and a significant percentage of such patients may have considerable hepatic involvement. this challenges our current concept about immune clearance state of hbv infection, although much larger study is needed to draw any specific conclusion. background: hbv infection is common in bangladesh, but characteristics of young patients incidentally detected with chronic hepatitis b is yet to be studied in this country. methods: we did percutaneous liver biopsies of 88 chb patients aged between 8 to 20 years. results: significant necro-inflammation (hai-ni >3) was seen in 79.6% patients with hbeag positive and 56% patients with hbeag negative chb, while significant fibrosis (hai-f >2) was seen in 20.3% and 17.6% patients in these two groups respectively. serum alt (cut off 42 u/l) was raised in 37% hbeag positive and 38.2% hbeag negative patients, while in these two groups 87% and 26.5% patients respectively had high hbv dna load (>10 5 copies/ml). conclusion: hbeag negative chb is an entity that can also be seen in young population. a significant percentage of both hbeag positive and negative patients may have considerable hepatic involvement. profile of hbeag +ve chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: inactive hbv carriers constitute the major reservoir of hbv. present management guidelines provide inadequate treatment modalities. they are recommended for regular check-up; treatment is only recommended when patients exhibit evidence of liver damage. this is due to lack of information about their extent of liver damage. aim of this study was to assess extent of liver damage in hbeag +ve patients, unaware of their infection. methods: in this retrospective study, records of 206 hbeag +ve chb patients from our pool of 561 chb patients were reviewed. they were tested for hbsag, hbeag, hbv dna, anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 78.2% (161/206) patients were males and 21.8% (45/206) females. they were between 8-45 years of age. alt was raised >2times unl in 17% (35/206). 92. 2% (190/206) patients had high hbv dna (>10 5 copies/ml), while low hbv dna (<10 5 copies/ml) was seen in 7. 8% (16/206) . in high hbv dna group, significant necro-inflemmation (hai-ni >7) was seen in 48. 9% (93/190) and significant fibrosis (hai-ni >3) in 24. 7% (47/190) . figures were 37.5% (6/16) and 31.3% (5/16) respectively in low viral load group. none tested positive for hcv infection. conclusion: study indicates that machinery should be developed to characterize undetected hbv carriers in developing countries by conducting multi-center clinical studies. we have shown that considerable number of patients, unaware of their hbv infection, suffer from progressive liver damage. the overall strategy of management of chronic hbv infection should also be revisited. high viral load does not necessarily represent significant liver damage in patients with chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: in general, it is assumed that patients with chronic hepatitis b virus (hbv) infection with high viral load exhibit increased liver damages. treatment guidelines also emphasize on reducing viral load. these observations were mainly accumulated from developed countries. >80% chronic hbv carriers live in the developing nations, but little is known about relationship between hbv viral load and extent of liver damage in these countries. in this study, we addressed this issue. methods: in this retrospective study we reviewed records of 306 chb patients from our pool of 561 patients. all had high hbv dna (>10 5 copies/ml). 62. 1% (190/306) were hbeag +ve and 37.9% (116/306) hbeag -ve. they were alsotested for anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 51.1% (97/190 ) hbeag +ve patients with high hbv dna had non-significant hepatic necro-inflammation (hai-ni <7); this figure was 53.4% (62/116) in hbeag -ve patients. non-significant hepatic fibrosis (hai-f <3) was observed in 75. 2% (143/190) and 69.8% (81/116) in hbeag +ve and -ve patients respectively. none tested positive for hcv. conclusion: correlation doew not exist between viral load and liver damage in chb in bangladesh. many with both hbeag +ve and -ve chb with high hbv dna do not have significant hepatic necro-inflammation and fibrosis. further study may be needed to find out influence of other factors on liver damages in chb in bangladesh. most of these patients have not been characterized and treatment modalities have not been defined for them. background/aims: expression of intrahepatic hepatitis b core antigen (hbcag) is related to the immunopathogenesis of hepatitis b virus (hbv) infection. the role of hbv genotype and basal core promoter (bcp) mutation in expression of hbcag was investigated. methods: seventy hbeag-positive chronic hepatitis patients (genotype b in 52 and c in 18; bcp t1762/a1764 mutation in 16) were enrolled. clinical, virologic and histologic features were compared with regard to localization and expression of intrahepatic hbcag. the effects of hbv genotype and bcp t1762/a1764 mutation on the expression of hbcag were further evaluated by in vitro assays. results: cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic hbcag were found in 38 (56.7%), 25 (37.3%) and 4 (6.0%), respectively. fifty-eight (80.6%) of these patients expressed a high level of hbcag. in multivariate analysis, cytoplasmic localization of hbcag correlated only with low serum viral load (p=0.045) and bcp mutation (p=0.04). high expression level of hbcag also correlated with high serum viral load (p=0.015) and bcp wild-type sequence (p=0.037). in vitro assays supported that hbv bcp mutant had lower subcellular expression of hbcag compared with bcp wild-type strain. conclusions: hbv bcp mutation and viral load but not genotype contributes to the expression of intrahepatic hbcag. hepatitis b virus (hbv) genotypes show distinct geographical distributions and virological and clinical differences. in some of genotypes, specific substitutions and mutations have been described in association with hepatitis b e (hbe) protein expression and viral replication. in this study, genetic characteristics of hbv genotype e (hbv/e) were investigated using clinical samples obtained from 12 hepatitis b e antigen (hbeag)-positive, and 11 anti-hbe-positive asymptomatic carriers (ascs) in west-africa. full-genome analysis of isolated hbv strains revealed strong association between precore (pc) mutation and hbeag to anti-hbe seroconversion. furthermore, using 53 partial genome sequences, correlation among hbeag/anti-hbe status, viral load and key mutations were analyzed. the data showed that pc mutation is associated with hbeag seroconversion and enhanced viral replication efficiency. comparison between hbv/e and hbv/d strains reveals these two genotypes to have an identical sequence in their core-promoter-upstream and basic core promoter (curs/bcp) regions. it has been known from the previous phylogenetic studies, that hbv/d and hbv/e cluster together in trees reconstructed on x and precore/core orfs. in addition, this study, demonstrates that in spite of the high sequence similarity of curs/bcp region, the seroconversion-related mutation patterns are different between hbv/e and hbv/d in asc. further studies are needed to clarify the clinical significance of the regulatory sequence similarity between hbv/e and hbv/d. necro-inflammation and fibrosis p. siddappa 1 , p. kar 1 , b. das 2 , r. gondal 1 , m. asim 1 1 maulana azad medical college, 2 icpo, new delhi, india background: chronic hepatitis b(chb) is an important cause of morbidity and mortality. methods: pilot study involving 30 patients of chb, were equally randomized to receive either adefovir or lamivudine for 6 months. quantification of serum and hepatic hbv dna levels by real time pcr and liver biopsy done at start and end of 6 months. results: after 6 months there was significant and comparable reduction in serum and hepatic hbv dna viral load and liver biopsy showed significant reductions in hai scores in both the groups. serum alt which was elevated to 2 or more times normalized in both the groups. in the adefovir group 2 patients became hbeag negative and 2 patients who were hbeag negative at the start of therapy remained so. in the lamivudine group one patient became hbeac negative and 2 patients who were negative at the start of therapy remained so. in the adefovir group 4 patients became hbv dna (qualitative test) and in the lamivudine group 2 patients became hbv dna negative. there was strong correlation between serum and hepatic hbv dna levels both before and after the completion of therapy. conclusion: both the drugs bring about biochemical, histological and serological improvement with significant reduction in viral load in serum liver after 6 months without complete clearance of virus. there was not enough evidence to show therapeutic advantage of one drug over the other. the serum and hepatic hbv dna levels correlate well with eachother before and after treatment. aim: assessing efficacy and safety of treatment of chronic hepatitis b in children with pegylated ifn. materials and methods: 13 children (9 boys and 4 girls) aged 11-17 years with chb treated with peg-ifn alfa-2a, 100 g/m 2 /week during 48 weeks, 5 hbeag-positive and 8 hbeag-negative children, 4 previously treated with recombinant interferon. no child had liver disease greater than grade 2, stage 2. serum hbv dna was quantified at baseline, tw 4, ("rvr") tw 24, tw 48 (etr) and w 72 (svr) with rt pcr method (roche taqman). alt activity, haematology and adverse events were monitored. results: after 4 weeks treatment median hbv dna level decreased from 7.8x10 3 iu/ml at baseline to 1.43x10 2 iu/ml (p<0.01). "rvr" -undetectable hbv dna at tw4 was observed in 6/13 children and associated with lower pretreatment alt levels <25 iu and pretreatment viral load <750 iu/ml. all children with "rvr" were hbeag-negative pretreatment. at tw 24 and tw 48 seven children including all with "rvr" had undetectable hbv dna. 5 children achieved svr (undetectable serum hbv dna in w 72), among them 3 with "rvr". in 2/6 children with "rvr" hbsag disappearance was observed since tw 48. leukopenia was reported in 7 children, thrombocytopenia in 3. no adverse events were observed following dose modifications. conclusions: 1. peg-ifnalfa-2a is a good therapeutic option for children with chb, in particular with hbeag-negative chb 2. low pretreatment viral load and "rvr" seem to be predictive factors of efficient therapy. control by investigating the sanitizing modes among appliances used in the public service places (psp) and hbsag among appliances and practitioners worked in those places. methods: 63 beauty parlors, barber shops and bathing centers selected by stratified randomization sampling, 682 workers were investigated in questionnaire. the hbsag in appliances of psp and employee was detected by ria. results: the rate of hbsag among appliances of psp was 2.13%. the rate of hbsag in large-, medium-and small-sized appliances was 0.63%, 2.67% and 3.70%. the rate of hbsag has different( 2=6.68 p 0.05). the rate of hbsag among appliances of beauty parlors, barbering shops and footbath inns was 2.97%, 0.61% and 3.42%. different appliances had different rate of hbsag, such as the rate of acne needle and the forceps was 5.13% and 4.17%. the positive of hbsag amongworkers in psp was 7.13%. the rate of hbsag among workers in large-, medium-and small-sized psp was 7.34%, 8.33% and 2.94%. the rate of hbsag among workers in beauty parlors, barbering shops, footbath inns and bathing centers was 9.01%, 6.37%, 4.35% and 7.29%. the hbsag rate among workers was different in different works, the rate was higher in tattoo workers (13.33%), pedicures workers (12.68%), massagists (8.03%). conclusions: it is important to enhance the sanitizing management in psp and improve workers kap) of hepb. and we should promote health education to enhance the knowledge of hepatitis b control and build up supervision consciousness. background: integration of hepatitis b virus (hbv) dna into host chromosomes is often found in chronic liver disease and hepatocellular carcinoma, which is likely an early event of hbv-related carcinogenesis. however, the molecular mechanism of integration remains unclear. here we describe a potential mechanism of hbv integration and identify that ku70 and ku80, the gatekeepers of non-homologous end-joining (nhej) repair pathway, can serve as targets for anti-hepatitis virus integration. methods: using i-sce endonuclease-based system, we induced a dna double-strand break (dsb) in human hepatoma cell line huh-7. the cells were then incubated with serum from patients with chronic hbv infection. pcr amplification and direct sequencing were used to detect the inserted sequence in the site of dsb. finally, we employed taqman-based real-time pcr assay to quantify the integrated hbv dna and evaluate the effects of shrna on hbv integration. result: when huh-7 were exposed to viral serum and incubated for several days, hbv dna was detected in integrated form at the exact site of dna damage. furthermore, small interference rna (sirna) targeted against gatekeeper genes for nhej can down-regulate nhej repair and even the frequency of hbv integration. conclusion: thus, this project provided us with the first direct evidence that dna double-strand breaks are potential targets for hbv integration. the study has also shown that shrnas targeted against gatekeeper genes for nhej can regulate the frequency of hbv integration. objective: to screen proteins of human pancreas cdna library interacting with hbsag protein. methods: the library was amplifed, purified and evaluated, and then the puried library plasmids were transformed into yeast strain y187. the reconstructed plasmid pgbkt7-hbsag was transformed into yeast strain ah109 and screened on the nutrient deficiency medium sd/-trp. the transformed ah109 mated with y187 containing the library plasmid. the diploid yeast cells were plated on nutrient deficiency medium sd/-trp/-leu/-his/-ade and sd/-trp/-leu/-his/-ade containing x--gal for selecting. the plasmids in diploid yeast cells were extracted and electrotransformed into e.coli dh5 . the plasmids in dh5 were extracted, sequenced and analyzed by bioinformatic methods. results: sixteen proteins interacting with hbsag were founded. conclusions: these results show that hbsag protein may be related with metabolism of glucose and lipid. comparison of the sensitivity and specificity of the elecsys ® hbsag ii assay with other available assays in china for detection of hbsag j.d. jia 1 , l. wei 2 , x.x. zhang 3 , y.l. mao 4 , l.l. wang 5 , z.l. gao 6 , j.l. hou 7 , j. zhang 8 , w. melchior 9 , w. van der helm 9, 10 1 beijing friendship hospital, beijing, china, 2 beijing people hospital, beijing, china, 3 ruijin hospital, shanghai, china, 4 beijing 302 hospital, beijing, china, 5 west china hospital, chengdu, china, guangzhou, china, 7 guangzhou nanfang hospital, guangzhou, china, 8 shanghai public health clinical centre, china, 9 roche diagnostics ltd, rotkreuz, switzerland, 10 conclusions: in this patient population the prevalence of hbsag positive and anti-hcv were much higher than reported in community studies. genotypes 1 and 6 accounted for most of hcv. these very high rates of viral hepatitis in a hospital setting challenge to healthcare providers in terms of patient management as well as caregiver's prevention. hepatitis b is one of the major diseases of mankind that kills about one million persons each year in the world. accoring to primary study about 3% of iranian population is chronic hbv carriers. among iranian cirrhotics, 70-84% has evidence of exposure to hbv and 51-56% is carriers. because increase demand of blood transfusion, high blood dependent patients and long term window period of hbv infection, any controlling hbv infection program in blood donors can enhance the blood safety and public health. pe078 in this descriptive study included all the blood donors that referred to dezful blood transfusion center during 2005-2008. all the blood donors screened for hbs ag by using enzyme immuno assay and repeatedly reactive (r.r) samples confirmed by hbc-ab or confirmatory (neutralization) tests. the data analyzed by using spss 11.5. we found that in the first year 1.052 % were repeatedly reactive and 1.045 % confirmed. the results for other years as the followed: 0.782 %(r.r) and 0.770 % confirmed and in the last 0.725 % (r.r) and 0.700 % confirmed. the repeated blood donors increase in this period (42.11 %, 50.15 % and 51.68 % respectively). aim: we aimed to evaluate the cost-effectiveness of telbivudine versus entecavir with reference to lamivudine by roadmap model. methods: decision analysis model was used to study the incremental cost-effectiveness ratios (icer), i.e. the additional cost (in usd) required to achieve undetectable hbv dna or hbeag seroconversion for a patient at 2 years in america and hong kong. entecavir was used as a continuous monotherapy. lamivudine and telbivudine would be shifted to entecavir if hbv dna was detectable at month 6 and continued otherwise with drug resistance treated by add-on adefovir. weighted event rates based on previous reports were estimated for analysis. according our study, although the prevalence was higher than other region in our province, the hbv prevalence showed good decrease after stablishment strategies such as of repeated blood donor recruitment , improvement the donor selection and other educational programs . good following up those strategies to enhance the blood safety recommended. results: telbivudine was generally cheaper than entecavir to achieve an incremental case of undetectable hbv dna from lamivudine at 2 years. entecavir was least effective and most costly for hbeag seroconversion. conclusions: telbivudine is a cost-effective alternative to entecavir particularly when its cost is low in hong kong. h. tang 1 , g.l. zhang 1 , y.x. li 1 , r.q. tian 1 , m. liu 1 , x. li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china micrornas (mirnas) are single-stranded noncoding rnas of 18 to 25 nucleotides that play critical roles in a wide spectrum of biological processes. we investigated whether the mirnas-silencing machinery influences hbv replication or antigen expression. on the basis of elisa and mtt, the effect of 328 mirnas on the hbsag expression and cell proliferation was examined. three micrornas efficiently inhibited hbsag expression without significant effect on the proliferation of hepg2 2.2.15 cells compared to lacz control. subsequently, bioinformatics analysis were used to predict targets for the three mirnas, and the prediction results were conformed by cdna microarray analysis. the target region in hbv genome and the 3'utr region of one cellular gene were identified by fluorescent reporter assay, semi-quantitative rt-pcr and western blot. the results demontrated that mirna may play an important role in replication and gene expression of hbv. hepatitis b virus (hbv) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. although considerable progress has been made, the pathogenesis of hbv infection is still elusive. there's an urgent need to elucidate the mechanisms of hbv-host interactions, to discover novel biomarkers for diagnosis and prognosis and to develop therapeutic targets for anti-hbv treatment. herein, we applied a two-dimensional gel electrophoresis and maldi-tof/ms based comparative proteomics approach to globally analyze the host response to hbv by using an inducible hbv-producing cell line hepad38. of the 23 differentially expressed proteins identified, glucose regulated protein 78 (grp78) was one of the most striking proteins elevated by hbv replication, which was confirmed by real-time pcr and western blotting. knockdown of grp78 expression by rna interference resulted in a significant increase of both intracellular and extracellular hbv virions in hbv-transfected hepg2 cells. reversely, grp78 overexpression led to hbv suppression. the expression levels of hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) were determined by enzyme linked immunosorbent assay (elisa). immunofluoresce further revealed a positive correlation between the expression levels of grp78 and hbsag in both hbv-transfected hepg2 cells and hbv-infected human liver tissues. altogether, these data demonstrate for the first time that grp78 is an endogenous anti-viral factor in hbv-transfected hepg2 cells and may serve as a potential prognostic indicator of viral status in anti-hbv therapies. background/aims: to evaluate the predictors of response to long-term treatment of adefovir dipivoxil (adv) in patients with emerging lamivudine (lam)-resistant hepatitis b e antigen (hbeag)-positive chronic hepatitis b (chb) patients. methods: one-hundred-thirty-four lam-resistant hbeag-positive chb patients were treated with adv for a median of 28.0 months (range, 18 -54 months), following lam therapy for a median of 25.5 months (range, 5 -66 months). 65 patients (48.5%) were switched from lam to adv monotherapy, 43 (32.1%) were switched to adv with 1 month of lam overlap therapy, and 26 (19.4%) were switched to adv with 3 months of lam overlap therapy. the influence of baseline parameters on treatment response to adv in patients with lam-resistant hbeag positive chb was analyzed. result: during the follow-up period, 28 (20.9%) of 134 patients achieved complete response, defined as normalization of alt level, negative hbv dna by a digene hybrid capture assay and achievement of hbeag loss. sixteen (11.9%) patients achieved hbeag seroconversion. twenty-eight (20.9%) patients developed adv-related mutations during adv treatment. in multivariate analysis, virological response at 3 months (or=9.70, 95% ci: 32.63-35.78, p=0.001), defined as serum hbv dna levels less than 5 log10 copies/ml or a reduction in serum hbv dna levels greater than 2 log10 copies/ml after 3 months of adv therapy, independently predicted complete response. conclusions: virological response at 3 months was the strongest predictor of adv response in lam-resistant hbeag-positive chb patients. background/aims: to explore the effects of hbv dna level hbv genotype/subgenotype on the pathogenesis of severe liver diseases in chongqing. methods hbv dna level was analyzed in patients with severe liver diseases in retrospect,and hbv genotype/subgenotype hbv dna level and hbeag were determined in patients with hepatocellular carcinoma (hcc,n=78 ), liver cirrhosis(lc, n=58),chronic hepatitis b(chb, n=20) and acute on chronic liver failure(aclf, n=42). results hbv level from high to low with chb were lc, aclf and hcc in turn(p 0.05). hbv genotype was mainly genotype b.the rate of genotype b and c were 51. 3% and 47.4 respectively in hcc patients, 60.3% and 39.7 in lc patients, 55% and 40 in chb patients, 55% and 40 in aclf patients. the percentage of genotype b/c in aclf patients was higher in compared with other groups. but the distribution of hbv genotype among groups was not statistically different(p 0.05).subgenotypes of genotype b were almost ba but one. subgenotypes of genotype c were mainly ce in chongqing area, and there was no statistical difference among the 4 groups (p 0.05). conclusion: hbv dna level seems not to be a determining factor at end point of severe liver disease. both genotype b and c of hbv can lead to severe liver diseases, and there are more mixed infections by different genotypes in aclf. the efficacy of switching to entecavir (etv) monotherapy in japanese lamivudine (lvd)-experienced patients. background: this study aims to determine the efficacy of switching to 0.5mg etv daily in chronic hepatitis b (chb) patients previously treated with lvd. method: retrospective analysis of chb patients (n=134) previously on 100mg lvd daily and switched to 0.5mg etv daily. results: lvd-experienced patients were divided into three groups based on hbv viral load at time of switching to etv (<2.6 log10 copies/ml; 2.6-5.0 log10 copies/ml and >5.0 log10 copies/ml). detection of lvd-resistant virus at the time of switching was higher in the group with hbv dna 2.6 log10 copies/ml (76% in both 2.6-5.0 and >5.0 log10 copies/ml groups versus 23% in <2.6log10 copies/ml group) and was higher in patients treated with lvd for 3 years (52% versus 24% for patients on <1 year of lvd). a year after switching to 0.5mg etv daily, hbv dna undetectable rates were 100% (42/42), 94% (17/18) and 43% (6/14) for <2.6, 2.6-5.0 and >5.0 log10 copies/ml groups, respectively. alt normalization occurred in more than 90% patients at the end of the first year of switching to etv for all three patient groups. only one patient in the 2.6-5.0 log10 copies/ml group, who had lvd-resistant mutants at the time of switching, developed etv resistance during follow-up. conclusion: switching from lvd to etv maintains or improves viral suppression and alt normalization, especially in patients with viral load <5.0 log10 copies/ml. background/aims: we investigated the association between on-treatment hbsag decline and sustained response in patients treated with pegasys±lamivudine. methods: hbsag levels were measured retrospectively pre-treatment and at weeks 12, 24, 48 and 72 using the abbott architect hbsag assay in sera from 542 patients (83% asian) treated with pegasys alone (180 g qw; n=271) or combined with lamivudine (100mg qd; n=271) alone for 48 weeks as part of a large multinational trial. response was measured 6 months post treatment. results: more patients treated with combination therapy had >1 log decline in hbsag from baseline to week 48 ( figure) . hbsag level <1500 iu/ml at week 12 was associated with higher rates of response to pegasys±lamivudine 6 months post treatment ( figure) . data comparing hbsag and hbv dna as on-treatment predictors of response will be presented. conclusion: on-treatment hbsag monitoring may be useful for predicting response in patients treated with pegasys. y. wakui 1 , j. inoue 1 , y. ueno 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university graduate school of medicine, sendai, japan background/aim: chronic hepatitis b patients are clinically treated with nucleot(s)ide analogues and ifn-. nucleot(s)ide analogues have problems including drug resistance in continuous treatment, and ifn-has disadvantages of limited effectiveness and side effects. therefore, novel antiviral drugs are still needed. in this study, the suppressive effect to the replication of hbv was examined in vitro by using bezafibrate and rosiglitazone, which are ligands of peroxisome proliferator activated receptor (ppar) and , respectively. methods: the cytotoxicity of bezafibrate and rosiglitazone to hepg2 cells was examined with mts assay, and the concentration of 50% cytotoxicity (cc50) was calculated. hepg2 cells were transiently transfected with the plasmid containing 1.3-fold hbv genome of a genotype b strain. after 24 hours of transfection, rosiglitazone and bezafibrate was added to the cells. using the medium at day 3 after the addition of drugs, hbv dna was quantified with real-time pcr. results: the cc50 of bezafibrate and rosiglitazone in hepg2 cells were 250 m and 150 m, respectively. the amount of hbv-dna in the medium was decreased when the density of bezafibrate was over 200 m, but the density demonstrated considerable cytotoxicity. in contrast, rosiglitazone of 5 m, which showed no cytotoxicit, decreased the amount of hbv dna. the 50% effective concentration (ec50) was calculated to be 9.8 m. conclusions: in this study, it was suggested that the replication of hbv was inhibited by rosiglitazone of the density without cytotoxicity. the mechanism is uncertain and being investigated now. q. zheng 1 1 center for liver diseases, the first affiliated hospital, fujian medical university, fuzhou, p. r. china background: the objective of this study was to evaluate the early virologic response for prediction of achievement of hbeag seroconversion and hepatitis b virus (hbv) dna negativity after two years of lamivudine treatment in chronic hepatitis b (chb) patients. methods: in this retrospective study, adult patients with chronic hepatitis b (255 hbeag-positive and 122 hbeag-negative) were treated with lamivudine (100 mg/day), and followed-up up to 72 months. response and resistance to the treatment were assessed during the treatments with lamivudine. results: it was found that gender, age, baseline levels of alt and hbv dna, serum hbv dna at week 24 (p =0.019 , or = 0.442) were closely related to the achievement of hbeag seroconversion, undetectable hbv dna level and emergence of drug resistance after 2 years of lamivudine treatment. hbeag positive patients with baseline serum hbv dna in 10 6 -10 8 copies/ml and serum hbv dna 10 3 copies/ml at week 24 showed high response rate of alt normalization rate (91.7%), undetectable hbv dna rate (84.5%), hbeag seroconversion rate (55.2%), as well as low drug resistance rate (25.4%) after 2 years of treatment. similarly, hbeag negative patients with serum hbv dna 10 3 copies/ml at week 24 could achieve high 2-year response rate of alt normalization rate (72.41%), undetectable hbv dna rate (82.3%), and low drug resistance rate (15.5%). conclusion: serum hbv dna 10 3 copies/ml at 24-week provide the best prediction of 2-year lamivudine treatment response. background/aims: unlike oral antivirals, a finite course of (peg)interferon can induce sustained post-treatment response in patients with chronic hepatitis b (chb), with increasing rates of hbsag clearance observed in patients who respond during post-treatment follow-up. hbsag clearance is considered to be the closest outcome to a cure, being associated with improved histological outcome, reduced incidence of hcc and increased survival. methods: in a randomised multinational study, patients (hbeag-negative) received 180µg pegasys+placebo (n=177); 180µg pegasys+100mg lamivudine (n=179); or 100mg lamivudine (n=181) for 48 weeks, and were assessed 6 months post-treatment. from this initial study, 230 of those who had received pegasys±lamivudine and 85 patients who had received lamivudine monotherapy participated in a long-term observational study to investigate post-treatment response. hbsag clearance at yearly post-treatment follow-up visits up to 5 years post-treatment was analysed. results: hbsag clearance in patients treated with pegasys±lamivudine increased post-treatment (3% at 1 year to 6%, 8%, 11% and 12.2% at years 2, 3, 4 and 5). at year 5, 28 pegasys-treated patients (12.2%) had cleared hbsag compared with 3 (3.5%) of lamivudine-treated patients (p=0.022). 16/28 pegasys-treated patients had anti-hbsag (hbsag seroconversion). detailed analysis of the 5-year follow-up data will be presented. conclusion: the ability of a finite course of pegasys to induce sustained response with increasing hbsag clearance rates in responders during post-treatment follow-up supports its use as first-line therapy in hbeag-negative patients with chb. background/aims: recent studies suggest that quantification of hbsag levels early during treatment can be used to predict post-treatment response to pegasys. elecsys ® hbsag ii (roche) is a sensitive assay for the detection of hbsag. this assay can be used for the quantification of hbsag levels using a simple dilution algorithm. we compared results obtained using the elecsys ® hbsag ii method with those of a commonly used quantification assay. methods: hbsag levels obtained using the elecsys ® hbsag ii assay were compared with those obtained using the abbott architect ® hbsag assay for a total of 40 samples from patients infected with hbv genotypes a (n=8), c (n=1), d (n=29) and f (n=2). samples were diluted 1:100 in diluent provided by the manufacturer. samples with hbsag levels >250 iu/ml were retested at a final dilution of 1:1000. samples with hbsag levels <0.05 iu/ml were retested undiluted. results: overall, hbsag levels measured with the two assays correlated well (r 2 =0.9718) over a wide range (3-3x10 5 iu/ml). discrepancies in hbsag levels >±20% were reported for a minority of the samples (n=15), mainly distributed evenly above and below the ideal line (n=11). in the four low titre (range 3-9x10 3 iu/ml) samples with greatest discrepancy elecsys ® underestimated values (in two cases by >50%). conclusion: the elecsys ® hbsag ii assay provides a simple and reliable means for determining hbsag levels. this simple assay format could be used to provide useful information during on-treatment monitoring of hbsag levels in patients with chronic hepatitis b undergoing therapy. conclusions: hbv/a has been increasing in chb patients in japan as the consequence of ahb, spreading in the younger generation through promiscuous sexual contacts, thrust by an inclination of hbv/a to induce chronic hepatitis. the spread of hbv/a infection in japan should be prevented by universal vaccination programs. introduction: chronic viral hepatitis is common in end-stage renal disease (esrd), from endemic hepatitis b (chb) and nosocomial hepatitis c (chc). reduced outcomes post-renal transplant were reported thus chb and chc cirrhosis became contraindications to listing. however, these predated effective anti-viral therapies. we reviewed outcomes of patients with chronic viral hepatitis following assessment for renal transplantation. methods: prospective database of esrd patients with viral hepatitis referred for renal transplantation was reviewed. results: 110 patients were assessed. 23 patients underwent kidney transplantation. two were cirrhotic and had liver/kidney transplantation; both died within 6 months. 21 were non-cirrhotics, of whom 20 are alive. 14/20 have functioning allografts; predictors were normal alt and low viral load. of the 87 non-transplanted, 31 had cirrhosis; 17/31 received anti-virals. mortality was 35% -7 liver-related (3 hepatoma,1 bacterial peritonitis,3 sepsis -5 inactive cirrhosis); 4 non-liver related (2 cerebral,1 haemorrhage,1 renal -3 inactive cirrhosis). 12/20 surviving cirrhotics received anti-virals. in non-transplanted non-cirrhotics, mortality was 18%; 80% of survivors had inactive disease. 23 chb patients received lamivudine; 11 adefovir (lamivudine resistance). 11 chc patients received ifn-based therapy. conclusion: excellent outcomes are achieved in esrd patients with chb/chc post-renal transplant, in absence of cirrhosis. normal alt/non-detectable viral load can predict graft function. however, cirrhosis is associated with high mortality on dialysis whereas non-cirrhotics with inactive disease do well. the role of kidney transplantation in cirrhotics with suppressed viral replication needs to be reassessed. the truncated hbc149 interferes with replication of hepatitis b virus j.c. han 1,2 , x.b. pan 1,2 , l. wei 1,2 , k. deng 1,2 1 institute of hepatology, 2 peking university people's hospital, china hepatitis b virus (hbv) capsids play an important role in production of progeny virus and other elements of the virus life cycle. misdirection of capsid assembly and formation of aberrant particles may be an effective approach to interfere with virus replication. hbv capsids can be assembled in vivo and vitro from the dimeric hbv core protein (hbcag). the interaction of single and dimeric hbcag with some truncated hbcag is verified in vitro. the truncated hbcag consists of the first 149 amino acids and lacks the c-terminal, 34-residue rna-binding domain. method: we transiently transfected hepg2.2.15 with pcdna3.1hbc149 by fugene6.after 48 and 96h, hbvdna, hbeag and hbsag in culture supernatant were detected and cell subjected to southern blot and immunofluorescence analysis. result: the level of hbsag and hbeag had gentle change, we found that hbvdna decreased at 96h after transfection( 10 3 10 4 copies/ml p<0.01) ,but replication intermediates obviously decreased from 48h. some positive signal of hbcag located around the nuclear and conglomerated in cytoplasm compared to the control. conclusion: the truncated hbc149 can inhibit replication of hepatitis b virus. misdirection of capsid assembly and formation of aberrant particles could be an important cause. y.p. li 1 , r.c. li 1 1 objective: to assess the long-term efficacy of recombinant yeast derived hepatitis b vaccine in infant s born to hbsag and hbeag carrier mother. methods: a total of 273 neonates born to hbsag, hbeag both positive mothers were vaccinated with 5 ,5 ,5 g doses of recombinant yeast derived hepatitis b vaccine by 0 ,1 , and 6 months schedule. they were all followed for 129 years after the primary vaccination. results: twelve infant s (6.63 %) become hbsag positively conversed in 9 year after primary vaccination ,and the positive rate of hbsag in 1-9 year was 0.72 %-6.8 % ,17.18 % of child in no/ lowly respond become hbsag positively. at the ninth year, the positive rates of anti-hbs were 60 % above. anti-hbs positive rates and immunity level were higher at 3-5 year old by repetition immunity than others. conclusion: the recombinant yeast derived hepatitis b vaccine have good immunogenecity and long-term protective efficacy to hbv interruption of perinatal transmission , a booster dose seems necessary in aged 3-4 years to the mother with hbsag and hbeag.it is high risk tobecome hbsag positively in the baby of norespones to hepatitis b vaccine. chb patient group-initiated programme to improve awareness, adherence and treatment outcomes in asia pacific n. leung 1 1 founding chairperson of asiahep background: worldwide, over 400 million people live with chronic hepatitis b (chb); 300 million in asia pacific. regional survey data from 1,500 patients in 10 countries showed a lack of knowledge and understanding of chb, its severity and impact on quality of life. this initiative aims to coordinate patient groups in the region and devise programmes to improve knowledge and healthcare outcomes. methods: the patient groups met in hong kong in may 2008 and identified common needs to: (1) improve educational resources; (2) raise awareness; (3) increase diagnostic yield; and (4) enhance treatment compliance through education about the need for sustained viral suppression to reduce long-term complications. results: a patient engagement programme was developed for people with newly diagnosed or known chb. the programme comprises: -detailed information about chb -a health-tracking tool for self-monitoring of blood tests and treatment progress -detailed information for carers/family -a patient-physician communications video (including role-play) -mobile phone text messages providing advice and compliance/appointment reminders conclusion: this programme was developed to address the needs of patients and clinicians. improved knowledge and long-term support, particularly for patients on antiviral medication, is expected to improve quality of life. the programme encourages clinicians and patients to develop enduring therapeutic partnerships to promote optimal outcomes. acknowledgement: the chb patient group meetings and the patient engagement programme are supported by an unrestricted educational grant from glaxosmithkline. serum hbv rna level reflects the potency of nucleos(t)ide analogue y.w. huang 1,2 , k. chayama 3,4 , m. tsuge 3,4 , s. takahashi 3,4 , t. hatakeyama 3,4 , m.y. lai 2,5 , h.l. you 1 , j.t. hu 1 , c.j. liu 2,5 , p.j. chen 2,5 , d.s. chen 2,5 , s.s. yang 1 , j.h. kao 5, 6 1 liver unit, cathay general hospital medical center, 2 background and aims: serum hbv rna is detectable in patients treated with lamivudine (lmv) or entecavir (etv) (hatakeyama, 2007 and huang, 2008) . the aim of this study was to determine the clinical significance of serum hbv rna levels in patients treated with nucleos(t)ide analogues of different potency. methods: serum hbv rna was serially determined in 20 patients treated with nucleos(t)ide analogues for 48 to 52 weeks (9 with adefovir (adv), 5 with lmv, and 6 with etv). serum hbv rna was quantified by reverse transcription of hbv nucleic acid extract with subsequent real-time pcr. results: hbv rna was detectable in 11 patients as follows: 2 of 9 in adv (22%), 3 of 5 in lmv (60%), and 6 of 6 in etv (100%) (p = 0.002). mean log serum hbvdna levels at baseline were 10.1 ± 0.6 for adv, 6.6 ± 2.0 for lmv, and 9.5 ± 0.9 for etv, which were comparable between less potent adv and most potent etv (p = 0.14). during antiviral therapy, peak log hbv rna level of patients with etv was significantly higher than that of those with adv or lmv (3.2 ± 0.9 vs. background: in the phase iii clinical trials, clevudine 30mg for 6 months showed potent antiviral activity along with a marked post-treatment antiviral effect. the objective of this study is to compare the anti-hbv activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis b (chb) patients in a randomised way. methods: the patients are received clevudine for 24 weeks and then combination of clevudine and vaccine for another 24 weeks or clevudine alone for 48 weeks. eligible patients were treatment-naïve hbeag(+) chb patients with hbv dna levels 500,000 copies/ml. the primary endpoint is the proportion of patients with hbeag loss. preliminary results are presented here. results: thirty-one patients have completed week 24 visits and from them, 15 patients (11 in clevudine alone and 4 in combination group) have completed week 36 visits. at week 24, 26% of patients had hbeag loss. at week 36, 46% in clevudine alone and 25% in combination group (3 months on combination after clevudine monotherapy) had hbeag loss. at week 24, 63% of patients had negative hbv dna by amplicor pcr (<300 copies/ml). at week 36, all of patients in both groups had negative hbv dna by pcr and 73% in clevudine alone and 80% in combination group had normal alt. conclusion: clevudine demonstrated good serologic response as well as significant viral suppression and alt normalization. with this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. background/aims: to determine the reasonable number of clones for hbv quasispecies analysis. methods: 16 chronic hepatitis b patients were enrolled with hbvdna levels range from 4~10 log copies/ml. hbvdna was extracted. hbv reverse transcriptase (rt) gene encompassing the overlapping surface s gene was amplified by polymerase chain reaction, then cloned and sequenced. ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. the characteristics of hbv quasispecies including shannon entropy and genetic distance were calculated. results: the shannon entropy and genetic distance of 10 clones group was higher than those of 20 and 30 clones group, either in rt gene or in s gene (p<0.01). while the shannon entropy and genetic distance of 20 clones group showed on difference with those of 30 clones group, neither in rt gene nor in s gene (p>0.05). the number of different quasispecies detected in 30 clones group was higher than that of 20 and 10 clones group (p<0.01). the shannon entropy and genetic distance in three different clones group had no correlation with hbv dna levels (p>0.05). conclusion: although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than 20. the information contained in 20 clones per sample could well represent the quasispecies characteristics. the clone number was not necessary modulated according to different hbv dna levels. background: recent studies reported that basal core promoter mutation (a1762t and g1764a) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and hcc. but the effect of the double mutations on the activity of enhancer ii/basal core promoter is still uncertain. objectives: to evaluate the influence of nt1762 a/t and nt1764 g/a mutations on hbv enhancer ii/basal core promoter activity. methods: the pcr fragments of hbv enhancer ii/basal core promoter (nt1601 to nt1815) from the serum-derived genotype b hbv dnas of one hbv carrier aged 66 and one hbv related hepatocellular carcinoma patient aged 26 were introduced into the pgl3-basic-vector from promega via restriction sites of xho i and hind iii. the nt1762 a to t and t to a, the nt1764 g to a and a to g mutations were carried out by genetailor site-directed mutagenesis system from invitrogen. the promoter activity was evaluated by comparing firefly luciferase measurement with renilla luciferase as the internal control using the dual-luciferase reporter assay system from promega. results: the luciferase reporter assay results indicated that the 1762 t to a combined with 1764 a to g mutations increase (p<0.001) while the 1762 a to t combined with 1764 g to a mutations decrease (p<0.05) the hbv enhancer ii/basal core promoter activity significantly. conclusions: associated with increased risk of hepatocellular carcinoma, a1762t and g1764a double mutations of hepatitis b virus reduce the enhancer ii/basal core promoter activity. background/aims: a substantial proportion of chronic hepatitis b (chb) patients with mildly elevated alanine aminotransferase (alt) have significant fibrosis. we evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. methods: one hundred five chb patients with alt less than two times the upper limit of normal underwent liver biopsy. multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. results: there were 27 patients in the low normal alt group, 21 in the high normal alt group, 37 in the low elevated alt group, and 20 in the high elevated alt group. fifty eight patients (55.2%) had significant fibrosis ( stage 3) and 35 (21.0%) had significant inflammation ( grade 3). the age, platelet count and grade of inflammation were factors associated with significant fibrosis. progressive liver disease was observed in 23 (27.4%) of the 84 followed-up patients. the stage of fibrosis, alt group and antiviral therapy were significant predictive factors for progressive liver disease. conclusion: liver biopsies should be recommended in patients over 35 years with mildly elevated alt levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. background: four nucleos(t)ide analogues (nas) are currently approved for the treatment of hbv infection in china. however, long-term benefits are limited by the emergence of drug-resistant viruses. methods: patients accepted the examination based on physician's instruction. hbv reverse transcriptase gene was amplified from serum via nested pcr and sequenced directly. results: well-recognized drug-resistant mutations were detected in 567 of 2,000 patients. in patients receiving na monotherapy, corresponding drug-resistant mutations were detected in 214/381 for lamivudine (lam), 35/333 receiving adefovir (adv), 0/57 for entecavir (etv), and 9/17 for telbivudine (l-dt). the mutations were detected in 272/615 patients receiving 31 kinds of sequential/combined usages of the 4 nas. m204i (32%), m204v+l180m v173l (32%), and m204i+l180m (21%) were identified as major mutant patterns of lam monotherapy. n236t a181 substitution was the dominant adv-resistant mutation. t184 substitution was the dominant etv-resistant mutation always accompanied with lam-resistant mutation. l-dt-resistant mutation was m204i l180m exclusively. adv-resistant mutation was frequently seen in lam-resistant patients receiving adv sequential therapy rather than those receiving adv add-on therapy. controversial lam/adv-resistant mutations including a181t, v214a, q215s and i233v were detected in some patients singly or with the well-recognized drug-resistant mutations. interestingly, the drug-resistant mutations were also observed in a few of patients naïve to nas. conclusions: the exploration of hbv drug-resistant mutation profile in large clinical samples furthers our understanding of hbv drug-resistant status in china with implications for administrating anti-hbv therapy more reasonably. toll-like receptor (tlr) 2, tlr4 and cd4+cd25+cd127low/-regulatory t cells correlate with hepatitis b virus infection y. zhang 1 , j.q. lian 1 , c.x. huang 1 , x. wei 1 , j.p. wang 1 , p.z. wang 1 , x.f. bai 1 1 center of infectious diseases, tangdu hospital, the 4th military medical university, xi'an, china background: tlrs play a crucial role in sensing and initiating innate antiviral response and tregs actively suppress immune response, contributing to viral persistence and chronic tissue damage. in this study, we determined tlr2 and 4 expression and treg frequency, as well as their function in the effect of hbv infection. methods: tlr2 and tlr4 expression on monocytes and circulating cd4 + cd25 + cd127 low/-tregs were determined by flow cytometry in 16 ahb, 42 chb, 22 asc and 20 nc. spearman correlation was performed to investigate associated variables on treg or tlrs. pbmcs were stimulated with hbeag or hbcag and the tlrs profile was examined. result: tlr2 expressions were up-regulated in chb and asc, while tlr4 were increasingly expressed in ahb and asc. treg frequency in chb was significantly higher than that in nc. in chb, the increased tlr2 negatively correlated with hbv dna loads and treg frequency negatively correlated with tlr4 expressions. tlr2 was up-regulated after hbeag stimulation in both nc and chb. conclusion: increased tregs may be associated with chb and there might be possible interactions between hbeag, tlr signaling and the innate immune response, which may partially explain the mechanism of hbv infection induced immuno-tolerance. (6.7±1.35) . hbv-dna was quantitatively determined by polymerase chain reaction (pcr) technique, and hbv genotype was determined by pcr microwave gene chip technique. antiviral efficacy was assessed using measuring the following scales: the alt normalization rate, hbv-dna negative conversion rate and the hbeag/anti-hbe seroconversion rate. results: among 55 serum specimen, hbv genotype distribution was 38 genotype c, 14 genotype b, and 3 genotype non-b or c respectively. in genotype b, alt normalization rate was 78.57%(11 cases), hbv-dna negative conversion rate was 35.71%(5 cases) and the hbeag/anti-hbe seroconversion rate was 14.28%(2 cases). in genotype c, alt normalization rate was 76.31% (29 cases), hbv-dna negative conversion rate was 47.37%(18 cases) and the hbeag/anti-hbe seroconversion rate was 18.42%(7 cases). the efficacy of adefovir dipiroxil showed no significant differences between genotype b and c in the treatment of chronic hepatitis b p>0.05 . conclusion: adefovir dipiroxil is an effective antiviral drug. hbv genotype is irrelevant to the antiviral efficacy of adefovir dipiroxil in treatment of patients with chronic hepatitis b. the effect of anti-hbv drugs on albumin and bilirubin levels, and platelet count h. yoshida 1 , h. taniguchi 1 , r. nagano 1 , k. sakitani 1 , e. seki 1 , t. serizawa 1 , y. ito 1 , h. mizuno 1 , y. mitsuno 1 , r. nakata 1 , m. omata 2 1 japanese red cross medical center, 2 university of tokyo, japan background/aim: we assessed the efficacy of anti-hbv drugs on the liver function. methods: patients with hbv-related disease followed at our center between 2005 and 2007 were enrolled. lamivudine (100mg), lamivudine (100mg) +adefovir (10mg), or entecavir (0.5mg) was administered to the patients with detectable hbv dna and elevated alt. liver function (alt, alb, and t.bil) and platelet count were observed. alt, alb, t.bil, and platelet count of treated group at pretreatment, year 1, and year 2 were compared with untreated group. results: eighty six patients with positive hbsag were enrolled between jan 2005 and dec 2007. seven patients (2 acute infection, 1 overlap infection with hcv, 7 lost of follow up) were excluded. in total 76 patients were followed up for a median follow up of 26 (range 2-39) months. of 76 patients, 32 received anti-viral treatment. twenty one patients were treated with lamivudine, 4 with lamivudine+adefovir, and 7 with entecavir. the mean of levels of pre-treatment-year1-year2 were alt:129-37-43 (u/l), alb:4.0-4.4-4.4 (g/dl), t.bil:1.0-0.8-0.7 (mg/dl), and plt:14.3-15.4-16.0 (x10 4 mcl) respectively. markers of untreated group (n=44) (at baseline-year1-year2) were alt:33-40-35 (u/l), , t. bil:0.8-0.8-0.9 (mg/dl), and plt:18.7-18.0-18.5 (x10 4 mcl) respectively. although all of four markers in treated group were significantly worse than untreated group at baseline, all of four markers did not showed significant difference from untreated group at year2. conclusion: treatment with anti-hbv drugs showed the efficacy not only transaminase levels, but also on albumin, bilirubin, and platelet count improvement-improvement of "hepatic reserve" which is valuable for prevention of cirrhosis. background: currently, hbeag-negative chronic hepatitis b(chb) is increasing. but there are still controversial on the treatment of hbeag-negative chb with alt 2×uln. we have investigated the clinical efficacy of nucleotide analogues(nas) in the treatment of hbeag-negative chb with alt 2×uln. methods: the data of patients who were treated by nas for more than 2 years and with alt 1 2×uln (n=87) , alt 1×uln(n=43) and alt 2×uln(n=88) were collected, and 12w and 24w virologic response, 48w and 96w complete response, virologic breakthrough and clinical resistance were analyzed. results: compared with the base line, hbv dna level in all three groups were significantly decreased (p 0.01), and there was no significant difference between alt 1 2×uln group and alt 2×uln group. the viral load was significant decreased in alt 1×uln group at 12w, 24w and 36w (p<0.05). virologic response at 12w and 24w complete response at 48w and 96w was 71.3%, 82.8% , 82.8% and 86.2% respectively in alt 1 2×uln group and was 51.2%, 60.5%, 65.1% and 74.4% respectively in alt 1×uln group. there was no significant difference between alt 1 2×uln group and alt 2×uln group. virologic response at 12w and 24w and complete response at 48w were significant decreased (p<0.05) in alt 1×uln group. there was no significant difference among the three groups in virologic breakthrough and clinical resistance. conclusion: hbv replication can be satisfactory inhibited by nas in hbeag-negative chb patients with alt 2×uln, which suggests that in these patients the indication of alt is different from hbeag-positive patients. quantitative hbeag assay as a predictive factor of hbeag seroconversion induced by peg-ifn -2a therapy to hbeag-positive chronic hepatitis b y.y. zhu 1 , j. dong 1 , y.t. chen 1 , j. chen 1 , j.j. jiang 1 1 liver diseases research center, the first affiliated hospital of fujian medical university, fuzhou, fujian, china rp, 350005 background: to find predictive factor for hbeag seroconversion in the treatment of hbeag-positive chronic hepatitis b (chb) by peg-ifn -2a. methods: hbeag-positive chb patients were given peg-ifn -2a treatment for 48 weeks. clinical data were collected every 3 months. receiver operator characteristic (roc) curve was employed to calculate positive predictive value (ppv), negative predictive value (npv), sensitivity and specificity. results: sixty-five patients completed peg-ifn -2a therapy. among them, 17 (26.15%) were found hbeag seroconversion and 19 (29.23%) were found hbeag loss at cessation of therapy. none of age, gender, alt level and hbv dna load at baseline had relationship with hbeag seroconversion. hbeag level of baseline was correlated to hbeag seroconversion, with p value as 0.003 (table 1) . according to roc curve, supposed auc as 0.705 and p value as 0.042, the ppv, npv, sensitivity and specificity of hbeag level as 61 at 12 week were 0.361, 0.862, 0.765 and 0.521, respectively. supposed auc as 0.828 and p value as 0.001, the ppv, npv, sensitivity and specificity of hbeag level as 15 at 24 week were 0.452, 0.912, 0.824 and 0.646, respectively. the hbeag level (s/co) and decreased degree (percentage) at 12 week and 24 week were significant related to hbeag seroconversion (table 2) . conclusion hbeag level at baseline and at 12th and 24th week and its decreased degree (percentage) during the treatment course could be used as predictive factor for hbeag seroconversion. background: it is well documented that perinatal transmission is the major cause of chronic hbv infection in china. the aim of this study was to evaluate the efficacy of interruption of hbv intrauterine infection with hepatitis b immunoglobulin (hbig) in pregnant women with hbeag positive. methods:: a prospective randomized controlled trial was adopted. each subject in the trial group (28 cases) was given 200 iu hbig intramuscularly every 4 weeks from 28-week of gestation, while each subject in the control group (24 cases) received placebo in the same way. the cord blood of newborns were collected for detecting hbsag, hbeag and hbv-dna. results: for newborns, hbeag positive rate in trial group was 21.4%(6/28).hbeag positive rate in control group was 79.2%(19/24). there was significant difference in hbeag positive rate of newborns between the two groups( p < 0.01, rr = 0.27). hbv-dna positive rate in trial group was 25%(7/28). hbv-dna positive rate in control group was 83.3%(20/24). there was significant difference in hbv-dna positive rate of newborns between the two groups( p < 0.01, rr = 0.30). hbv-dna load of 7 cases of newborns in trial group was lower than that of their mothers(t = 28,p = 0.02). there was no significant difference in hbv-dna load between women and their newborns after delivery in control group (t = 81.5,p > 0.1). conclusion: it is effective and safe to prevent hbv intrauterine infection with hbig from the 28(th) wk in pregnant women with hbeag positive. ), especially, the cirrhosis and hcc cases obviously more in both hbeag and anti-hbe patients are negative than hbeag-negative but anti-hbe positive patients (.36.7% vs 26.7%; 7.9%vs5.5%, p ) .the prevale nce of pre-core g1896a mutate have no significant difference regardless of hbv serum marker status or the state of illness. conclusion: recent 5 years the hbeag-nagative chronic hepatitis b patients are gradually increasing in yunnan province. while the hbeag disappear but no anti-hbe serum transfer, and the virus still active replication -it may be a crucial phase determined the diseases outcome, which should be pay more attention by physicians. the clinical significant of pre-core g1896a mutate remain unknow. efficacy of interferon for chronic hepatitis b patients with normal or paranormal alt z. liu 1 , j.z. guo 1 , y.j. lin 1 , y.j. zhang 1 , z.w. lang 1 1 beijing ditan hospital, beijing, china background: we reported interferon treatment for 4 cases with normal or paranormal alt but in which liver histologic exam showed g2-4 and/or s3-4. methods: 4 patients were male with an average age of 28 years.mean alt was 46.7 iu/l and hbv dna level was 3~5 log10copies/ml. two patients were hbeag positive; one patient was both negative for hbeag and anti-hbe and one patient was anti-hbe positive. liver biopsy showed g2~g4 and s3~s4 respectively. 3 patients were treated with ifn-alpha, liver biopsy was repeated after 1 year.only one patient had received combination therapy with ifn and adefovir after 10 months treated ifn monotherapy and liver biopsy was taken after 1.5 years. results: all patients got normal alt after 1 year treatment. hbv dna was undetectable in 3 patients. 2 patients with initial positive hbeag cleared. but 3 patients still were anti-hbeag negative.liver biopsy showed change fromg4 s3-4 to g2 s2-3 in 1 patient; fromg2 s3 to g3 s4 in 1 patient and no change in the other 2 patients. conclusions: though alt and hbv dna improved after 1 year treatment, histological improvement is not satisfying. 1 patient's improvement in liver histology may be due to seroconversion before treatment and adding adefovir after 10 months of interferon therapy. after 8 months of combination therapy we did liver biopsy again. the other 3 patients were hbeag negative, but hbeab were also negative, liver biopsy was taken 1 year later without combination of nucleoside analogs. evaluation of long term efficacy of hepatitis b vaccination r.c. li 1 , j. gong 1 , j.y. yang 1 1 objective: to evaluate the long term effectiveness of preventive hbv infection and to monitor the incidence of hepatitis b in children to see possible impact on the program of long an that was launched in 1986. methods: (1) set up a surveillance systemof hepatitis ,to evaluate the possible impact on incidence of hepatitis b. (2) to serologically evaluate the effect of the program, a stratified random sampling of 1000 subjects in 1987 birth cohorts was recruited for long term follow up at the age 1-20 years. (3) cross-sectional seroepidemiolgical survey was carried out in the county in 1985 before the program and 20 years later. hbsag , anti-hbs and anti-hbc were tested by ria. results: the average coverage of hepatitis b vaccine was 89. 1 %. at 20 years after vaccination, the seropositivity for hbsag in population of 1-20 years has decreased from 16. 0 % to 2. 3 %, the annual effectiveness was 89. 7 %. hbv accumulated infection rate was 3. 5 %, protective rate was 96. 2 %. the incidence of acute hepatitis b was 1. 60 per 100,000 in population aged 1 -20 years , it decreased by 91.3 % as compared with the incidence of 18.38 per 100,000 in same age group in 1985 -1987. conclusion: mass hepatitis b vaccination program in long an county has proved to be effective in control of hbv chronic infection and incidence of acute hepatitis b. background and aims: although the evolution of viral quasispecies may be related to the pathological condition of disease, little is known about this in hepatitis b virus (hbv), especially during hbeag seroconversion. methods: nucleotide sequences of hbv precore/core genes from 5 time points were analyzed in four cohorts of chronic hepatitis b, interferon-induced seroconverters (is, n=9), interferon non-responders (in, n=9), spontaneous seroconverters (ss, n=9) and non-seroconverters (sn, n=9), followed during 60 months on average. only patients with genotype c were used. viral diversity was then estimated after nucleotide genetic distance was assessed and phylogenetic trees were constructed. results: analysis of 1800 nucleotide sequences showed that the nucleotide genetic distance of seroconverters (is and ss; 9x10 -3 substitutions/site and 8.5x10 -3 subsititutions/site, respectively) was similar to that of non-seroconverters (in and sn; both 7x10 -3 substitutions/site) before seroconversion. compared to that of nonseroconverters (in and sn; substitutions/site and 7.5x10 -3 substitutions/site, respectively) the viral diversity of seroconverters (is and ss; 13x10 -3 substitutions/site and 12x10 -3 substitutions/site, respectively) was significantly higher after seroconversion (p<0.05) and it was higher after seroconversion in seroconverters compared with that berore seroconversion (p<0.05) while it almost didn't change in non-seroconverters irrespective seroconversion. phylogenetic trees also showed that complex trees appeared in secoconverters and relatively simple in nonseroconverters. conclusions: the distinctly higher viral diversity after seroconversion in hbeag seroconverters could be related to increased hbv-specific t-cell responses and escape mutant which arise from stronger selective pressure caused by host immune activity. adefovir dipivoxil 10mg (adv) resistance at 5 yrs in chinese hbeag+ve chronic hepatitis b (chb) j.l. hou 1 , y.z. wang 2 , x.q. zhou 3 , j.q. niu 4 , y.m. wang 5 , h. wang 6 , y.m. mao 7 , k.f. barker 8 1 nanfang hospital, guangzhou, prc, 2 jinan infectious disease hospital, jinan, prc, 3 ruijin hospital, shanghai, prc, 4 1st hospital of jilin university, changchun, prc, 5 xinan hospital, chongqing, prc, 6 people's hospital, beijing, prc, 7 renji hospital, shanghai, prc, 8 glaxosmithkine r&d, london, uk background: long term adv provides clinical and histological improvement in chb, but may lead to emergence of treatment associated resistant mutations. we report on adv resistance data from chinese hbeag positive subjects treated for 5 years. methods: 480 hbeag positive chb subjects were randomized in an initial 52 weeks controlled adv study (with a 12 weeks placebo period in half of patients) and then offered open label adv treatment for a further 208 weeks. a total of 474, 456, 443, 421 and 390 subjects completed the 1 st , 2 nd , 3 rd ,4 th and 5 th yr, respectively. at the end of each year samples were analysed from those subjects with protocol-defined hbv dna breakthrough for the rtn236t or rta181v adv mutations associated with resistance. sera from subjects with breakthrough were analysed at all subsequent yearly timepoints whenever possible. results: at the end of the 1 st yr, none of the 45 subjects with hbv dna breakthrough had either mutation. sera were available for analysis from 137, 199, 228 and 187 subjects with viral breakthrough at the end of the 2 nd , 3 rd , 4 th and 5 th yr, respectively, with new mutations identified in 6, 20, 24 and 20 subjects at the same timepoints. of the cumulative 70 subjects at the 5 th yr analysis 31 had rtn236t, 29 had rta181v, and 10 had both mutations. conclusion: treatment with adv in chinese hbeag positive chb subjects for up to 5yrs resulted in a cumulative rate of 14.6% (70/480) adv resistance-associated mutations with hbv dna breakthrough. background and aims: to evaluate the predictive significance of rapid virologic response (rvr) for achieving an end-of-treatment virologic response (er) or hbeag seroconvertion and the predicting indicator of nonresponse (nr). methods: 205 patients with chronic hepatitis b were treated with adv and prospectively observed to 48 weeks. we assessed the values of virus load reduction at weeks 4, 8, and 12 weeks to predict the er and hbeag seroconversion. the association between less reduction of viral load at 12 and 24 weeks and nonresponse was also analyzed. results: of 146 etv-treated patients enrolled in etv-901, 98 met criteria for inclusion into 5 year etv treatment analyses. the proportion of patients achieving efficacy endpoints through 5 years of etv therapy is presented the table. results: after 48 weeks of therapy, serum hbv dna levels decreased with a median 4.07±2.47 log10 copies/ml. twenty-three(11.2%) of patients had er. twenty-six (12.7%) patients achieved hbeag seroconversion. hbv dna <4 log 10 copies/ml at week 4 predict both er and hbeag seroconversion. hbv dna> 4log10 copies/ml at 4 weeks but decline to <4log10 copies/ml at 12 weeks or 24 weeks both can predict er and hbeag seroconversion. less than 2 log 10 hbv dna reductions at 24 weeks might predict nr. conclusions: the majority of patients experienced durable serum hbv dna suppression (94%) and alt normalization (80%) after 5 years etv therapy. conclusions: the virologic response within 4 weeks could be useful for prediction of er and hbeag seroconversion of adefovir therapy. failing to evr might not predict nr. objectives: to determine the accuracy of hbcigm in diagnosing ahb and the correlation between hbcigm and liver inflammation (alt), bilirubin & biosynthetic functions (albumin,pt). result: a total of 233 patients were included: in 40 patients, adv was added on lam (add-on therapy), and in 193 patients, lam was switched to adv (switch therapy). during 16.5 months of follow-up, 25 patients developed adv resistance (rta181v and/or rtn236t) and all had undergone switch therapy. the cumulative probability of adv resistance at the 24th month was 17.5%. although add-on therapy induced no adv resistance, it failed to show significant superiority over switch therapy (p=0.220). in multivariable analysis, female (odds ratio [or], 2.75; 95% confidence interval [ci], 1.22-6.17; p=0.014), liver cirrhosis (or, 2.39; 95% ci, 1.07-5.33; p=0.033), and age >45 yr (or, 3.31; 95% ci, 1.14-9.66; p=0.028) were independent risk factors of adv resistance. methods: a retrospective cross-sectional study involving patients with hbcigm positivity between june 2006-december 2007, satisfying the definition for ahb and chbf,and fulfilling the exclusion criteria was performed. hbcigm test were done by using microparticle enzyme immunoassay (meia) and results were expressed as an index value.hbcigm positivity was defined as index value of >1.00 results: 74 patients were positive for hbcigm and 60 fulfilled the criteria(33 ahb, 27 chbf).hbcigm was significantly higher in ahb compared with chbf(median 3.46 vs 1.70;p <000.5).the hbcigm arbitary index value of 2.76 was highly sensitive(97%) and specific(85%) in diagnosing ahb with high accuracy(auroc 0.964;95% ci:0.925-1.003;p<0.0005).among patients in both groups, there was a weak, but significant negative correlation between hbcigm and pt above control(r = -0.309,p =0.016).however, among patients with chbf,the negative correlation between hbcigm and pt above control was moderately strong(r = -0.557,p =0.003).there was also a weak, but significant positive correlation between hbcigm and albumin in with chbf(r = +0.434,p =0.024). conclusion: adv add-on therapy developed no adv resistance during the observation period. therefore, add-on therapy is recommended to lam-resistant chb patients with genotype c who have any risk factors for development of adv resistance: female, liver cirrhosis, and age >45 yr. hbcigm-hepatitis b core igm antibody ahb-acute hepatitis b,chbf-chronic hepatitis b flare alt-alanine transaminase,pt-prothrombin time pe117 detection of emerging drug resistance mutations associated with major approved hbv antivirals using a novel line probe assay (lipa). j. doutreloigne 1 , f. shapiro 1 , r. maertens 1 , e. van assche 1 , e. sablon 1 1 hepatitis diagnostics unit, innogenetics nv, belgium background: in study etv-022, etv demonstrated superior virologic, histologic and biochemical benefit compared to lamivudine (lvd). this study (etv-022/-901) presents efficacy and safety results for patients who received 5 years continuous etv treatment. background/aims: an increasing number of antiviral drugs are being used to treat chronic hepatitis b virus (hbv)-infected patients. however, induced viral escape mutants -some potentially cross-resistant -lead to viral non-responsiveness and treatment failure. effective treatment strategies must therefore take possible drug resistance (dr) into account with respect to monitoring and selection of alternative drugs. we evaluated the use of an updated inno-lipa hbv dr v2+v3 reverse hybridization assay versus sequence analysis to detect resistance mutations. methods: the study evaluates etv-treated nucleoside-naïve hbeag (+) patients who completed etv-022 and enrolled into etv-901 with a treatment gap 35 days. the proportion of patients with hbv dna <300copies/ml, alt normalization, hbeag loss or hbeag seroconversion was evaluated at week 240. background: etv resulted in improved liver histology compared to lvd at 1 year. histologic data for patients on etv for a median of 6 years is evaluated. methods: 273 clinical samples (from untreated hbv patients or treated with different antivirals; hbv genotypes a-h) were tested for mutations with the lipa assay and sequencing. for lipa, samples were extracted with the qiaamp® dna blood mini kit (qiagen), and then tested on the lipa strips. sequencing-derived reference data were subjected to phylogenetic analysis (kodon version 3.10 applied maths, neighbour joining, with kimura-2 parameter). sequential samples from 9 patients were evaluated as well. methods: etv-treated patients completing etv-022 or etv-027 received etv (1.0mg daily) in etv-901. primary endpoints included 2-point decrease in knodell necroinflammatory score, no worsening of knodell fibrosis score and improvement in ishak fibrosis score (ifs) ( 1-point decrease) vs. baseline. secondary endpoints included proportions with hbv dna<300 copies/ml, alt normalization, and ifs normalization in patients with advanced fibrosis/cirrhosis. results: quasi-perfect concordance (> 99.6%) was obtained between the two assays for the samples tested. no indeterminate results were observed. for one sample, lipa provided additional information (wild-type/mutant mix), whereas sequencing showed only wild type. for sequential samples, lipa was clearly able to detect emerging treatment-resistance mutations associated with viral breakthrough. results: etv treatment led to significant histological improvements and improved ifs in 96% (55/57) and 88% (50/57) of patients respectively. of 10 patients with baseline fibrosis/cirrhosis (ifs 4), all demonstrated 1-point improvement in ifs (median change of -3). conclusions: lipa accurately detects the complex quasispecies nature of hbv and can help unravel the dynamics of emerging hbv resistance during treatment with different antiviral drugs. like its predecessor, it is useful for the monitoring and early detection of drug resistance. conclusions: long-term etv therapy in nucleoside-naïve chb patients results in durable virologic suppression, continued histologic improvement and regression of fibrosis/cirrhosis. precore (pc, g1896a) and basal core promoter (bcp, a1762t and g1764a) mutations of hbv are important for predicting the risk of hepatocellular carcinoma (hcc). we developed a new mass spectrometry-based assay using restriction fragment mass polymorphism (rfmp) to detect a1896 and t1762/a1764 mutations, and applied it to analyze their clinical significance in type b liver diseases (n=336), including 35 hccs, 118 liver cirrhosis (lc), 157 chronic hepatitis b (chb), and 25 hbsag-positive with low level viremia (inactive hbsag carrer, ihc). we devided patients into 4 major groups according to the presence of wild (w) or mutant (m) genes in bcp/pc regions; w/w, w/m, m/w and m/m gene types. each proportion was 9.5%, 3.6%, 41.4% and 23.5%, respectively. mixed infection (x) was also found as minority; 4 w/x, 28 m/x, 15 x/w, 4 x/m and 13 x/x. disease distributions (hcc, lc, chb and ihc) in each group were as follows; [w/w (n=32)] 3.1%-6.3%-90.6%-0; [w/m (n=12)] 0-16.7%-58.3%-25%; [m/w (n=139)] 9.4%-42.5%-46%-2.2%; [m/m (n=79)] 11.4%-45.5%-20.1%-13.9%. these results suggest that, in korea where only genotype c has been identified, bcp dual mutation is predominant (>73.2%), while bcp wild alone is only 13.1%. especially, a1896 mutation alone without bcp mutation (w/m type) is uncommon, while bcp mutation alone without a1896 mutation (m/w type) is most common. it might be suggested that prognosis of wild type in bcp and pc region (w/w type) is much better than that of m/w or m/m types. background/aims: entecavir is a potent inhibitor of hbv dna polymerase, which has been shown to be safe and effective for the treatment of chronic hepatitis b (chb) patients. the aim of this study was to evaluate the virologic, biochemical, and serologic responses of entecavir through 1 year in chb patients. methods: from 2007 may to 2008 october, we reviewed 82 patients (mean age 46±12 years, male:female=57:25) who were diagnosed as chb patients (hbeag (+) 64). forty-seven patients (57.3%) had been treated with 0.5 mg of entecavir and 35 (42.7%) with 1 mg of entecavir, respectively. mean follow-up period was 25±17 weeks. hbv dna was quantified by bdna assay with a lower limit of detection of 141,500 copies/ml. results: median hbv dna levels before therapy was 7.64 log10 copies/ml and the median decreases from baseline in hbv dna were -4.19, -4.31, -5.09, -5.31, and -5.59 log10 copies/ml at 4 (n=39, p<0.001), 12 (n=62, p<0.001), 24 (n=42, p<0.001), 36 (n=22, p<0.001), and 52 (n=15, p =0.053) weeks of follow-up, respectively. at baseline, overall median alt was 106 iu/l and the proportions of patients with normal alt were 13%, 48%, 56%, 71%, 67%, and 69% at baseline (n=82), 4 (n=42), 12 (n=63), 24 (n=44), 36 (n=24), and 52 weeks (n=16) after entecavir therapy, respectively. thirteen cases (15.9%) of hbeag seroconversion were noted. background: hepatitis b virus (hbv) infection is a major risk factor for the progression of liver diseases. because its clinical course varies, it is difficult to detect the predictive factor for the prognosis of patients with hbv infection. the aim of the present study was to determine the risk factors for the occurrence of hcc. methods: a total of 620 patients who tested positive for hepatitis b surface antigen and were referred to chiba university hospital between february 1985 and march 2008 were included in the study, and their following characteristics were analyzed: age, gender, the status of hbeag, alt, hbv-dna level, and plt. result: hcc was detected in 30 cases during the follow-up period (5.4 ± 5.1 years). multivariate analysis revealed that age [compared with young patients: odds ratio (or) = 1.07, 95% confidence interval (ci) = 1.03-1.11] and plt level (compared with patients with low plt level: or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. in patients with age more than 35 years, the hbv-dna level (compared with <5.0 log copies/ml: or = 3.29, 95% ci = 1.40-11.5) and plt level (or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. conclusion: advanced age and low plt level were the risk factors for hcc occurrence in patients with hbv infection irrespective of the plt level at baseline. in patients with age more than 35 years, viral load was also a risk factor for hcc. results: before treated by lps, the total mapk p38 level of pbmcs have no significant difference among the healthy control group, different stage groups with hbv infection, however, after treated with lps, the phosphorylated mapk p38 (ptpy180/182) in healthy control group are significant elevate than hbv infected groups(120.0±52 vs 80.2±84.8, p<0.05). in the two groups which hbsag, hbv dna are positive, alanine aminotransferase elevate than normal and hbsag positive, but hbv dna lower under the detect limited level, after treated by lps the ptpy180/182 although lower than healthy control yet, but significant elevated than themselves before treated by lps(86.6±38.6 vs117.8±21.3, 63.3±24.7 vs 93.1±21.8; p<0.05).otherwise, in the group of both hbsag and hbv dna are positive, but alt is normal, before and after treated by lps, the level of ptpy180/182 have no significant difference. conclusion: mapk p38 is a important signal transduction pathway which involving in inflammation and immune response, especially, mapk p38 activated up-regulate the ifn-gamma mrna. according to the result shown, we propose a hypothesis, hbv infection and virus active replication inhibit the mapk p38 activated, consequent on host immunotolerance and hbv persistence, thus, mapk p38 may be as a potential therapeutic target to break immnotolerance and establish host anti-viral states. van der helm 10 1 siriraj hospital, bangkok, thailand, 2 ramathibodi hospital, bangkok, thailand, 3 phyathai hospital, bangkok, thailand, 4 singapore general hospital, singapore, 5 national university hospital, singapore, 6 changi general hospital, singapore, 7 cheil general hospital, korea, 8 hwasun jeonnam university hospital, korea, 9 st mary hospital, korea, 10 roche diagnostics ltd, rotkreuz, switzerland background/aims: hepatitis b virus (hbv) surface antigen (hbsag) is one of the most important markers for diagnosis of acute and hbv infection. high sensitivity of hbsag assays can reduce the diagnostic window during course of disease. in addition, the presence of hbv mutants may be affected by the performance of the hbsag kit. therefore, the technical performance of the elecsys ® hbsag ii assay was explored, using samples (including recombinant mutants), at multiple sites in three countries. methods: nine hbsag screening centers in thailand, korea and singapore compared the sensitivity of elecsys ® hbsag ii assay with that of their routine testing procedure -abbott architect ® (7 centers), abbott axsym ® (1 center) and bayer advia ® centaur hbsag assays (1 center) using preselected seroconversion panels (n=5), recombinant hbv mutant panels (n=13) and routine clinical practice samples (n=1,863). results: the sensitivity of elecsys ® in seroconversion samples was equivalent to the architect ® assay, but more sensitive than the axsym ® and advia ® centaur assays (26 vs 23 and 24 vs 18 positive bleeds, respectively). there was concordance between the elecsys ® and architect assay results with respect to potentially cross-reactive samples (99.74%). the elecsys ® and architect ® assays detected all recombinant mutant samples, whilst axsym ® and advia ® centaur failed to detect three and nine samples, respectively. conclusion: elecsys ® hbsag ii assay was not only highly sensitive and specific when compared with established hbsag screening assays, but also reliably detected hbsag mutants. therefore, this attractive assay is suitable for hbv diagnosis and assessing safety of blood products. background: recent studies have shown a higher rate of adefovir-resistant mutation in lamivudine-resistant chronic hepatitis b (chb) patients treated with switch-to therapy than those treated with add-on therapy. we compared the clinical efficacy of adefovir monothrapy and lamivudine-adefovir combination therapy in lamivudine-resistant chb. methods: a prospective cohort study was performed in 49 patients with lamivudine-adefovir combination therapy and 77 patients with adefovir monotherapy for lamivudine-resistant chb over 18 months. result: biochemical response was achieved in 41 patients (83.7%) treated with combination therapy and in 77 patients (92.8%) treated with monotherapy (p=0.101). virologic response was observed in 19 patients (38.8%) in combination therapy and in 26 patients (31.3%) in monotherapy (p=0.383) and treatment periods for virologic response was significantly shorter in patients with combination therapy than in monotherapy (8.9±4.8 months vs. 13.9±5.0 month, p=0.001). cumulative rate of virologic response was significant higher in patients with combination therapy than monotherapy (p=0.033). hbeag loss was found in 6 patients (16.2%) in combination therapy and 17 patients (20.5%) in monotherapy (p=0.485). biochemical breakthrough was found in 17 patients (20.5%) with monotherapy significantly more frequent than 3 patients (6.1%) with combination therapy (p=0.026). genotypic resistance to adefovir was developed in 1 patient (2.0%) in combination therapy and 9 patients (10.8%) in monotherapy conclusion: to achieve a complete virological response and reduce the risk of adefovir-resistant mutants in lamivudine-resistant chb patients, adefovir in combination with lamivudine is preferable. background/aims: adefovir dipivoxil (adv) effectively inhibits both wild-type and lamivudine (lam)-resistat chonic hepatitis b virus (chb) replication. the aims of this study were to determine the factorts associated with antiviral effect of adv in lam-resistant chb. methods: one hundred-eighteen lam-resistant chb patient (74.6% hbeag-positive) were treated with adv plus lam (n=96) or adv monotherapy (n=22) for a mean of 33.0 months. restriction-fragment mass polymorphism analysis was used for detection ymdd and adv mutants. results: fifty-eight patients (49.2%) achieved complete response(cr) defined as hbv-dna levels <2000 copies/ml and alt normalization. twenty-eight patients (23.7%) achieved initial vilologic response(ivr) defined as hbv-dna levels <10 4 copies/ml within the first 6 month of treatment. 47 (53.4%) of 88 hbeag-positive patients exhibited hbeag loss and 31% seroconverted to anti-hbe ab. five (4.23%) patients developed adv-related mutations. factors associated with ivr were pretreatment level of alt (p=0.00), ast (p=0.00), pretreatment hbv dna level (p=0.03), hbeag negativity (p=0.01) and hbeab positivity (p=0.00). factors associated with cr were ivr (p=0.00), hbeab positivity (p=0.01), pretreatment level of alt (p=0.01), ast (p=0.02) and y-glutamyl transferase (p=0.04). age, sex, presence of liver cirrhosis, pretreatment hbv dna level and the type of ymdd mutants were not related to an cr during adv treatment. conclusions: adv therapy achieved cr in more than 49% of lam-resistant chb. factors associated with cr were ivr, hbeab positive status, high base line alt, ast, ggt levels. q.j. sheng 1 , y. ding 1 , x.g. dou 1 1 department of infectious disease, shengjing hospital affiliated to china medical university, shenyang, 110004, china objectives: to study a kinetics of hepatitis b virus during 12-week and 24-week of treatment with enticavir (ent); to compare the detecting results of hbvdna levels from different detection reagents. methods: thirty-seven cases of chronic hbv infections were selected randomly, treated with daily dose of ent 0.5-1.0mg (0.5mg for nucleoside-naïve patients, 1.0mg for lamivuding-refractory patients). evaluation indexes: serum hbvdna, hbv serological markers, and liver function tests. hbvdna levels were measured by pcr assay, using both domestic reagents and roche cobas amplicor,. the lower limits of measure level of hbvdna were 1000 copies/ml and 300 copies/ml, respectively. results: mean baseline of hbvdna was 5.24log10copies/ml for detection using domestic reagents and 5.70log10copies/ml for that using roche cobas amplicor, (p>0.05). the ratios of cases with undetectable (<1000 copies/ml) hbvdna at week-12 and week-24 were 46.2% and 72.9%, respectively. the ratios of cases with undetectable (<300 copies/ml) hbvdna at week-24 were 54.5%. among the cases whose hbvdna were lower than 1000 copies/ml(using domestic reagents), the ratio of hbvdna lower than 300 copies/ml(using roche cobas amplicor) was 94.7%. conclusions: ent can suppress hbv dna rapidly no matter the patients with alt elevation or not. there is a concordance on hbvdna levels detection between domestic reagents and roche cobas amplicor. background the study on the effect of nucleoside analogue therapy on the quantity of hepatocellular cccdna and tdna and sera hbv dna, hbsag to probe reliable marks for evaluation of therapy endpoint. methods the quantity of hepatocellular cccdna and tdna and sera hbvdna were assayed by fq-pcr, and sera hbsag by elisa in 4 chb patients over 2 years nucleoside analogue therapy satisfied the china criteria of therapy endpoint (therapy group)and 12 chb patients without antiviral therapy and sera hbvdna<10 3 copies/ml(control group). results: the quantity of hepatocellular cccdna, tdna and sera hbvdna, hbsag in therapy group were lower than that of control group,but low level hepatocellular cccdna in therapy group could be detected. conclusion: long term nucleoside analogue therapy may consume hepatocellular cccdna with decreasing of hepatocellular tdna and sera hbvdna, hbsag; although the patients have satisfied the china criteria of therapy endpoint, low level of hepatocellular hbvcccdna were detected, cessation of therapy may cause relapse. peptides that lead nuclear entry of nucleocapsid of hepatitis b virus in hepg2.2.15 cells x.b. pan 1,2 , l. wei 1,2 , j.c. han 1,2 , k. deng 1,2 1 peking university hepatology institute, 2 peking university people's hospital background: the nuclear entry of nucleocapsid is a key step for the hbv life cycle and the formation of covalently closed circled dna (cccdna). it has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (nls). whereas hbcag was primarily distributed in cytoplasm and no marked cccdna was detected in hepg2.2.15 cells. methods: we designed peptides containing a cell-penetrating sequence (rrrrrrr) and a nucleocapsid binding sequence (gsllgrmkga) with/without a classic nuclear localization sequence (pkkkrkv) and these sequences were linked by a soft linker acp. hepg2.2.15 cells were treated with the peptides at levels of 0 m, 25 m and 50 m for 4 days. results: compared with that of control cells, the results showed hbv dna levels in culture medium decreased at least 2 log10 both in 50 m of peptide rrrrrrracpgsllgrmkga treatment group and rrrrrrracpgsllgrmkgaacppkkkrkv treatment group; whereas hbsag and hbeag increased at 1.34+0.21 folds and 2.15+0.37 folds respectively. the signal strength of cytoplasmic hbcag increased at about 2.5-fold in both groups. in rrrrrrracpgsllgrmkgaacppkkkrkv treatment group, nuclear hbcag increased about 3.2-fold and obvious cccdna signal was detected by southern blot. conclusion: our results implied that the nls of core protein likely does not expose to surface of nucleocapsid in hepg2.2.15 cells, the artificial peptide containning nls binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccdna. our study presents a tool for study on cccdna formation and nuclear entry of nucleocapsid. b. tang 1 , j. xia 1 , y.m. wang 2 , h.f. wang 1 1 liver failure treatment and research center, 302rd military hospital, 2 the dept. aims: to establish a reliable real-time fluorescence quantitative (rfq) pcr method to quantify hbv cccdna, basing on lightcycler system and taqman probe. methods: hbv genotypes a-g were aligned to obtain a conserved sequence, crossing rcdna gap, which was used to design cccdna primers and taqman-mgb probe. also another pair of primers for quantify hbv total dna (tdna) was designed, utilizing the same probe. to increasing specificity, we added plasmid-safe atp-dependent dnase (psad) digestion step just before cccdna pcr amplification. a standard curve from 9 standard plasmid samples, from 2.0×10 9 to 2.0×10 1 copies, was created to examine our system. 50 hbv cccdna samples with known-amount were quantified by creating standard curve from 5 standard samples. results: the standard curve had clear log-phase and excellent parallelism, which means nice and equal amplification efficiency in all reaction capillarys. the slope (regression coefficient) of standard curve was -3.141, mean square error was 0.0990 and regression coefficient was -1.0. all of these key indexes measured up. in 50 tests, we got right results if the starting templates of cccdna copies were between 10 2~1 0 9 copies. the range was superior to commercial hbv kits. by quantifying 16 samples containing different amounts of cccdna and rcdna, digested or undigested, psad digestion would eliminate 10 7 rcdna molecules, leaving 10 2 cccdna molecules untouched. the test specificity was maintained up to 1:100,000 ratio of cccdna:rcdna. conclusions: the rfq-pcr based on lightcycler system for hbv cccdna quantification is reliable, sensitive, with high specificity and low cost. background: to study the changes of toll-like receptor (tlr)3 on dendritic cells derived from peripheral blood mononuclear cells(modc) and its role in the pathogenesis of chronic hepatitis b(chb) and chronic severe hepatitis b(cshb). methods: the expressions of tlr3 on 10000 modc were stimulated by poly i:c, and then were determined by flow cytometry in 20 healthy controls, 28 patients with chb and 30 patients with cshb.the level of interferon ifn-was determined by elisa. the differences of expression of tlr3 on modc and serum ifn-among the three groups of study subjects were determined by student-t test.the correlation between tlr3 and ifn-were determined by linear correalation test. results: the values of mean fluorescence intensity(mfi) of tlr3 on modc of the healthy controls, patients with chb and cshb were 1593.00±349.65,1369.56±287.08,and 1203.96±192.40.the serum ifn-(pg/l) of respective groups was 172.66±37.96,107.98±31.15 and 72.06±29.58. there was a gradual decrease of these values from the group of healthy controls to the group of patients with chb and cshb .significant positive correlations between tlr3 and serum ifn-were found. conclusion: tlr3 may have a role in the pathogenesis of chb and cshb. background/aim: to evaluate the efficacy and safety of entecavir treatment in patients with hbeag-positive chronic hepatitis b who had not previously received a nucleoside analogue. methods: fifty-five patients received 48-week entecavir 0.5mg/d therapy. serum hbv dna load was measured with quantitative real-time-pcr. alanine aminotransferase (alt) activity, hbeag, anti-hbe-antibodies, hbv dna level in serum were evaluated at baseline, week 2, 12, 24 and 48 during therapy. evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. results: hbv dna levels declined sharply by around 3 log10 copies/ml during the first two weeks, with a highly significant reduction (p<0.0001) at week 2 and thereafter, as compared to those at baseline; 31%, 51% and 78% of the patients had undetectable serum hbv dna levels at week 12, 24 and 48 respectively. highly significantly decreasing serum alt (p<0.0001) occurred during the first 2 weeks of the study. at week 48, alt levels were normalized in 84% of the patients. hbeag seroconversion (hbeag negative, hbeab positive) was achieved in 7.3% and 14.5% of patients by 24 and 48week. at the end of 24 th and 48 th weeks, complete response (alt normalization and hbv dna and hbeag loss) was observed in 11% and 15%, respectively. there was no evidence of drug resistance or adverse effect in chb patients treated for up to 48 weeks. conclusion: entecavir treatment through 48 weeks was well tolerated and resulted in continued benefit for patients with hbeag-positive chronic hepatitis b. aim: to assess the associations of single nucleotide polymorphisms of the mxa gene promoter and sustained treatment response of chronic hepatitis b or c patients with interferon treatment by meta-analysis of individual dataset from all studies published till date. methods: to clarify the impact of mxa gene promoter polymorphisms on sustained treatment response of chronic hepatitis b or c patients with interferon treatment, we performed a meta-analysis of the published data from eight studies comparing the frequencies of mxa gene promoter polymorphisms at nt -88 g/g, -88 g/t, -88 t/t and nt -123 c/c, -123 c/a , -123 a/a alleles in individuals with interferon treatment. as we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. results: the sustained treatment response rate was higher in patients with the nt -88 g/t and nt -123 c/aalleles in the mxa promoter snp . the meta-analyses yielded summary estimatesodds ratio (or) were 2.07 [95%ci (1.58, 2.7), p <0.00001] and 1.9 [95%ci (1.32, 2.73), p =0.0006] of the nt -88 g/t and nt -123 c/a alleles, respectively. conclusion: mxa gene promoter polymorphisms at nt -88 g/t and nt -123 c/a may be useful as a marker to predict the sustained treatment response of chronic hepatitis b or c patients with interferon treatment, and further investigation regarding their real significance is warranted in a large series of patients. background: to determine whether hbv with the same characteristics causes dissimilar mutations in different hosts. methods: full-length hbv genome was amplified and linked with pmd t18 vector. positive clones were selected by double-restriction endonuclease digestion (ecori and hindiii) and pcr. twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (s)]. bioeditor, clustal x and mega software were used to perform phylogenetic and mutational analysis. potential immune epitopes were determined by the stabilized matrix method (smm), smm-align method and emini surface accessibility prediction. result: all of the 27 sequences were genotype c, the inner-divergence for the mother and son was 0%-0.8%.13 specific nucleotides differed from the other pubished genotype c isolates were co-exist in the mother and her son. aa 1-11 deletion in pres1 was the dominant mutation in the mother (14/18). the 1762t/1764a double mutation existed in all clones of the mother, 3 of them were also coupled with g1896a mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential hla class i epitopes and one b cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the p gene. conclusion: the son was infected hbv from his mother, and discrepant mutation occurred in the mother and her son during infection. background/aims: nucleos(t)ide analogues have been recognized as an effective treatment for chronic hepatitis b. this randomized, double-blind trial compared the efficacy and safety of telbivudine and lamivudine after 104-week therapy in patients with compensated chronic hepatitis b in taiwan. methods: we analyzed 114 taiwanese patients from globe trial receiving telbivudine 600mg (n=58) or lamivudine 100mg (n=56) once daily for 104 weeks. the primary efficacy endpoint was therapeutic response with serum hbv dna <5 log10 copies/ml and either hepatitis b e antigen (hbeag) loss or alanine aminotransferase (alt) normalization. results: the therapeutic response at week 104 was 74.8% in telbivudine group versus 50% in lamivudine group (p=0.005). more patients with telbivudine achieved nondetectable serum hbv dna (<300 copies/ml) (p=0.024) and alt normalization (p=0.021) at the end of treatment. the cumulative resistant rate was significantly lower in those with telbivudine treatment (p=0.0032). the rate of hbeag seroconversion was comparable in both groups (p=0.407). although a lower percentage of patients in lamivudine group (83.9%) reported adverse events than those in telbivudine group (89.7%), the difference was not significant. conclusions: telbivudine demonstrates a significantly greater efficacy and a lower resistant rate than lamivudine in treatment of chronic hepatitis b in taiwan. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. aim: to define the potential role of pd-1/pd-lpathway in different hbv infection status; we examined the expression of pd-1on cd8+ t cells in pbmc of patients with chb and aehb infection. methods: the pd-1 level on cd8+ t lymphocytes and the number of hbv specific cd8+ t lymphocytes in patients and healthy controls were analyzed by flow cytometry. pcr was used to measure the serum hbvdna levels. results: the level of pd-1 expression on cd8+ t cells in chb patients was higher than that in aehb patients and healthy individuals. compared to aehb patients, lower frequency of hbv-specific cd8+ t cells was detected in chb patients. there was an inverse correlation between the strength of hbv-specific cd8+ t-cell response and the level of pd-1 expression. besides, there was a significant positive correlation between hbv viral load and the percentage of pd-1 expression on cd8+ t cells in chb and aehb subjects. however, pd-1 expression was not associated with alt levels. conclusion: our results confirm previous reports that hbv specific cd8+ t-cell response in the peripheral blood is more intense in patients with aehb than in chb with persistent viral infection. moreover, there is a negative correlation between the level of pd-1 and the intensity of virus specific cd8+ t cell response. observed in 10.9% of patients with a mean age of 51.8±12.9. the ethnic composition was 53.1% chinese; 27.3% malay; 14% indigenous sabahans; 2.9% indigenous sarawakians; 1.8% indians and 0.8% others. chinese patients were on average, older (mean 45.6±14.5 years), indians patients had higher mean alanine transaminase and indigenous sarawakian patients had the highest rate of cirrhosis (p<0.0001). during the study period, 20.7% of patients were on treatment and they were significantly older than those who were not on treatment (mean age 44.6 ± 14.5 vs 40.7 ± 14.3). lamivudine was the first agent used in 86.9% of cases. conclusions: in malaysia, chb remains a public health issue and significantly afflicts males in the productive age groups and of chinese ethnicity. the observed differences among ethnic groups could point to different disease severity which needs to be addressed in the local treatment guideline and policy. background/aims: liver stiffness measurement (lsm) has been validated for predicting fibrosis stage in patients with chronic hepatitis c. however, studies on lsm for chronic hepatitis b (chb) are few, and the relationship between histologic findings and liver stiffness needs to be further elucidated. this study was conducted to assess the association of histologic activity on liver stiffness in addition to fibrosis in patients with chb methods: thirty three patients who had taken liver biopsy and lsm at korea university ansan hospital between march 2008 and october 2008 were enrolled. necroinflammatory activity and fibrosis stage were assessed by metavir system. activity, fibrosis, and the sum of both score were included for the correlation analysis with lsm results: among 33 patients, 29 (87.9%) were male, and median values were as follows: age, 42 (20~54); ast, 49 iu/l (19~627); alt, 62 iu/l (8~778); total bilirubin, 1.03 mg/dl (0.4~2.39); lsm, 10.1 kpa (4.2~43.5). fibrosis stages were f1 in 4 (12.1%), f2 in 9 (27.3%), f3 in 12 (36.4%), and f4 in 8 (24.2%) patients. spearman correlation coefficient with lsm were 0.457 (p=0.008) for activity, 0.694 (p<0.001) for fibrosis stage, and 0.724 (p<0.001) for the sum of activity and fibrosis. in linear regression analysis, only the sum of activity and fibrosis remained to be significant. conclusions: not only fibrosis but also activity was an important factor for determining lsm for chb. it would be more appropriate to consider both activity and fibrosis for interpretation of lsm in patients with chb background: hbeag seroconversion is a key goal of chb therapy. hbeag kinetics may predict hbeag seroconversion during treatment. we aim to develop a robust hbeag quantitative method as the value of hbeag quantitation is undefined and data is limited. methods: we evaluated two commercially available qualitative hbeag assays (abbott architect, siemens centaur) for their linear range and validated them against paul-ehrlich institute (pei) standards. hbeag levels were determined from samples of untreated and telbivudine-treated chb patients. results: as a pre-requisite for quantitative use, the linear range for the architect (0.5-43 peiu/ml) and centaur (0.05>5 peiu/ml) assays were defined. architect was selected for further investigation. hbeag levels of 44 untreated patients (mean hbv-dna 9.8 log10 copies/ml, mean alt 166.7 iu/ml) varied from 0.4 to 1073.5 peiu/ml (median 161.7 peiu/ml). in 23 patients (mean hbv-dna 9.9 log10 copies/ml, alt 166 iu/ml) treated with telbivudine for 12 weeks, baseline hbeag levels varied from 1.6 to 664 peiu/ml (median 150.7 peiu/ml). after 12 weeks of telbivudine treatment, median hbeag level was 4.4 peiu/ml, with 76% decline from baseline (median decline 95.1 peiu/ml, range 1.4-657.6 peiu/ml). individual hbeag decline from baseline varied but occurred in all patients and was not correlated to baseline or decline from baseline hbvdna. conclusion: hbeag quantitation is feasible and robust with architect hbeag assay. hbeag decline occurred in all telbivudine-treated patients, and was not correlated to hbvdna. whether the magnitude of hbeag decline is predictive of future hbeag seroconversion merits further investigation. experience from the combined globe (nv-02b-007/cldt600a2302) and 015 (nv-02b-015) study clinical safety database. c. avila 1 , r. laeufle 2 , w.b. bao 3 1 novartis pharma ag, fabrikstrasse 6, basel, switzerland, 2 novartis pharma ag, basel, switzerland, 3 novartis pharma, east hanover, us background: creatine phosphokinase (ck) is a commonly used marker of muscle damage and is elevated by many factors (e.g. exercise, injury, drugs). normal ck levels are affected by muscle mass and elevated levels are described during the natural course of chb. 22% of patients in the globe study had pretreatment grade 1-4 ck elevations. methods: we reviewed data from this combined study clinical safety database, and describe the experience of ck elevation and its relationship to adverse event reports of muscle related symptoms. results: the frequency of new onset of grade 3-4 ck elevations in telbivudine-treated patients (combined database itt population) was 1.8% (15/847), 6.3% (53/838), 3.2% (27/826) and 5.1% (40/786) from weeks 0-24, 24-52, 52-76 and 76-104 respectively. the frequency of grade 3-4 ck elevations for all patients from week 0-104 was 12.6% (107/847). the majority of grade 3-4 ck elevations were asymptomatic, rarely resulted in discontinuation or interruption, spontaneously declined within 1 or 2 visits and were not associated with more frequent muscle-related adverse events. cumulative data from this combined database showed no relationship of the degree of increased ck to acute or persistent muscle disease. conclusion: ck elevations are associated with hbv disease and were also common during the globe and 015 trials and were not predictive of the development of muscle related symptoms. onset of muscle-related symptoms should prompt clinical and treatment review, including concomitant medications. backgrounds: leptin plays a crucial role in the regulation of energy balance and body weight control by activating the long form of the leptin receptor (ob-rl). epidemiologic studies showed that obesity is one of the factors associated with hbv related hepatocellular carcinoma. methods: huh7 cells were transiently transfected with 1.3 copies of hbv-replicon plasmid. after 48h, cells were harvested and total rna of the cells were extracted and reverse-transcribed into cdna. long form and short form leptin receptor (ob-rl, ob-rs) mrna transcription levels were assayed by real-time pcr respectively. and mrna transcription levels and protein expression of ob-rl and ob-rs in hepg2.2.15 cells were also detected. results: after transfected by 1.3 copies hbv-replicon plasmid, the mrna transcription level of ob-rl was inhibited significantly (**p<0.01), but the mrna transcription level of ob-rs did not change, and the ob-rl protein expression was reduced. in hepg2.2.15 cells, the mrna transcription level of ob-rl was also significantly lower than the mrna transcription level of ob-rl in hepg2 cells, while the mrna transcription of ob-rs in hepg2.2.15 and hepg2 cells didn't show significant difference. besides, the protein expression level of ob-rl in hepg2.2.15 was also lower than it in hepg2 cells. conclusion: hbv replication down-regulated the expression of long form leptin receptor in cell cultures, which could in part explain the clinical observation of obesity in association with development of serious sequelae in hbv infections. the results of entevavir treatment in patients with chronic hepatitis b s. kose 1 , g. akkoclu 1 , m. turkeri 1 , a. gozaydin 1 1 the ministery of health tepecik training and research hospital, izmir purpose: we evaluated the short and long term effectiveness of entecavir. patients and methods: those patients had received diagnosis of chronic hepatitis b. their pretreatment transaminases, hbsag, anti-hbs, hbeag, anti-hbe, hbv-dna were checked and a liver biopsy and a resistance test for lamivudine (lam) and adefovir (adv) were performed. a total of 44 patients who were taking entecavir for at least 24 weeks were included in the study. findings: the biochemical and virologic response were observed in 88.8 % at 3 and 6 months and in 75 % at 12 months. in 9 hbeag positive patients who had received therapy previously, the biochemical response was observed in 88.8 % at 3 and 6 months and in 100 % at 12 months. the virologic response in 77.7 % at 3, in 88.8 % at 6, and 100 % at 12 months. posttreatment hbeag seroconversion did not develop. in 9 hbeag negative patients the biochemical and the virologic responses were observed in 88.8 % at 3 months and 100 % 6 and 12 months, respectively. in 17 hbeag negative patients had received therapy previously, the biochemical response was observed in 76.4 % at 3, in 100 % at 6 and 12 months. the virologic response in 94.1 % at 3, in 100 % at 6 and at 12 months. conclusion: in our study, a higher therapy-response rate was achieved, especially in hbeag negative patients. in hbeag positive patients biochemical and virologic response rates were high. background/summary: patients with liver disease are known to have a higher prevalence of glucose intolerance. preliminary studies suggest that viruses can be an additional risk factor for the development of diabetes mellitus. individuals with type ii diabetes have an increased prevalence of cirrhosis, and a proportion of patients with acute and chronic liver disease develop diabetes mellitus. there is now emerging epidemiological data to suggest that hepatitis c virus (hcv) infection may also contribute to the development of diabetes reported to be higher than expected compared with the general population. while these investigations suggest an epidemiological association between hcv infection and diabetes, large controlled studies are required to observe association between hbv infection and diabetes. the present study was designed to study the relative proportion of diabetes mellitus in patients suffering from hepatitis b virus (hbv) infection. background: in easl 2008, we reported significant liver disease among hbeag negative patients with serum hbv-dna level <4log10copies/ml (i.e. 2.0x3log10iu/ml) and alanine aminotransferase (alt) <55iu/l. this reflects fluctuating nature of these levels. the aim of this retrospective study is to demonstrate the frequency of fluctuation among this group of chb patients. methods: clinical records of hbeag negative treatment naïve chb patients with at least one serum hbv-dna <4log10copies/ml were reviewed. results: there were 194 (62.5% male, median age 48.0 years) chb patients with negative hbeag and hbv-dna <4log10 copies/ml (roche cobas amplicor pcr assay, lod<300copies/ml). 32 had serial hbv-dna measurements within 2 years; 7 of them (21.9%) had increase serum hbv-dna level by >1log10 copies/ml; 2 patients had associated serum alt elevation from normal (normal range <55 iu/l), 4 had persistent normal alt, and one had persistently raised alt. 5 (15.6%) had serum hbv-dna level decrease by >1log10 copies/ml. another 20 patients had hbv-dna levels fluctuating within 1log10 copies/ml. 159 hbeag negative patients with single hbv-dna measurement showing <4log10 copies/ml had serial serum alt measurements within 3 years. 35 (22.0%) patients had intermittent / persistently raised alt; while 124 (78.0%) patients had persistently normal alt. conclusions: hbeag negative chb is common among chinese. serial serum hbv-dna and alt measurements are necessary to detect fluctuating levels and progressive liver disease that may require antiviral therapy. background: to determine the best vaccination strategy, a model that reflects the country-specific infection profile is needed. methods: a model was built in order to obtain the age-specific infection frequency q(t) for neonates(n), infants(i), children(c), and adults(a). the infected group can either become hbsag(+) or anti-hbs(+)* based on f(t). q(t) can be found from p(t) = [q(t) x (1 -f(t)) x crs + q(t) x f(t) x (1 -cras)], where p(t) represents the proportion of the late anti-hbs(+)** group and crs/cras denote natural conversion of hbsag/anti-hbs. to test the model, cross-sectional serologic marker data in korea were used. because f(t), crs, and cras were known(f(n)=0.1, f(i)=0.5, f(c)=0.8, f(a)=0.95, crs=0.015, cras=0.02), in order to determine q(t), only p(t) values were needed, which were evaluated from logistic modeling using the glm() function of s-plus. results: the infection frequencies during neonate, infant, children, and adult periods in non-vaccinees were 15.4%, 33.3%, 16.6%, and 34.7%, respectively. each group's likelihood of infection compared to adults was then: neonates 170.2 times more likely, infants 33.5 times, and children 0.9 times, making a strong case for neonatal and infantile vaccination for the studied region. conclusions: the hbv infection model can be used for determining the most cost-effective strategy for hb vaccination in nations where longitudinal data are not available. and where longitudinal data are available, it can be used to determine the appropriate time of transition of vaccination strategy to maintain cost-effectiveness. the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b x.c. pan 1 , f. yang 1 , m. chen 1 1 objective: to investigate the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b. methods: 36 patients with hbeag positive chronic hepatitis b were recruited and receiving telbivudine treatment for 9 months. before and during 3 6 9 months of treatment , flow cytometry was used to detect the proportion of peripheral blood tregs; real-time pcr was used to detect the levels of hbv dna in surum, markers of hepatitis b virus infection were detected by elisa assay and levels of alanine aminotransferase in serum were measured. results: the proportion of peripheral blood tregs in patients with chb was significantly higher than that in healthy controls and decreased over 6 or 9 months of treatment to a level comparable to that of healthy controls. after 3 months of treatment, the rate of alt normalization in patients which the proportion of peripheral blood tregs was unreduced was significantly lower than that in patients which the proportion of peripheral blood tregs was reduced (p<0.01). 3, 6 or 9 months of telbivudine treatment resulted in negative hbeag in 4(11%) patients, 7(19%) patients or 9(25%) patients respectively. within 9 months of treatment, 7 (19%) patients seroconverted from hbeag to anti-hbe , in which the proportion of peripheral blood tregs had decreased to a level comparable to that of healthy controls over 3 or 6 months of treatment. conclusion: during antiviral treatment with subsequent reduction of the viral load or alt levels, the proportion of tregs decreased to a level similar to that of normal healthy controls. in addition, seroconversion from hbeag to anti-hbe was prone to be established in patients which the proportion of tregs decreased quickly at the early phase of antiviral treatment with telbivudine. background: in china a part of patients with alt <1.5×uln and hbv dna >10 5 copies/ml will advance into hepatic cirrhosis even hepatoma. so these patients should not only be monitored but also be treated. this study was made to determine the safety and efficacy of combining therapy of pegylated interferon alpha 2a (peg-ifn -2a) and entecavir in treating naive patients with alt <1.5×uln and hbv dna>10 5 copies/ml. methods: nine patients with hbsag positive over 6 months and alt<1.5×uln hbv dna >10 5 copies/ml were taken as research subjects. before treatment,liver biopsy was used to assess histological damage. patients were treated with peg-ifn -2a 180 g /week for 48 weeks, and in the first 12 weeks entecavir 0.5 mg/day was applied, then it was stopped. results: 1 liver biopsy showed that 7 patients had mild inflammation. 2 after 12 weeks' treatment , hbv dna level in all patients decreased to less than 10 4 copies/ml, and after 24 weeks' treatment(12 weeks after entecavir was stopped)hbv dna in all patients was less than 10 3 copies/ml. 3 normal alt was seen in all patients after 12 weeks' treatment and 24 weeks' treatment. 4 none of the patients had peripheral neuropathy with combining treatment. conclusions: 1. bulk of patients with alt <1.5×uln and hbv dna>10 5 copies/ml had mild inflammation and need treatment. 2 combing treatment of peg-ifn and entecavir was safe and effective to this group. 3 it proved that it was safe for patients to stop treatment with entecavir after short time use. background & aims: quantification of serum hbv dna levels is important to monitor viral replication in chronic hepatitis b (chb) patients. both abbott realtime hbv and roche cobas amplicor hbv monitor are updated fully automatic commercial assays for hbv dna quantification. the aim of this study is to compare the performance of these two assays on the hbv dna quantification in chb patients. methods: serial serum samples from 30 chb patients were collected at the baseline and at days 4, 7, 10 and 14 and weeks 3, 4, 8 and 12 after the commencement of therapy. genotype was determined by sequence alignment. abbott and roche assays were employed for hbv dna extraction and quantification according to the instructions of manufactories. results: hbv dna quantification results of abbott assay was significantly correlated with those of roche assay (r=0. 972, p<0.0001). for genotype c, the difference in hbv dna levels [median (range): 1.22 (-0.16-2.36) log units] measured by these two assays was significantly higher than that for genotype b [0.66 (-0.52-1.63) log units, p<0.0001]. moreover, the difference in serum hbv dna levels after 12 weeks antiviral treatment [1.18 (-0.21-1.76) log units] measured by these two assays was significantly higher than that in baseline serum hbv dna levels [0.68 (-0.12-1.24) log units, p<0.0001]. conclusion: the quantification results of abbott realtime hbv showed a good correlation with those of roche cobas amplicor hbv. but the performances of these two assays have significant difference in the quantifications of serum hbv dna levels in genotype c patients and in patients after 12 weeks antiviral therapy. background: guidelines suggest hepatitis b virus (hbv) vaccination to all hepatitis c virus (hcv) infected patients and healthcare workers. we attempted to find out hbv vaccination status in our hcv infected population, and healthcare workers. methods: prospective survey of 100 consecutive hcv infected patients and also doctors and paramedical staff in our hospital. results: major sources of viral infection in study patients (58 males; average age 40 years -range 18 to 65 yrs) were reused syringes (38 pts). twenty had a household member infected with hcv. twenty were co-infected with hbv. eighty five of hcv infected patients were not vaccinated.against hbv. twenty five of them (29%) had financial reasons and 45 patietns (52%) had lack of awareness. out of 30 doctors, 12 and 15 did not know about their hbv and hcv status respectively, but none was known to have either of these infections. four (13%) were not vaccinated against hbv. out of 29 paramedical staff, 1 was hcv positive, 11 each were unaware of their hbv and hcv status, and remaining were negative for these markers. thirteen of them (44%) were not vaccinated against hbv. conclusion: thirty eight percent hcv infected patients were infected by reuse of syringes. eighty five percent were not vaccinated against hbv, out of which 52% had no awareness about it, whereas 29% could not financially afford it. a significant number of paramedical staff and some doctors were also not vaccinated background: early prediction of efficacy could decrease unnecessary interferon exposure of patients with chronic hepatitis b. methods: a multi-center clinical study. patients were injected interferon alpha 2b 5 million iu subcutaneously every other day for 24 weeks and 24-week follow-up was followed. results: 53 patients (44 male) were enrolled, 27.5 ±9.1 years old. 24 hours after administration, hepatitis b virus (hbv) load decreased significantly (6.96±1.03log copies/ml, p<0.05) from baseline (8.00±0.93 log copies/ml). hbv load was 4.94±1.47 and 5.25±1.58 log copies/ml at week 24 and 48, respectively. at the two points upwards, complete response rate was 4.2%(2/48) and 7.9%(3/38), partial response rate was 35.4%(17/48) and 34.2%(13/38), respectively. at week 4 and 12, hbv dna levels of complete responder and partial responder were lower than those of non-responder (p<0.05) at week 24. at baseline, on hour 12, day 2, 3, week 2, 4 and 12, hbv dna levels of complete responder were lower than those of non-responder at week 48 (p<0.05). multiple linear regression showed that baseline hbv dna was the independent variables to predict the response at week 24 and 48. conclusions: interferon alpha 2b was effective in treating patients with hbeag positive chronic hepatitis b. it could decrease the hbv dna level rapidly. early hbv dna levels were predictive to response at the end of treatment and follow-up. baseline hbv dna level was the independent predictor of the response at the end of treatment and follow-up. aim: to investigate features of pd-1 expression on peripheral tcells and pd-l1 expression in liver in chronic hepatitis b (chb) patients in immune clearance phase. methods: pd-1 expression on total peripheral t cells were evaluated by using flow cytometry. immunostaining was performed according to the envision chemmate methods. the degree of pd-l1 expression was scored and assessed according to the percentage and staining intensity of positive cells. results: compared to health control, the percentage of total peripheral t cells expressed pd-1 was elevated in chb with repeatedly increasing alt level. no specific association between the percentage of pd-1 positive and the mean fluorescence intensity mfi of pd-1 expression on total t cells with serum viral load were found. but alt level was correlated with the mfi of pd-1 expression on total cd8+t cells significantly. pd-l1 is up-regulated on hepatocytes by viral infection, and high expressed in fibrosis section. conclusion: the mfi of pd-1 on cd8+t cells plays important role in regulating the immune-host interaction in chb in immune clearance phase. and pd-1 expression on t cells is correlated with high immune inflammatory refection. aim: to study the quantity, characteristic of hbv-specific t-cell and the extent of liver damage in chronic hepatitis b (chb) patients with different hbeag status. methods: 103 chb patients were enrolled and divided into two groups according to the hbeag status, and the liver damage index were analyzed. the frequency and foxp3 expression of cd4 + cd25 + regulatory t cells (treg) were measured, as well as the frequency and phenotypic molecules expression of hbv-pentamer+ t-cell. hbv specific t-cell responses including cellular proliferation and ifn-production, with or without anti-pd-l1 and/or anti-ctla-4 blocking, were also observed. results: the demographic characters, serum alt, ast levels, the frequency and foxp3 expression of cd4 + cd25 + treg were similar, while the serum hbv dna levels were higher in hbeag+ patients (p <0.05). the liver necroinflammation was comparatively more severe in hbeag-patients (p =0.056), but the median percentage of liver cirrhosis was much higher in hbeag+ patients (p <0.05). the difference of hbv-specific t-cell frequency was not significant between two groups, while the expression levels of pd-1 and ctla-4 on hbv-specific cd8 t cells were significantly higher in hbeag+ patients (p both <0.05). combined using of anti-pd-l1 and anti-ctla-4 mab significantly increased the cellular proliferation in either hbeag+ or hbeag-patients, but only markedly enhanced the ifnproduction in hbeag+ patients. conclusion: hbeag persistency could probably induce higher expression of pd-1 and ctla-4 on the hbv-specific t cells and result in t-cell impairment, high hbv dna load and high percentage of liver cirrhosis in hbeag+ chb patients. hepatocyte apoptosis in patients with chronic hepatitis b y. liu 1 , k. wang 1 1 background: to investigate the relationship between hepatocyte apoptosis and the level of inducible nitric oxide synthase (inos) in hepatic tissue in the patients with chronic hepatitis b chb . methods: we observed 37 cases with chb and 10 normal controls. transferase-mediated-utp-biotin nick-end labling ( tunel) technique was used to detect apoptosis cells and immunohistochemical staining were also performed to investigate the expression of inducible nitric oxide synthase (inos) in biopsy samples .the serum level of alt hbv-dna grading of necroinflammatory activity and staging of fibrosis were also assessed. results: hepatocytes in all chb liver tissues were positively stained by tunel in various degree. in contrast, control tissues did not show dna fragmentation. a significant correlation was seen between apoptosis index (ai) and necroinflammatory grading ((r=0.404, p=0.015) and serum inos level r=0.465, p=0.004 . it did not correlate with fibrosis stage and serum alanine aminotransferase level. conclusion: the oxidative stress.in patients with chb may reflected the apoptosis of hepatocyte. apoptosis involves in liver injury of chb,but with no significant correlation to serum level of alt. objectives: to investigate the genotype-dependent development of lamivudine resistance in hepatitis b virus (hbv). methods: 215 patients with chronic hepatitis b who had been treated with lamviudine for more than 1year, and become lamviudine resistance were analysed for the hbv genotypes and cumulative rate of rt region mutant with standard dna sequencing technology. results: among the 215 patients, 60 patients were infected with hbv genotype b (hbv/b)(27.9%), and 155 with genotype c (hbv/c)(72.1%). in the hbv/b patients, 16/60(26.7%) were of subtype ba, and 44/60(73.3%) were of of subtype bj. the cumulative type and ymdd mutation rates in patients with genotype c were showed as l180m+m204v (67/155,43.2%) > l180m+m204i (52/155,33.5%) > m204i (36/155, 23.3%), while in patients with genotype b as l180m+m204v 36/60(60.0%) > m204i(24/60, 40.0%), none of l180m+m204i. conclusions: our results indicated that in patients with lamivudine resistance, hbv genotype c (hbv/c) were higher than genotype b (hbv/b). in both genotypes the combined mutations (180+204 sites) were found more than the single 204 site, showed some significance for monitoring lamivudine resistance. background & aims: il-35, a novel identified inhibitory cytokine specifically produced by regulatory t cells (tregs), is an ebi3-il-12 heterodimer encoded by epstein-barr-virus-induced gene 3 (ebi3) and interleukin-12 alpha (il12 ). the aim of the study is to determine the expression levels of il-35 in peripheral blood mononuclear cells (pbmcs) of chronic hepatitis b (chb) patients in different phases. methods: a total of 36 treatment naïve chb patients, including 17 in immune-tolerant phase [group 1, alt: 21 (12-51) u/l, serum hbv dna: 2.48 x 10 9 (6.30 x 10 6 -1.20 x 10 10 ) copies/ml] and 19 in immune-clearance phase [group 2, alt: 221 (71-1530) u/l, serum hbv dna: 5.10 x 10 8 (1.62 x 10 6 -1.77 x 10 10 ) copies/ml] were enrolled in the study. the relative mrna expression levels of ebi3, il12 and foxp3 were determined by semi-quantitative pcr. results: the significant correlations were observed between the expression of ebi3 and il12 (r=0.661, p<0.001), ebi3 and foxp3 (r=0.388, p<0.05), il12 and foxp3 (r=0.431, p<0.01). the relative expression levels of ebi3 and il12 in pbmcs were significantly higher in group 2 when compared with those in group 1 (1.46 ± 0.23 vs 0.80 ± 0.10 and 1.26 ± 0.19 vs 0.65 ± 0.09, p<0.05, respectively). furthermore, the relative expression levels of ebi3 and il12 in group 2 were significantly correlated with alt levels (r=0.473, r=0.474, p< 0.05, respectively), but not with serum hbv dna levels. conclusions: the expression levels of il-35 in pbmcs were significantly higher in chb patients in immune-clearance phase than that in immune-tolerant phase. increased il-35 expression levels were associated with liver injury. background: there are a number of oral antivirals approved for chronic hepatitis b. lamivudine, the first oral nucleoside analog, is associated with increased rates of drug resistance with prolonged use--from 20% at one year to 50% at three years. therefore, an alternative or add-on treatment is necessary. adefovir, an oral nucleotide analog, is used either in combination with lamuvudine or as monotherapy in lamivudine-resistant chronic hepatitis b. we did a meta-analysis to compare the efficacy of adefovir in combination with lamivudine versus adefovir alone in the treatment of lamivudine-resistant chronic hepatitis b infection. methods: a comprehensive literature search was performed using the following databases: medline, cochrane, and embase. a total of 3 randomized controlled trials were retrieved and analyzed. outcomes measured were virologic response, biochemical response and resistance rates. results: meta-analysis on virologic response showed that combination treatment with adefovir and lamivudine is as effective as adefovir monotherapy (or 1.04, 95% ci 0.50-2.15, p=0.92). likewise, in terms of biochemical response, both regimens were equally effective (or 1.06, 95% ci 0.47-2.40, p=0.90). one study showed statistically significant increase in adefovir resistance rate in the monotherapy arm compared to combination arm (p= 0.0182) after the first year of therapy. conclusion: in patients with lamivudine-resistant chronic hepatitis b infection and compensated liver disease, adding adefovir to lamivudine is as effective as switching to adefovir alone in terms of virologic and biochemical response. r. safadi 1 , q. xie 2 , y.g. chen 3 , y.k. yin 4 , l. wei 5 , s.g. hwang 6 south korea, 7 bnai zion medical center, haifa, israel, 8 beijing friendship hospital, beijing, china, 9 novartis pharma ag, basel , switzerland background: ldt produces greater viral suppression than lam. we investigated whether patients receiving lam can benefit from switching to ldt. methods: hbeag positive and negative persistently viraemic patients (median hbv dna 5.0 (ldt), 5.3 (lam) log 10 copies/ml) and lam treated for 3-12 months, were randomized to either switch to ldt or continue lam. we report the benefit of ldt switch assessed by primary treatment failure (tf, <1 log hbv dna decline) and viral breakthrough (vb, >1 log above nadir). results: 17% (21/122) of the ldt switch and 15% (18/124) continuing lam patients had pre-existing m204 mutations at screening. tf was 5% (ldt) versus 21% (lam, p<0.05). in patients with >24 weeks prior lam treatment, tf was 10% (ldt) versus 41% (lam). 83% ldt tf (5/6) was associated with resistance at screening versus 56% lam tf. in ldt switch with <24 weeks prior lam, no ldt tf occurred versus 12% lam. in hbeag positive, tf occurred in 6% (ldt) versus 29% (lam). among hbeag positive with >24 weeks prior lam treatemtn, vb was 19% (ldt) versus 44% (lam, p<0.05). differences were not significant for hbeag positive with >24 weeks lam or for hbeag negative regardless of duration of prior lam treatment. conclusions: early switch to ldt is associated with better virological outcomes in these patients. persistent viraemia for >6 months on lam treatment is associated with a high risk of tf and vb. for these patients, genotypic analysis is recommended prior to screening. objective: the aim of this study is to evaluate the proper endpoint in the treatment of chronic hepatitis b with antivirals by investigating the viral rebound ratio after one year's nucleosides or (three months) sustained treatment with lamivudine, adefovir, entecavir, or interferon when viral response and seroconversion response have been finished . methods: eag positive chronic hepatitis b naïve patients with alanine aminotransferase (alt) more than 2 uln were assigned to receive 100 mg of lamivudine, 10mg of adeforvir, or 0.5mg of entecavir once daily, respectively. patients in the interferon group were administrated with 5,000,000 iu of 2a interferon on every other day, and the therapeutic duration lasted for another three months after eag-ab seroconversion appeared. hbv dna and eag-ab in the serum were tested during the off-treatment period of 12 months. results: thirty four patients in lamivudine group of 148 cases got eag-ab seroconversion after treatment with 20±5 months of average duration, and the viral rebound ratios in the off -treatment 6 an 12 months follow up period were 20.6 7/34 and 40.1 15/34 , respectively. in adeforvir group were 19 4/21 and 33. 7/21 . in enticavir group were 20 3/15 and 46.7 7/15 . in interferon group was 16.2 6/37 in the off-treatment 6 months follow up period. conclusions: we conclude that eag-ab seroconversion in the treatment of eag positive chronic hepatitis b patients is the goal but not an endpoint of therapy physicians should aim at. to gain everlasting effect, longer duration of treatment may be needed. background: universal hepatitis b(hb) vaccination of hbsag negative people (especially infants) is widely recommended and practised. objective: to assess whether there is robust evidence of protective efficacy to back such practice. methods: this cochrane review included randomised trials identified from six databases through detailed electronic searches. trials comparing hb vaccine versus placebo/another vaccine, in hbsag negative persons were included without any restrictions. the primary outcome was hb infection (developing hbsag or anti-hbc). robustness of evidence was assessed through comparison of available-case analysis versus intention-to-treat(itt) analysis using four different models: (i)assuming unfavourable event for all missing data, (ii)assuming favourable outcome for all missing data, (iii)best-case-scenario and (iv)worst-case-scenario results: twelve trials were eligible among 2964 citations; all were methodologically poor (high risk of bias). data from four trials could be included in meta-analysis. efficacy of vaccination varied with the type of data analysis. available-case analysis suggested efficacy in reducing risk of developing hbsag (rr=0.17;95%ci=0.09-0.31;n=1341) and anti-hbc (rr=0.42;95%ci=0.31-0.57;n=1235). itt analysis results varied depending on the model chosen (table) , but liberal approaches suggested high efficacy, whereas conservative approaches did not. the available evidence on efficacy of hb vaccination in hbsag negative people is not robust; there are serious limitations in quality and quantity. background: open-label rollover study (etv-060) assessed histologic improvement in chb patients on at least 3 years etv therapy. methods: 100% nucleoside-naïve patients and 98% lamivudine (lvd)-refractory patients from etv-053 and etv-052 studies, respectively, entered etv-060 study and received etv at 0.5/1mg for greater than 96 weeks. improvement in knodell necroinflammatory (ni) score and knodell fibrosis score at weeks 48 and 148 were studied. results: at week 148, 95% of nucleoside-na ve patients and 56% of lvd-refractory patients achieved hbv dna <400copies/ml. furthermore, 95% of nucleoside-na ve patients and 93% of lvd-refractory patients had normalized alt levels. mean platelet counts in both naïve and lvd-refractory patients were improved at weeks 48 and 148 compared with baseline. conclusions: naïve and lvd-refractory chb patients showed significant improvement in liver histology after 3 year etv therapy, and improved dna and serum alt levels. results: 84.7 percent of patients were younger than 30 years old, 15.3 percent were older than 30 in this study. 48.0% patients' mothers were hbsag positive. high levels of serum hbv dna were founded in all patients, >10 7 copies/ml were 78.6%. only 5 cases (5.1%) whose liver inflammation grade were g0, the rest patients were mild inflammation, in which g1 were 64 cases (65.3%), g2 were 29 (29.6%); there were 56 patients (57.1%) had no signifecant liver fibrosis, the rest 42 cases (42.9%) had different fibrosis, among those s1 were 23 cases (23.5% , s2 were 14 14.3% , s3 were 5 5.1% , none of patients had cirrhosis. the fibrosis stages of higher alt level were markedly severer than lower alt in patients with normal alt p < 0.01 . conclusions: most of patients with chronic hepatitis b virus in immune tolerant phase present mild inflammation in liver, part of them have already appeared fibrosis, so some patients determinated by clinics are actually not in immune tolerant phase. although alt testing are in the normal range, but the possibility of liver fibrosis is increased in patients with relative higher alt level, so liver pathology should be recommended to judge illness correctly. background/aims: hepatitis b virus infection (hbv) is a global health problem. in bangladesh, 5-7% of people are hbsag positive. this study was carried out to evaluate the efficacy and safety of peginterferon alfa-2a in chronic hepatitis b patients. methods: a total of 60 patients with chronic hepatitis b, 32 (53.3%) were hbeag positive (group a) while 28(46.7%) were hbeag negative (group b) were included in this study after meeting the following criteria: age 18 to 60 years, hbsag positive for more than 6 months, serum hbv-dna was >5 log(10) copies/ml and alt more than two times the upper normal limit. they were given peginterferon alfa-2a (180 microgram once weekly) for 24 weeks and followed for an additional 24 weeks. results: after 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hbvdna levels below 20,000 copies per milliliter was significantly higher in hbeag positive patients (59 percent and 43 percent, respectively) than among hbeag negative patients (45 percent and 33 percent). loss of hepatitis b surface antigen occurred in 3 patients in group a, as compared with 1 patients in the group b (p<0.01). adverse events including pyrexia, fatigue, myalgia, headache and haematologic abnormalities were similar in both groups. conclusions: patients with hbeag positive chronic hepatitis b had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a. background: the effect of hepatitis b vaccination on individuals with isolated anti-hbc in endemic areas is not clear. we investigated the prevalence of individuals positive for anti-hbc only and their antibody response after hepatitis b vaccination in a single healthcare center. methods: the study included 1,812 healthcare workers. after screening for hbsag and anti-hbs, the individuals negative for both hbsag and anti-hbs were examined for anti-hbc and were vaccinated with a recombinant hepatitis b vaccine at 0, 1, and 6 months. the serum anti-hbs level was measured after the vaccination. results: of the subjects, 334 (220 females) were negative for both hbsag and anti-hbs. forty (2.2%) subjects had isolated anti-hbc, including more males (60.0% vs. 30.6%) and older people (45.7±8.2 vs. 37.3±8.5 years), compared with individuals negative for all of the viral markers. the anti-hbs seroconversion rate and anamnestic response in the individuals with isolated anti-hbc after the first vaccine injection were 60% and 27.5%, respectively. in the 294 persons who were negative for all hepatitis b viral markers, the seroconversion rate after the first vaccination was 52.6%. the anti-hbs seroconversion rate did not differ between the isolated anti-hbc positive individuals and those negative for all hepatitis b markers (89.5% vs. 96.6%) after the full course of vaccination. conclusions: serum hbsag and anti-hbs tests are sufficient for screening before hepatitis b vaccination, especially in healthcare workers. objective: to understand the quantity and distribution of cd83 + mature dendritic cells in patients with hepatitis b virus in immune tolerant phase. methods: there were 30 immune tolerant phase patients with hepatitis b virus infection (fibrosis stages were s0), 10 immune clerance phase patients, 10 non-active status patients and 5 healthy controls involved in our research. the quantity and distribution of cd83 + mature dendritic cells in liver were determined by immunohistochemical staining. result: the liver inflammation grades were between g1-g2 in patients who in inmmune tolerant phase and non-active status, moreover, patients in immune clerance phase were between g2-g4. there were a small amount of cd83 + dendritic cells in healthy liver tissue, scattered in portal areas and hepatic lobules. the quantity and distribution of cd83 + dendritic cells in patients who in inmmune tolerant phase and non-active status were similar to the healthy, and the quantity were no difference among them p 0.05 .the number of cd83 + cells in patients of immune clerance phase was significant increased compared with other groups, there were differences among them p 0.05 , the cd83 + cells mainly distributed in portal areas infiltrated with inflammatory cells and hepatic lobules with inflammatory necrosis. conclusion: cd83 + mature dendritic cells are involved in liver immune response in patients of inmmune clerance phase, is likely to related to hepatitis b virus clearance. lack sufficient mature dendritic cells may be one of the mechanisms of immune tolerance. background: local hospitals provide obstetric services including antenatal care to women normally living in the mainland china, whose prevalence of hepatitis b carrier is unknown. objectives: compare prevalence of hbv carrier of pregnant women from the mainland china with local counterparts and discuss the implications of results. materials and methods: antenatal serological results were retrieved from corporate laboratory information system databases. pregnant women from the mainland china were identified by a specific set of temporary-allocated identity number during january 2007 -october 2008. results: 7491 pregnant local residents and 1397 pregnant women from the mainland china underwent antenatal serological tests for hepatitis b surface antigen. positive hepatitis b surface antigen results were more frequent in pregnant women from the mainland china (12.7%) than in local pregnant women (8.17%) (p<0.001). discussion: because infected pregnant women can transmit the hepatitis b virus to the infant at delivery, specific management could entail maternal medication, injection of hepatitis b immune globulin to the infant at birth and immunization later on. however, early repatriation to the mainland china, which is common, will make completion of immunization program difficult. these babies will be at a higher risk to be infected by hbv, particularly when breast-fed by hbv carriers. their return to hong kong later will dilute the effects of local immunization program. the volume of work derived from the provision of obstetric services to women from the mainland chinese is larger with regard to medication, counseling and immunization for babies born to hbv carriers. immunosuppressive or anticancer therapy k. hirano 1 , t. kodani 1 , s. sato 1 , y. narita 1 , t. kikuchi 1 , t. genda 1 , k. iijima 1 , k. ogawa 1 , t. ichida 1 1 background/aim: we compared the prevention of hbv reactivation in (hbsag)-positive patients with hbsag-negative patients who were positive for antibody to (anti-hbc) and/or (anti-hbs) undergoing immunosuppressive, anticancer or molecular target therapy. methods: from sep 2004 to nov 2008 hbsag-positive patients and 22 anti-hbc and/or anti-hbs-positive patients were enrolled in this study. we compared with 2 groups about background disease, age, blood examination, and nucleoside analogues. results: in hbsag-positive patients mean age were 56.6±10.2 years old, median ast levels were 30 (18-706) iu/l, and median alt levels were 36 (13-544) iu/l for 8 (47%) haematological disease and 9 (53%) collagenosis disease. in anti-hbc and/or anti-hbs-positive patients mean age were 71.3±9.6 years old, median ast levels were 20 (9-106) iu/l, median alt levels were 16.5 (5-247) iu/l for 17 (77%) haematological disease and 5 (23%) collagenosis disease. serum hbv-dna levels >5.0 log copies/ml were 8 (47%), 2.6~5.0 were 7 (41%), <2.6 were 2 (12%) in hbsag-positive patients, and serum hbv-dna levels <2.6 were all cases in anti-hbc and/or anti-hbs-positive patients. 14 (82%) of hbsag-positive patints received nucleoside analogues (7 lam and 7 etv), and 12 (55%) of anti-hbc and/or anti-hbs-positive patients received nucleoside analogues (8 lam and 4 etv). mean duration of treatment for 14.2 months in hbsag-positive patients, and for 4.1 months in anti-hbc and/or anti-hbs-positive patients, the resistance virus occurred to 3 (75%) of 4 hbsag-positive patients treated with lam for collagenosis disease more than two years. conclusion: when hb carriers of collagenaous disease undergoing immunosuppressive therapy required the nucleoside analogues more than two years, we recommended treatment to prevent hbv reactivation with etv. background: adefovir dipivoxil is used for the initial treatment of chronic hepatitis b or rescue treatment of lamivudine-resistant chronic hepatitis b, and exhibits excellent antiviral activity. however, the presence of resistance to adefovir dipivoxil was more frequently in lamivudine-resistant chronic hepatitis b patients than in lamivudine-naïve patients during adefovir dipivoxil monotherapy. but the rate of adefovir resistance related mutations is little known in lamivudine-resistant patients before adefovir dipivoxil treatment. the aim of this study was to investigate the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus in lamivudine-resistant patients not treated with adefovir dipivoxil. methods: the existence of adefovir resistance-related mutations was examined in 240 lamivudine-resistant chronic hepatitis b patients with breakthrough hepatitis and 100 antiviral-naïve chronic hepatitis b patients. both polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) and directly sequencing of pcr product were used to detect resistant viruses. results: rta181t mutants were detected in only two sera of lamivudine-resistant patients, while none in the antiviral-naïve chronic hepatitis b patients. there was no rtn236t detected in the two groups. conclusion: our results suggest that the rta181 mutant virus were present in a few lamivudine-resistant chronic hepatitis b patients before they have been treated with adefovir dipivoxil, but the rtn236t mutant was not detected in any of the two groups. the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus was low in such lamivudine-resistant patients before adefovir dipivoxil treatment. objective: in this study, we tried to detect and identify the special protein of hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. to find new opinion on the developing of chronic liver disease. methods: the sera of health adult, hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were respectively detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms). the arrays of every group were analysised by clustering analysis and to establish disease predictive model. then the sample was eluted with different ph tris, trypsinization on-chip, mass determination and peptide database comparison. results: according cm10 chip we find 18 protein with obviously deviation (p<0.05) among hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. clustering analysis for the data from seldi-tof-ms confirmed 42 differentially expressed proteins. then we developed disease predictive mathematic models ( decision tree model, dt model ) with average validity up to 73.9 . the 5805 da protein peak was identified to be chondroitin sulfate synthase 2 (chss2), which is a potential molecule involved in the pathologic process and a potential serum marker for the hbv related hepatic diseases as well. conclusions: our results suggest that seldi-tof-ms is a usefull technique for differential expressed proteins screening and analysis in hbv related chronic liver disease. chss2 may be useful during the developing of hbv related chronic liver disease. backgound: clevudine is a new nucleoside analogue with potent antiviral activity in chronic hepatitis b patients. however, the efficacy and safety of clevudine in cirrhotic patients are not well recognized. this study was conducted to evaluate the early virologic and biochemical response rate as well as safety of clevudine in cirrhotic patients with chronic hbv infection. methods: 46 patients with chronic hbv infection who visited korea university ansan hospital and guro hospital between may 2007 and may 2008 were included. 24 patients had chronic hepatitis b (group a) and 22 had liver cirrhosis (group b). early virologic response was defined as hbv dna less than 200 iu/ml at week 12. early biochemical response was defined to be normalization of alt (<45 iu/l) at week 12. result: pretreatment hbv dna levels were higher in group a compared with group b (8.06 log iu/ml vs 7.09 log iu/ml, p=0.06). pretreatment alt levels were not significantly different between the two groups (166 iu/l vs 139 iu/l, p=0.725). the rate of early virologic response was significantly higher in group b compared with groups a (72.7% vs 33%, p=0.01). the rate of early biochemical response were not significantly different in both groups (75% vs 72.7%, p=0.725). conclusion: clevudine is considered to be safe and effective in cirrhotic patients with chronic hbv infection as well as chronic hepatitis b patients. long term safety and efficacy need to be evaluated in the future. objective: the aim of this study was to evaluate the role of nucleos(t)ide analogues against hbv reactivation in immunosuppression. methods: non-active hbsag carriers suffering from cancer, autoimmune diseases and needing the treatment of immunosuppressants or cytotoxic chemotherapy were enrolled in the study. the outpatients or in-patients from april 2007 to july 2008 were enrolled. the nucleos(t)ide analogues were used in cancer patients 1-2 weeks before chemotherapy, and the duration lasted 6-12 months according to patients' compliance after completion of chemotherapy. patients with other diseases used nucleos(t)ide analogues in 1-3 months before using glucocorticoids or other immunosuppressive agents, and continued to use for 6-12 months after accomplishing the course of immunosuppressant treatment. the characheristics and clinical manifestations about hbv reactivation were investigated. results: of the thirty two patients in prospective group, twenty two patients suffered from cancer, eight patients suffered from idiopathic thrombocytopenic purpura, two patients suffered from chronic nephritis. the amount of hbv dna was detected in the first, third, sixth and 12th month after the use of nucleos(t)ide analogues. after chemotherapy or immunosuppressant treatment, only 9.4% (3 / 32) of them suffered from hbv reactivation, which presented with hbv dna positive and abnormal liver function. conclusion: non-active hbsag carriers would appear potential incidence of hbv reactivation during use of chemotherapy or immunosuppressant. nucleos(t)ide analogues could be used in early phase as prophylaxis for reactivation of hepatitis b in immunosuppression and to improve clinical prognosis. background: hbv therapies are evolving toward combination antivirals. this study evaluated the combination of clevudine (clv), a potent nucleoside analog, with tenofovir dipivoxil (tdf). methods: a phase i, single-arm, multi-dose study in 18 healthy adult volunteers to evaluate pharmacokinetic and safety interactions between clv and tdf. subjects received 21 days of clv 30mg followed by 7 days of clv 30mg +tdf 300mg. pk profiles were obtained on days 1, 21 and 28. clv auc and cmax were compared on days 21 and 28. day 28 tenofovir pk was compared to historical data. safety assessments were conducted throughout. results: 18 subjects were enrolled (13m/5f);17 completed the study. the mean (range) age was 37y (22-49) and body mass index (kg/m2) was 27.6 (22.9-31.9). 22 aes were reported by 6 subjects, with 10 aes reported during clv-only dosing and 12 aes reported during clv+tdf dosing. aes included nausea (2) and pharyngolaryngeal pain (2). the majority of the aes were mild. there were no clinically significant changes in ecgs or laboratory parameters. comparisons of clv auc and cmax on day 21 and 28 revealed no significant impact of tdf upon the plasma clv exposure (d28/d21 auc ratio=0.98, d28/d21cmax ratio=0.89). there is no significant effect of clv on tenofovir when comparing auc and cmax of tdf to historical values. conclusion: safety and pharmacokinetic results demonstrate that clv and tdf may be safely co-administered, supporting the further study of this drug combination for the treatment of chronic hbv infection. s. kuznecovs 1,2 , i. kuznecovs 1 , k. jegina 2 , g. kuznecova 1 1 background: dolichyl (dol), the main lipid intermediator of dolichyl phosphate cycle (dpc) has been reported to be elevated in urine of patients with multidrug resistance in cancer. drug resistance poses a major threat to nucleoside analogue-based therapies for chronic hbv infection. methods: with focus on a risk predictor for susceptibility to the development of hbv drug resistance the present study was carried out to estimate urinary levels of dol in chronic hbv infection. the samples obtained every week before and during the course of treatment from 42 patients with hbv. the occurrence of exacerbations of chronic hbv were registered for 2 years. dol in urine was assayed by hplc method. results: the normal amounts of dol in healthy persons urine (n=1500) are 6,0 + 10,0 mkg/mmol creatine. during the period of observation 36 (86%) of patients treated with nucleoside analogue-based therapies were diagnosed with exacerbations due to resistance of hepatitis b virus to antiviral drugs. from this group of hbv patients 35 (98%) have had elevated urinal dol excreation (45,8±5,2 g/ml vs . 8,2±1,9 g/ml, p<0.0001) in more than 3 months of observation. conclusion: there is a reason to suggest that elevated urinal dol detected in patients with exacerbations during hbv treatment may evidence of possible defect of host mechanism of drug resistance development to nucleoside analogue-based therapies. the interest drawn to the employment of dol as a predictor for exacerbation of chronic hbv is explained by the role of dpc in p-glycoprotein regulation in human hepatocytes. background: a significant proportion chb patients treated with adv have a suboptimal response, increasing the risk of disease progression and development of resistance. we report clinical results from patients who either failed or relapsed following adv therapy and were subsequently switched to etv. methods: study etv-079 was a randomized, open-label study comparing antiviral efficacy of etv (0.5mg/day) vs adv (10mg/day) in nucleoside-naïve hbeag-positive patients. after up to 96 weeks of treatment in etv-079, 24 patients treated with adv (13 suboptimal responders) rolled over into study etv-901 (1.0mg/day). hbv dna viral suppression and safety was evaluated during 48 weeks of etv treatment. results: at entry to etv-901, the median hbv dna was 5.72log10 copies/ml. median exposure to etv (1.0mg) in etv-901 was 46 weeks and 18 patients currently remain on study therapy. at week 24, the mean reduction in hbv dna was 4.5log10 copies/ml and 8/16 (50%) reached hbv dna levels <300 copies/ml. nine patients have achieved week 48 and all have achieved hbv dna <10 4 copies/ml and 8/9(89%) had hbv dna levels <300 copies/ml. no patients experienced virologic breakthrough on etv. the safety profile of etv in adv-treated patients remained consistent with the previously reported experience. conclusions: the majority of patients who were suboptimal responders or virologic rebounders following adv treatment in study etv-079, experienced rapid reductions in hbv dna levels when switched to etv. hbv dna levels continued to decline to undetectable levels with 48 weeks of etv treatment. y. li 1,2 , t. han 1 1 tianjin third central hospital, 2 aim:to quantify hepatitis b virus (hbv) total dna and covalently closed circular dna (cccdna) in liver biopsies and sera which from chronic hepatitis b(chb) liver cirrhosis of hepatitis b(lc) and hepatitis b relevance hepatocellular carcinoma(hcc) patients, and analysis hbv replication under the circumstances of different diseases. methods:total hbv dna and cccdna in serum and liver biopsy samples were measured in 21 chb 23 lc and 25hcc patients by the real-time pcr assay. results: the levels of total hbv dna in serum,intrahepatic total hbv dna, intrahepatic cccdna, as well as the proportion of intrahepatic cccdna in total hbv dna decreased progressively in chb,lc and hcc ,moreover chb had significantly higher levels of total hbv dna in serum and liver biopsy samples than lc (log [total serum hbv dna] p = 0.024;log [total intrahepatic hbv dna] p = 0.034); chb and lc had significantly higher levels of intrahepatic cccdna and the proportion of intrahepatic cccdna in total hbv dna than hcc(p <0.01); cccdna couldn't be detect in all patients'serum. in chb ,the levels of serum's total hbv dna,intrahepatic total hbv dna and cccdna in hbeag-positive group had significantly higher than the hbeag-negative group(p <0.01) ,but in lc only intrahepatic total hbv dna had statistical difference between hbeag-positive and negative group (p=0.026) , no statistical difference between hbeag-positive and negative group in hcc. conclusions: the replication activity of hepatitis b virus in chb,lc were higher than hcc, hbv reproduction reduced significantly in hcc. duplication of hbv in lc was lower than chb but had no statistical difference. the levels of hbv reproduce in hbeag-positive group was higher than hbeag-negative group of all three desease. background: clevudine is a pyrimidine analogue with potent and sustained antiviral activity against hbv in the 24 week therapy. the present study assessed the efficacy and viral resistance of 48 week clevudine therapy in patients with chronic hepatitis b. method: a total of 42 patients (26 hbeag positive and 16 hbeag negative) who were received clevudine 30 mg once daily for 48 weeks were included in this analysis. serum hbv dna was quantified by real time pcr assay. result: at week 48, median reductions of serum hbv dna from baseline were 4.98 log10 iu/ml (5.00 log10 iu/ml for hbeag positive and 4.95 log10 iu/ml for hbeag negative) and 76.2% of patients showed undetectable serum hbv dna (<60 iu/ml) (65.4% for hbeag positive and 93.8% for hbeag negative). the normalization of alt levels (<35 iu/l) was achieved in 81.0% (88.5% for hbeag positive and 68.8% for hbeag negative). 15.4% of hbeag positive patients showed hbeag loss or seroconversion. hbv dna negativity at week 48 was associated with hbeag negativity (p =0.037), hbv dna <2,000 iu/ml at weeks 4 and 24 (p =0.019 and 0.001, respectively). two hbeag positive patients showed viral breakthrough with m204i mutation during 48 week. conclusion: clevudine therapy in patients with chronic hepatitis b showed potent virologic responses at week 48, especially in those with hbeag negativity and complete early virologic response (hbv dna <2,000 iu/ml at weeks 4 and 24). but clevudine resistance can occur in hbeag positive patients. background: serum alanine aminotransferase (alt) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. current standards for "normal" alt level were defined by using populations that included persons with subclinical liver disease. there is no study regarding normal level of alt and its modulating factors in healthy thai people. objective: to definitions of normal ranges for serum alt level in thai people. design: prospective observational study setting: phramongkutklao hospital and army institute of pathology pramongkutklao medical center(a.i.p.), bangkok, thailand participants: 200 persons who were first-time blood donors from august through december 2007 were negative for anti-hepatitis c virus(hcv), negative hbsag(hbv), and had no contraindications to donation. measurements: univariate and multivariate analyses examined associations between clinical and laboratory factors and alt levels. normal ranges for alt were computed from the population at lowest risk for liver disease. results: serum alt activity was independently related to body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. normal ranges for serum alt level in thai people upper limits for men 26 u/l and for women 21.67 u/l. conclusion: in men serum alt is strongly associated with body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. the normal range of alt should be defined for male and female separately. background: the open-label rollover study etv-060 was conducted after etv phase ii clinical study etv-047 for nucleoside-na ve adult chb patients in japan. in this analysis, we report etv long-term efficacy and safety in patients who were switched from 24-week lvd treatment to etv therapy. methods: ninety-seven percent (33/34) of lvd-treated patients from etv-047 were rolled over into etv-060 treated with 0.5mg of etv. thirty patients completed 96 weeks of etv therapy and were evaluated for hbv dna level, alt normalization, hbeag seroconversion, resistance and safety. results: comparing to baseline before switching to etv, after 96 week of etv treatment, the proportion of patients achieving undetectable hbv dna (<400 copies/ml) increased from 21% to 90%. increases were also observed for alt normalization (81% to 90%) and hbeag seroconversion (10% to 19%). three patients had detectable hbv dna at week 96 after etv treatment and samples from two were tested for resistance. neither demonstrated substitutions associated with etv or lvd resistance. five patients had grade 3-4 laboratory abnormalities, including increased ast/alt and increased lipase levels. conclusions: switching patients from lvd therapy to etv resulted in increased proportions of patients achieving hbv dna suppression, alt normalization and hbeag seroconversion, with no evidence of etv resistance. etv was well tolerated during treatment. backgrounds/aims: hepatitis b virus (hbv) reactivation in patients undergoing chemotherapy hampers an adequate administration of cytotoxic agents and even causes an treatment failure. prophylaxis failure occasionally results from viral breakthrough or withdrawal flare. the aims of this study were to identify predictors of anti-viral prophylaxis failure and to determine the optimal strategy for anti-viral prophylaxis. methods: cancer patients with positive hbsag who underwent cytotoxic chemotherapy in a tertiary medical center from january 2005 to june 2008 were included. prophylactic lamivudine was started with initiation of chemotherapy, continued during the chemotherapy, and discontinued within 6 months after the completion of chemotherapy. all patients were followed up even after withdrawal of lamivudine. results: 115 patients were enrolled. twenty-nine patients (23.7%) had hematologic malignancies and eighty-six (76.3%) had solid tumors. median follow-up duration was 15.9 months and twenty-six patients (22.6%) experienced the prophylaxis failure: viral breakthrough (11 patients, 9.6%), withdrawal flare (15 patients, 13.0%). ymdd mutation developed in four patients. withdrawal flare occurred at a median 2.5 months after discontinuation of lamivudine. using log-rank test and cox multi-variate analysis, our results showed that the type of underlying malignancies (hr 2.46, 95% ci, 1.11-5.43; p=0.026) and baseline hbv dna titer (hr 3.91, 95% ci, 1.63-9.39; p=0.002) were significant independent risk factors for antiviral prophylaxis failure. conclusion: cancer patients with high viral load of hbv and hematologic malignancies may need more prolonged and potent anti-viral prophylaxis to avoid interruption or delay of chemotherapy. back ground: the usefulness of hepatitis b virus (hbv) dna and hbv core-related antigen (hbcrag) was evaluated for timing hepatitis flare after viral breakthrough or withdrawal of antiviral treatment in chronic hepatitis b. method: a total of 32 events of hbv reactivation due to withdrawal of lamivudine (lam) or emergence of mutants resistant to lam or adefovir dipivoxil (adv) virus were analyzed in 25 patients with chronic hepatitis b (20 men, median age 56 years [range: 30-66]). they were followed monthly for serum alt, hbv dna and hbcrag before, during and after the treatment. result: high alt flare (alt > 100 iu/ml) after viral breakthrough or withdrawal was related with baseline hbeag positivity (p=0.016), hbcrag level at hbv dna elevation (p=0.038) and duration from hbcrag elevation to salvage therapy (p=0.044). in multivariate analysis, hbcrag > 4.0 log u/ml (or 22.9, 95%c.i. 1.7-304.1, p=0.018) and salvage therapy after 8 weeks from hbcrag elevation (or 10.6, 95%c.i. 1.6-71.4, p=0 .015) were selected as related factor with high alt flare. after appearance of resistant-virus or withdrawal of lam or adv therapy, hbv dna re-elevated without increase of hbcrag, then hbcrag elevated with hbv dna. re-elevations of alt occurred in 27 of the 32 (84%) events. in 23 of the 27 (85%) events, alt re-elevated within 8 weeks from the start of hbcrag increase. conclusion: hbcrag was useful for timing the re-elevation of alt after hbv dna re-elevation induced by drug-resistant virus or withdrawal of lam or adv therapy. background and objectives: the aim of the study was to observe the efficacy of a patient's therapy for switching lamivudine + placebo to adefovir dipivoxil (adv), and modeling the viral dynamics. methods: the studied object was a chinese chb patient with lamivudine mutation. used the lvd + placebo for 12 weeks' therapy. then switched adv for another 95 weeks. after that stopping the treatment and following up for 24 weeks. based on our modified basic virus infection model, we introduce a personalized model consisting of four variables: x, y, v, e , representing uninfected cells, infected cells, free virus, and ctl cells, respectively. results: selected the model parameters, the simulation data of hbv dnas of our model are good in agreement with the clinical ones. observe that after 24 weeks' treatment cessation, the benefit (hbv dna < 250 copies/ml) for suppressing hbv replication can still be kept. numerical simulation show that if the patient's immune functions can be kept after therapy stops, it needs 9 years to replace all infected cells by normal ones. conclusion: for lvd mutation patients, lvd+ placebo to avd therapy scheme may help patients to suppressing hbv replication. further researches are promising. acknowledgments: this work is jointly supported by the nnsf of china background: g-a-1896 pre-core mutants (p-c-mt) cause hbeag-negative chronic hepatitis (chb) in genotype d infected mediterranean adults. we studied their emergence during chronic hbv infection in children. methods: eighty consecutive hbsag carriers (50/30 males/females, age 11y, range 0.2-17y) with vertical (66%) or intra-familial (16%) transmissions were followed-up for 12.5y (range 1-25 y). hbv genotype and hbeag status were determined at the admission, hbeag/anti-hbe every 2 years thereafter. during the follow-up, hbv-dna was measured in 185 sera (1-7 sera/patient) (cobas-amplicor, roche); p-c populations were characterized by direct sequencing (ds), by oligo-hybridization (oha) and allele-specific-pcr (as-pcr) with 30%, 10% and 0.1% sensitivities, respectively. results: seven children were genotype a and 73 d; 70 (87.5%) were hbeag-positive. fifty-five (78.6%) underwent hbeag/anti-hbe seroconversion (median age 13y, range 1.3-27y). baseline hbv-dna (cp/ml) was lower in seroconverters (7.9+1.9 vs 9,4+0,6, anova p=0.012). ds/oha p-c-mt were 4.2% at the admission and 45.8% after follow-up; as-pcr p-c-mt 33.3% and 50% respectively. after seroconversion 47 (85.5%) became inactive carriers, 6 (14.5%) lost hbsag (5 genotype d/p-c-wt); 8 p-c-mt had chb. hbv-dna (cp/ml) was lower in 31 p-c-wt than in 14 p-c-mt inactive carriers (3.42+1.05 vs 4.58+0.91; anova p=0.007). conclusions: in genotype-d infected children p-c-mt is selected progressively after hbeag/anti-hbe seroconversion to become predominant in hbeag-negative chb. early and efficacious immune control of hbv replication avoids p-c-mt selection and leads to hbsag loss. , a, , d, k, u, m, n , k1 ,k2 and k3. here k1, k2, and k3 are the rate of ctl production and dead, killing virus, respectively. results: the patients with hbv dna levels less than 1000 copies/ml were reported in 17.5% (7/40). a patient whose hbv dna levels were higher than 40000 copies/ml can keep treatment benefits even stopping the therapy for over ten weeks. the simulation data of our model are in agreement with the patient's hbv dna data. our simulation also shows that it needs to spend about 18 years for clearing all infected cells. conclusion: the simulation result implies that some chinese patients may need long term's therapy to clear all infected cells. patients' ctls assays are needed to confirm the effectiveness of the personalized modeling, and help doctors to decide whether stop the drug treatment even patients' hbv dnas are higher than undetectable levels. background/aim: recent reports have shown that programmed death 1(pd-1) expression is associated with t cell exhaustion and persistent viral infection. we studied longitudinally 28 chronic hepatitis b(chb) patients undergoing treatment with nucleos(t)ide analogues or pegylated interferon-(peg-ifn-) in 12-16 weeks to determine the relationship between pd-1 expression levels on t cells and early reduction of viral load induced by treatment. methods: our investigations were focused on three points: baseline (time point 1, t1), treatment weeks 4-6(time point 2, t2) and treatment weeks 12-16(time point 3, t3). pd-1 expression on total cd4 and cd8 t cells in chb patients during antiviral therapy was detected by flow cytometry. serum hepatitis b virus (hbv)-dna load was measured by real time polymerase chain reaction. results: between t1 and t2, pd-1 expression on total cd8 (p<0.01) and cd4 t cells (p<0.01) dropped concurrently with treatment-induced hbv-dna decline(p<0.01). between t2 and t3, however, only the hbv-dna levels reduced significantly (p<0.01). conclusion: early suppression of hbv replication induced by antiviral treatment results in a significant decrease in pd-1 expression on total cd8 and cd4 t cells in chb patients. c. zhao 1,2 , w. zhang 2,3 , x.c. tian 1 , c.y. fang 2,3 , h.j. lu 2,3 , p.y. yang 2,3 , y.m. wen 1,2 background/aims: hepatitis b virus (hbv) is still regarded as one of the major causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. the interactions between hepatitis b surface antigen (hbsag) and host cells still remain largely unknown and need to be explored in detail. methods: differential protein expression profiles of hepg2-s-g2( stably expressing hbsag cell line) and hepg2-neo-f4 ( control cell line) were compared using two dimensional gel based differential proteomic approach. cell proliferation assay and survival assay were used for further studies on the candidate protein. results: compared with the control down regulation of 44 proteins and up regulation of 38 proteins were found in hepg2-s-g2 cell. all these regulated proteins were identified by ms/ms and could be fell into several categories including metabolism-associated, immune-response-related, protein modification, signal transduction and others. among them, a group of proteins in putative pathways associated with apoptosis were found out and discussed, including glucose-regulated protein 78kd (grp78/bip), heterogeneous nuclear ribonucleoprotein (hnrnp), far upstream element-binding protein (fusebp), rho gdp dissociation inhibitor (gdi), cystatin b and some scaffold proteins. grp78, an important chaperone protein involved in multiple functions in host cells, was consistently decreased in hepg2-s-g2 and in huh7 cell transiently transfected with hbsag expression plasmid. decreased crp78 inducing by hbsag or blockage of rnai consistently led to the less resistance to staurosporine-induced cell death. conclusions: these results revealed a possible pathogenesis induced by hbsag via grp78. background/aim: to evaluate the efficacy of adefovir dipivoxil alone and in combination with lamivudine in treating patients with lamivudine-refractory hbeag-positive chronic hepatitis b. methods: eighty-five hbeag-positive patients who had received lamivudine treatment for various periods and had a lamivudine-resistant liver function abnormality, documented ymdd mutations and persistent viremia were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. the primary efficacy measure was virological response. the secondary efficacy measure was serological response (hbeag loss rate and hbeag seroconversion rate) and alt normalization rate. results: after 24 weeks of therapy, mean reduction of hbv-dna level, the percentage of patients with hbv-dna lower than 5 log10 copies/ml and the percentage of patients with hbv-dna level decrease of more than 2 log10 copies/ml in patients of adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups were significantly higher than those in patients of lamivudine group (2.58, 2.21 log copies/ml vs. 1.02 log copies/ml, 92.3%, 88.5% vs. 33.6%, 76.9%, 75.8% vs. 28.8%; p < 0.001, respectively). at the end of 52 weeks, mean reduction from baseline in serum hbv-dna level at was 0.08, 4.25, and 4.12 log10 copies/ml in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. alt normalization rates were significant hihger in adefovir dipivoxil/lamivudine and adefovir dipivoxil recipients than those in lamivudine recipients (62%, 55% vs. 8%, p < 0.001, respectively). a similar pattern was observed in hbeag loss among three groups. conclusions: adefovir dipivoxil is an effective treatment option for patients with lamivudine-refractory hbeag-positive chronic hepatitis b. aim: to find the prevalence of hbv virologic flare as defined by hbv dna viral load of > 2,000 iu/ml in inactive chronic hepatitis b (hbv dna less than 2,000 iu/ml), hbeag-negative patients who have not received any treatment and to identify if there are any predictors that can predict virologic flare. methods: we retrospectively analyzed medical records of the patients who have attended hepatitis clinic, siriraj hospital from january 1, 2002 to february 28, 2008 . the patients were eligible if they were naïve to any treatment and hbv dna less than 2000 iu/ml at entry. co-infection with hiv and/or hepatitis c virus were excluded. hbv dna measurement determine by roche amplicor ® (detection limit of 60 iu/ml). hbv virologic flare was defined as hbv dna more than 2000 iu/ml during follow up period. result: there were 84 patients with mean follow up time was 598 days with annual prevalence of hbv virologic flare of 12.8, 4.8, and 4.2% for the first, second and third year of follow up, respectively. initial hbv dna level was the only predictor that can predict reactivation. no patients with hbv dna at entry below detection limit developed flare and the patients with hbv dna above 740 iu/ml had 22 times higher chance to develop flare during follow up. conclusion: hbv dna flare is not uncommon in inactive chronic hepatitis b patients. most of the virologic flares occur in the first year. the most important predictor or virologic flare is higher hbv dna at beginning. background: multi-drug resistant hbv developed with multiple antiviral agents. there existed difficulty in dealing with multi-drug resistant hbv. methods: retrospective analysis of 23 consecutive patients who exhibited chronic hepatitis b associated with multiple drug-resistant mutations to lamivudine and adefovir during antiviral treatment. multiple drug-resistant mutations were detected in those patients by dna direct sequencing. result: before multiple drug-resistant hbv emerged, 20 patients accept sequential antiviral therapy, 3 patients accept na monotherapies. there were 16 cases of rta181t/v rtm204v/i mutation, 2 cases of m204v/i +n236t mutation, 4 cases of a181t/v+m204v/i +n236t mutation, 1 case of l180m+a181t/v mutation. 14 cases received rescue therapy of interfronand hbv dna level of 8 cases decreased; other 9 cases received combination treatment and hbv dna level of 4 cases decreased. conclusion: the main reason of multiple drug-resistant mutations was sequential antiviral therapy. another reason may be pre-exist drug-resistant mutation before nucleoside or nucleotide analogue treatment. de novo combination of antiviral agents should be recommended. combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant hbv. interfron may be one choice for hbv of multiple drug-resistant mutations. background: to furnish basis for an accurate evaluation of hbeag negative chronic hepatitis b (e chb), the present study studies the clinical features and hepatic pathology, and analyzes the relation between the data and the grade and stage of hepatic pathology in e chb. methods: a study is performed in 120 chinese e chb patients (106 men and 14 women; mean age sd, 34.3 9.4 years). the relationship between the clinical features and the grade and stage of hepatic pathology was analyzed by spearman's rank correlation test or kruskal-wallis test by applying stata 7.0 software. result: negative correlation is shown between the grade and leucocyte count methods: 80 patients with hbeag-positive compensated chb with hbv dna >6 log10 copies/ml, serum alt 2 x uln were divided two groups:one treated with telbivudine and the other treated with entecavir. results: baseline characteristics were well balanced between treatment groups. at 12wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 50 52.5 (p 0.05), the rates of hbeag negative were 10 0 respectively, the rates of hbeag seroconversion were 20 5 respectively; at 24wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 80 70 (p 0.05), the total rates of hbeag negative were 20 15 respectively, the total rates of hbeag seroconversion were 27.5 17.5 respectively(p 0.05).no adverse reactions were found in both groups conclusion: there was no significant difference in hbv dna undetectable rates between two nucleotide analogs in short-term (24 weeks).the telbivudine group has better effect in hbeag seroconversion rate than the entecavir group in early stage,but no statistical significance. j.w. song 1,2 , z. xin 1 , j.x. tang 1 , l. yao 1 , b. wu 1 1 sun yat-sen university, 2 zhuhai sinochips biosci. co.,ltd., china background: to develop a equipment free, and can be widely used in clinical practice biosensor-based microarray for hepatitis b virus pre-c/bcp mutation assay. methods: a thin film optical biosensor were applied for amplification the microarray signal in situ. and hbv sites 1762, 1764,1768,1770 and 1896 were selected as the targets and the microarray were be fabricated. the 5 mutated plasmids contained 1762, 1764,1768,1770 and 1896 sites and 30 hbv sera were be tested in our study and all the plasmids and sera pcr products were be assayed by really time pcr and sequencing. results: the biosensor based microarray signal can be easily record by digital camera or even by the naked eyes and the detection signal for positive discriminated from negative were sharply contrasted as whole yes or no and it looks be significantly superior to classical microarray technique; 2. the sensitivity of the detection limitation of sera hbv load is 2x10e3 copies/ml with 95% reproducibility. the concordance index of 50 times negative and mutated plasmids were 98%(kappa=0.932). 3. 30 sera samples of hbv>10e4 load and 20 sera of hbv negative tested by pcr fragment sequencing were showing very good agreement between sequencing with our biosensor based microarray and the concordance index kappa was 0.7619. conclusion: our biosensor-based microarray for pre-c/bcp mutation assay were a both sensitive and accurate method. and its advantages of equipment free, sharply contracted signal of positive vs negative and easily be perform in testing were make it be a promised assay for clinical application. objective: to investigate the frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b, we assayed the differences among hbsag-positive and healthy subjects by flow cytometry. the results might offer some experimental evidence to explain the high rates of hbv persistent infection in vertical transmission of hbv from hbv-infected mothers. methods: 12 newborns born from hbsag positive mothers were recruited , 10 healthy subjects being used as a control group. the cord blood and peripheral blood of mothers were collected respectively .frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b were analyzed by flow cytometric analysis. result: the number of cd4 + cd25 + regulatory t cells/pbmcs in the cord blood of newborns born from hbsag positive mothers 4.49 ±1.18 significantly exceeded that in normal controls 2.26 ±0.97 ,p 0.001 ;and newborns born from hbsag positive mothers presented a much higher fraction of cord blood cd4 + cd25 + /cd4 + 9.62 ±2.34 than those in normal controls 7.93 ±1.43 ,p p=0.0025 p 0.05 . conclusions: the results indicate that the proportion of cd4 + cd25 + regulatory t cells in hbsag positive mother cord blood was higher than those of healthy cord blood. objective: to study the clinical features of chronic severe hepatitis b with negative hepatitis b e-antigen (hbeag) and positive hepatitis b e-antigen (hbeag) methods: a total of 353 in-patients with chronic severe hepatitis b were recruited into the study and divided into two groups according to the hbeag status. the serological chemistry data, hepatitis b virus (hbv) dna quantification data were detected, and morbility of cirrhosis, its complications and prognosis were also studied. results: of the 353 in-patients, 236(66.8%) patients were hbeag-negative. 117(33.2%) patients were hbeag-positive. the ratio of hbeag-positive patients was significantly higher than that of hbeag-positive patients (p<0.05).the average age of hbeag-negative patients was older than that of hbeag-positive patients (p=0.048). the serum hbv dna level of hbeag-negative patients was significantly lower than that of hbeag-positive patients (5.49±2.02) vs(6.64±1.41) log copies/ml (p<0.01).the ratio of patients who had a serum hbv dna level less than 5log copies/ml in hbeag-negative patients was significantly higher than that in hbeag-positive patients (41.8% vs 11.9% ,p=0.000). there was no significant difference in serological chemistry data, morbility of cirrhosis and its complications on infections, ascites, hepatoencephalopathy, gastrointestinal hemorrhage, as well as prognosis of the patients between those two groups. conclusions: the study suggested that serological chemistry data, morbility of complications and prognosis of the disease of hbeag-negative patients mimics that of hbeag-positive patients. the hbeag-negative patients had a higher level of age, while a lower level of serum hbv dna. to reduce the incidence of liver failure, more frequent monitoring and earlier antiviral therapy prone to be reasonable for chronic hepatitis b patients with negative hepatitis b e-antigen. background: the emergence of lam-resistant virus greatly limits the efficacy of therapy and induces the liver injury. the aims of this study were to assess the related factors of lam-resistant mutation in hbeag positive chb patients. methods: thirty-five patients carrying lam-resistant with hbeag positive were enrolled in this study. all of them underwent percutaneous liver biopsy, histological findings and had detectable viral load. age, viral load, levels of alt, types of mutation and hbv genotype was monitored. result: the median year of mutation found was 23months. 85.71% were genotype c and 14.29% were genotype b. the mutation of l80i, l80v, g173l, l180m, m204v and m204i were detected. the emergence rates were 34.3%(12/35), 25.7%(9/35), 17.1%(6/35), 60%(21/35), 57.1%(20/35), 54.3%(19/35) respectively. the rate of patients with two or three mutation were much more than one or four mutation. 62.9% patients were found to have significant histological findings, even 5 had established cirrhosis. two had no histological finding. one had rtl80i and rtm204i. the other had rtl80v, rtl180m and rtm204v. the number of resistant mutation has no significant finding with histological finding, basic alt level and basic viral load. conclusions: the emergence rate of l180m, m204v and m204i were higher than that of l80i, l80v, g173l in hbeag positive chb patients with lam-resistance. most of them have two or three lam-resistant mutation regardless of histological finding severity, level of basic alt and viral load. we must select the efficacious method to treat the patients with lam-resistant. objective: to investigate the therapeutic efficacy of foscarnet sodium in the treatment of patients with severe chronic hepatitis b. methods: forty four patients were randomly divided into foscarnet sodium treatment and placebo groups.each group consisted of 22 patients, 22 patients in foscarnet sodium group were treated with foscarnet sodium twice daily 3.0g given by intravenous infusions ,in addition to general therapy for 28 days.the other 22 cases were treated without any form of antiviral therapy as control.all patients were followed up for 6 months.the hbv markers, quantification of hbv-dna, serological chemistry data were measured at baseline , during therapy period and the end of follow-up period . results: clinical symptoms were improved in two groups patients, meanwhile alanine aminotransferase (alt) and total serum bilirubin (tbil) decreased. compare alt and tbil at the end of trentment, there were no significant differences between the two groups (p>0.05). in foscarnet sodium treatment group, the level of serum hbv-dna descreased from (6.993±0.898) log copies/ml to( 4.033±1.286) log copies/ml (p<0.05), the rate of hbv-dna descrease of more than two log was 81.1% 18/22 . in the control group, the level of serum hbv-dna descreased from( 7.068±0.938) log copies/ml to (5.188±1.926 )log copies/ml, the rate of hbv-dna descrease of more than two log was 45.4% 10/22 .a comparison of serum hbv-dna showed significant differences between the two groups(p<0.05) conclusion: foscarnet sodium administered can inhibit hbv replication in treating severe chronic hepatitis b.it can rapid lower the level of serum hbv-dna obviously.but the relapse rate was 47.0% in foscarnet sodium treated at the end of follow up period objective: evaluation of efficacy and safety of five years trail of entecavir for chronic hepatitis bpatients failed with lamivudine therapy in the chongqing area. methods: thirty-two eligible patients were enrolled who had documented lvd failure.in the double-blind phase,patients were randomized(4:1)to etv1.0mg/d (n=28)and placebo (n=4) for 12 weeks.in the open-lable phase ,patients received etv 1.0mg/d for 240 weeks.hbv-dna level,liver function tests,hbv serology and safety assessments were conducted. results: the mean reduction in hbv dna levels at week 12 was 4 05 logl0 copies ml in etv group compared to 0.08logl0 copies ml in placebo group(p< 0 05). the mean of hbv dna levels after 240 weeks of etv treatment decreased to 2.58logl0 copies m1 the proportion of hbv dna<3log10copy/ml raised from 0 at baseline to 6.25% at week 8,to 15.6% at week 24,to 50% at week 96,and raised to 57.14% at week 240.there were two patients with hbsag seroconversion and four patients with hbeag seroconversion at the end of study. the mean of alt became normal at week 12 and remained normal throughout week 240.there was one patient who had a severe adverse event during the trail. conclusion: the findings from this study demonstrated the antiviral activity and safety of etv in adults with chb who have failed lvd pe198 showing delayed response on t cells as increased on day 7.the mrna expression of il-5 and il-2 showed no response to hbv vaccine but highly regulated in tt after day 7(p=0.00,0.001).myd88andtraf 6(p=0.042)upregulated in hbv vaccine group followed by of ifn-(0.012)no change of ifn found in tt conclusions: i) hbv vaccine stimulates innate response by day 3 which potentiates further cascade,peripheral dendritic cells plays significant role in generating immune flare follows myd88 pathway and releases ifn-.ii) whereas t cells marjory involved in tt showed delayed immune response.iii) identification of key factors at different time points may prove to be a novel model to study the initial events after vaccination. objective: to compare th1/th2 cytokines' dynamic change and its clinical significance in hepatitis b e antigen-positive patients treated with telbivudine. methods: twelve hepatitis b e antigen-positive patients treated with telbivudine.the blood sample was collected at baseline, week 4, week 8, week 12, week 24 and week 48 and stored at -70c; serum il-2, il-4, il-6, il-10, tnf-and ifn-were tested at each time point by cytometric bead array (cba), compare th1/th2 cytokines' dynamic change at different time point in each group and compare th1/th2 cytokines' dynamic change cross four different groups: complete response, partial response, non-response and break through . results:the level of th1 type cytokines in complete response group are obviously higher than the group of partial response non-response and breakthrough,but the level of th2 type cytokines are lower than the group of partial response, non-response and breakthrough. conclusions: th1/th2 cytokines is essential for the regulation of the immune function of the body. after treated with telbivudine, the level of th1-type cytokines in the complete response group increased significantly, while the level of th2 cytokines declined trend. a. soamni 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background: chronic infection with hepatitis b virus causes spectrum of manifestations ranging from asymptomatic carrier state (often inactive with low replication) to the development of cirrhosis-related complications.the characterization of asymptomatic state has not been done in this part of the country, which forms important objective of present study. methods: 61 incidentally detected asymptomatic hepatitis b surface antigen positive (idahs) subjects having hbsag positivity for >6 month presenting to our liver clinic were enrolled after appropriate consent. detailed clinical, laboratory and sonographic evaluation was done. they were divided into two groups according to presence or absence of e antigen. group a -hbeag + (n=48) group b -hbeag -(n=13) results: most of our patients (49%) were young adults (21-30 years) with male to female ratio of 3.6:1. approximately half of our patients were detected during routine medical checkup, followed by family screening of contacts. most of our patients were asymptomatic, and fatigue was most common symptom found in 16%. all demographic and biochemical parameters other than ast & alt were comparable in both groups. among hbeag negative 48 (79%) subjects, hbv dna level >10 5 copies/ml was found in 31%. subjects with positive hbeag as compared to non-replicative infection (antihbe positive and hbv dna negative) had more frequent elevation of transaminase levels (62% versus 31%, p<0.05). antihbe antibody was positive in all hbeag negative subjects. mean age of seroconverted (antihbe positive) individuals was a decade older than hbeag positive. conclusion: from our study we can suggest that ongoing liver disease is present in approximately one-thirds of incidentally detected asymptomatic hepatitis surface antigen positive subjects previously referred to as carrier state. hbsag testing should be mandatory in all routine medical checkup and family and sexual contacts of index case should be screened. background and objectives: this research was carried out to determine the prevalence of hbcab among the hbsag negative first-time blood donors who had referred to khorramabad and borujerd centers for blood donation. materials and methods: this study was established on a descriptive cross-sectional basis in which hbsag test (elisa) was primarily performed on all of the donors having referred to khorramabad and borujerd blood centers; then, out of all those referred 1000 subjects, who were first-time and hbsag negative, were selected for furthur investigation. the information concerning age, gender, job, blood transfusion, and hbv vaccine injection was included in the questionnaire of the study. hbcab (total & igm) and hbsab tests were performed on the selected donors. data were collected and finally the prevalence rate of hbcab was determined. results: the results of the study showed that out of 1000 hbsag-negative first-time blood donors, only 47 were hbcab+, from which 27 were hbcab (total)+, and 3 were hbcab (igm)+. 18 were both hbsab+ and hbcab+, and 53 were seropositive only for hbsab. conclusions: it was demonstrated that the first-time blood donors who are seronegative for hbsag marker will be easily identified through hbcab test if they are in the so-called core window period of the virus. meanwhile, this group of donors have been implicated as high-risk for transfusiontransmitted hbv infection. so, detecting this marker will remarkably reduce the chance of latent cases of hbv infection and help promote blood safety. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. method: 20 hbv transgenic mice were randomly divided into physiologic saline group and matrine injection group. another10 normal mice at the same species and age with hbv transgenic mice were regarded as the normal group. the mice in matrine injection group were administrated at dosage of 82.2 mgkg -1 d -1 by intraperitoneal for 30 days. the mice in physiologic saline control group and normal group were administrated normal saline with the same volume at same time. the contents of hbv dna in serum and liver were quantitated by pcr. and the spleens were separated for cultivating dendritic cells. the surface molecules of dendritic cells were tested by flow cytometry. ifn-mrna and tnf-mrna in liver were tested by rt-pcr. result: there was no significant difference of the serum hbv-dna level between physiologic saline and matrine injection groups. the content of serum hbv-dna after treatment showed a significant decrease in two groups. the content of serum hbv-dna in matrine injection dropped significantly as compared with that in the physiologic saline group. but there was no significant difference in the content of hbv-dna in liver between physiologic saline and matrine injection groups. the expression level of mhc-ii on dendritic and hepatic ifn-r mrna and tnf-a mrna showed a significant decrease in hbv transgenic mice than normal mice. in comparison with physiologic saline group the expression level of them in matrine injection group showed a significant increase. conclusion: matrine injection was effective on depressing hbv-dna in hbv transgenic mice. its antiviral action may be achieved through regulating mhc-ii on dcs surface and promoting the production of antiviral factor such as ifn-and tnf-. purpose: to stimulate non-specific immune response capacity as the main content of the study to explore the hbv-dna and non-specific immune responses in the relationship between the low response capability, methods: 190 cases of asymptomatic carriers, double-blind, randomized into mycobacterium fu 36, lamivudine and traditional chinese medicine for the treatment group, mycobacterium fu36 with traditional chinese and lamivudine with traditional chinese medicine were in the control group, a total of 12 weeks of treatment, follow-up six months after the termination of treatment. results: different treatment of hbv -dna effect of the existence of significant differences; p> 0.01, the performance of different types of asymptomatic carriers negative rate of hbv-dna there is a significant difference; p> 0.01, as well as the performance of the different types of asymptomatic carriers continued application a treatment plan presented hbv-dna rebound rate there is a significant difference; p> 0.01, conclusion: hbv-dna and non-specific immune responses in response to the lower capacity, anti-hbv therapy is not associated with non-specific immune response capacity or improve is the anti-hbv drugs alone can not solve the asymptomatic carriers in anti-hbv therapy where the cause of the problem, solve the asymptomatic carriers in the anti-hbv treatment although the need for anti-hbv drugs with non-specific immune activation synchronous drugs on the basis of the joint application , but the simultaneous combination of two drugs rather than as a result of hbeag and hbv-dna can hbeag-positive asymptomatic carriers receive hbv-dna negative effect of the results. background: adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine (lam)-resistant hepatitis b virus (hbv) replication and resistance to this drug is infrequent compared with lam. in this study, we tried to identify factors affecting the emergence of resistant mutants after adefovir monotherapy in lam-resistant chronic hepatitis b (chb) patients. methods: the subjects were 87 chb patients with lam-resistance who had received adefovir for more than 12 months (range 12-39 months). the initial viral response (ivr) was defined as hbv dna <4.0 log copies/ml. the adefovir resistant mutant was assayed at baseline and every 6 months during adefovir administration. results: ivr was observed in 38% of patients. the cumulative emergence rates of adefovir resistance were 2.6% at 6 months, 10.4% at 1 year, 14.6% at 2 years and 21% at 3 years. in univariate analysis, factors contributing to the emergence of adefovir resistant virus were baseline hbv dna > 6 log copies/ml (p=0.003) and ivr (p<0.0001). the presence of precore mutation and type of ymdd mutants were not related. in multivariate analysis, only ivr was an independent factors affecting the emergence of adefovir resistant virus (p<0.0001). conclusion: ivr is a useful predictor for emergence of adefovir resistant mutants after adefovir monotherapy in lam-resistant chb patients. for ivr-negative patients, the change of therapeutic options such as add-on lam or switch to other drugs should be considered because of the high incidence of the emergence of adefovir resistant mutants. background: elevated hbv dna is strongly associated with the risk of disease progression. this study investigated the early viral suppression effects of etv and lvd in nucleoside-naïve chinese patients with active hbeag (+) chb. methods: this open-label study was conducted in 5 major hospitals in china. at study entry all patients had hbv dna levels 10 7 copies/ml, elevated alt (1.3-10xuln) and compensated liver function. patients received either 0.5mg etv or 100mg lvd daily. hbv dna measurements were taken at baseline and at weeks 2, 4, 12 and 24 during treatment, using roche cobas amplicor assay (llod 300 copies/ml). results: a total of 97 patients were enrolled; 42/50 etv patients and 40/47 lvd patients completed 24 weeks of treatment. at baseline, mean hbv dna levels were 8.45 0.81 in etv group and 7.67 1.76 log 10 copies/ml in lvd group (p<0.05). the mean change in hbv dna from baseline (log10 copies/ml) was -2. 96±0 ngo's/funding-agencies representative at apasl2009-conference need to address-this-issue. we ngo-representatives from developing-nations need exposure to research treatments used by european/american experts. do we all failed in addressing socio-economic issues of cancer-sufferers? we need to address these socio-economic issues of affected population in resource-poor-nations. background: a garlic derivative s-allylcysteine (sac) has anti-cancer effect in human prostate and colon cancers. we aimed to investigate the effect of sac and combination of chemo-drug on tumorigenesis and metastasis of liver cancer. methods: the orthotopic liver tumor model using a metastatic liver cancer cell line mhcc97l labeled with luciferase gene was applied. sac was given at day 7 after tumor implantation at 1mg/g/day, or 1mg/g/day combined with low dose cisplatin for 5 weeks. tumor growth and metastasis were monitored by xenogen in vivo imaging system. hepatic stellate cell (hsc) activation and tumor-associated macrophage (tam) in the tumor tissue were detected by -sma and ed1/ed2 staining. tumor micro-vessel density (mvd) and apoptosis were also analyzed. in vitro functional tests including proliferation assay, cell cycle analysis and apoptosis analysis were performed. results: tumor growth was inhibited by sac combined with cisplatin treatment at different time points accompanied by lower incidence of lung metastasis compared with other groups. the observation of xenogen ivis was confirmed by histopathological examination. the hsc activation by -sma staining in the liver tumors was suppressed by sac and cisplatin treatment accompanied with less tam infiltration. consistent with in vivo study, in vitro functional study also demonstrated that sac not only induced cell cycle arrest, apoptosis, and inhibited tumor cell proliferation, but also sensitized the anti-cancer effect of cisplatin. conclusion: sac treatment inhibited liver tumor growth and metastasis by inhibiting tumor cell proliferation, inducing apoptosis and sensitization of chemotherapy. background: anti-angiogenic therapy would be a promising approach against hepatocellular carcinoma (hcc). although a sorafenib has survival benefits in patients at advances stages of hcc, there seem to be several serious concerns to employ this agent for chemoprevention against hcc. branched-chain amino acid (bcaa) reportedly inhibits the incidence of hcc in patients with insulin resistance (ir). however, the possible mechanism is still obscure. the aim of the current study was to examine the effect of bcaa on hepatocarcinogenesis under the condition of ir, especially in conjunction with angiogenesis. methods: the effect of bcaa on the development of liver enzyme-altered pre-neoplastic lesions and angiogenesis in the obese diabetic otsuka long-evans tokushima fatty rats was examined. we also performed an in-vitro study to elucidate the possible mechanisms involved. result: treatment with bcaa markedly inhibited the glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions along with suppression of neovascularization in the liver. the hepatic expression of the vascular endothelial growth factor (vegf), a potent angiogenic factor, was also attenuated. bcaa treatment significantly suppressed the glucose-and insulin-induced in-vitro angiogenesis in the presence of vegf. these results indicate that bcaa exerted a chemopreventive effect under the condition of ir via suppression of vegf-mediated angiogenesis. conclusion: since bcaa is widely used in the clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against ir-based hcc in the future. background: krüppel-like factor 8 (klf8) is a member of transcription factors. whether and how klf8 signaling pathways contribute to hepatocellular carcinoma (hcc) development and progression is unknown. this study investigated role of klf8 in hepatocellular carcinoma cell line hcclm3 proliferation, invasiveness and epithelial to mesenchymal transition (emt). methods: the expression of klf8 in different liver cell lines was detected by quantitative real-time pcr and immunocytochemistry. we used small interfering rna (sirna) to down-regulate klf8 expression in hcclm3. the change of proliferation and invasive ability of klf8 down-regulated hcclm3 was investigated by mtt reduction assay and trans-well invasive assay respectively. the change of proliferation, invasiveness and emt related gene in klf8 down-regulated hcclm3 was evaluated by quantitative real-time pcr. result: klf8 protein expressed predominantly in the nuclei of cancer cells and its expression is positively correlated with metastatic potential of these cell lines. hcclm3 has the highest klf8 level. decreased klf8 expression can notably inhibit the proliferation (p<0.001, n=6), mobility and invasiveness of hcclm3 (p<0.001, n=8). we found that the mrna level of n-cadherin, fibronectin and vimentin is much higher than that of e-cadherin in hcclm3. the expression of cyclin d1, focal adhesion kinase (fak) and fibroblast markers including n-cadherin and fibronectin was obviously suppressed in klf8 down-regulated hcclm3. conclusion: klf8 plays an important role in the process of hcclm3 proliferation, invasiveness and emt. background: insulin resistance (ir) has shown to play an important role in the progression of chronic liver diseases, including liver fibrosis development and hepatocellular carcinoma. the aim of this study was to elucidate the possible mechanisms of ir on the liver fibrosis development and hepatocarcinogenesis using obese diabetic otsuka long-evans tokushima fatty (oletf) rats. methods: to induce liver fibrosis, 1.0ml/kg of pig serum was injected twice a week for 4 weeks in the oletf and leto rats. in the hepatocarcinogenesis model, glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions were induced by a single injection of 200mg/kg of diethyl nitrosamine (den). we also performed in-vitro studies to examine the mechanistic insights. results: the liver fibrosis development and gst-p-positive pre-neoplastic lesions were both markedly accelerated in oletf. in the fibrosis experiment, -smooth muscle actin-positive activated hepatic stellate cells (hsc) also increased in oletf along with augmentation of the hepatic collagen content and transforming growth factor-b1. in the den model, the neovascularization was up-regulated in oletf almost in parallel with the pre-neoplastic lesions development and a potent angiogenic factor, the vascular endothelial growth factor. our in-vitro study showed that both glucose and insulin stimulated the proliferation of the activated hsc and augmented the neovascularization. conclusion: these results indicated that the ir status directly accelerated the liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated hsc proliferation and hepatic neovascularization, respectively, in the rat. n. wakui 1 , t. ikehara 1 , r. takayama 1 , m. takahashi 1 , k. shiozawa 1 , h. nagai 1 , m. watanabe 1 , k. ishii 1 , k. iida 1 , y. igarashi 1 , y. sumino 1 1 case: a 68 years old man diagnosed with chronic hepatitis c regularly visited our hospital. in april of 2005, ultrasonography revealed a tumor 20mm in diameter in s2 of the liver and another tumor 15mm in diameter in s6/7 of the liver. the patient was hospitalized for further examination. computer tomography (ct) revealed that the tumor localized in s6/7 presented a pattern of hypervascular hepatocellular carcinoma (hcc). for the tumor localized in s2, the following were revealed. 1) contrast-enhanced ultrasound findings: a tumor vessel passed from outside the tumor to the center of the tumor in the early vascular phase, then radiated in a wheel-like shape at the center of the tumor; parenchymal phase perfused imaging in the area produced a similar imaging obtained from the area surrounding the liver. 2) ct: no tumor was detected. 3) spio-mri (t2 weighted imaging): iso-low intensity images were obtained. although these imaging findings indicated fnh, the patient was hcv positive. in order to disprove the possibility of hcc, a biopsy was performed on the tumor at s2 in the liver. the resulting diagnosis was well-differentiated hcc. discussion: until now, a characteristic finding of fnh has been spoke-like vasculariation, which is considered diagnostically quite important. however, some recent cases of hcc have been reported to present fnh-like vascularization. from now on, when evaluating a tumor that presents spoke-like vascularization underlining chronic hepatitis, the possibility of hcc should be considered and a close examination may be needed. chronic infection with hcv is problem .clinical management of chronic hcv depend on extent liver fibrosis .liver biopsy gold stander an invasive procedure responsible for severe complications and sample variability interpretation. serum biomarkers for inflammation/fibrosis investigated to wave liver biopsy. diagnostic accuracy panel of non-invasive serum biomarkers for hepatic fibrosis (fibrosure , apri score, forn's score) versus liver biopsy. 20 hcv patients subjected for: apri, forn's , fibrosure scores pcr quantitative hcv-rnaliver functions .lipid profile cbc . ultrasound guided liver biopsy. forns score; auroc (0.917) with 95% ci(0.791-1.042) for(fof1) vs. (f2f3f4) while(0.688)with 95% ci(0.464-0.91)for(fof1f2) vs.(f3f4). cutoff(>6.9)sensitivity for significant fibrosis(f2f3f4)and extensive fibrosis (f 3f4)were (100%) and with low specificity ,with accuracy(40%) and (20%)respectively.-apri score; auroc(0.792)with 95% ci( 0.568 -1.015)comparing(f0f1) vs.(f2f3f4)while was(0.875)with 95% ci(0.703 -1.047)for( f0f1f2)vs.(f3f4).cutoff(<0.5) had low sensitivity and specificity(100%)with accuracy(60%)for significant fibrosis and(80%)for extensive fibrosis.-fibrosure(fibro-acti test); showed best auroc(1.00)in different fibrotic stages with 95 % ci (1.00-1.00).cutoff(>0.59) sensitivity(50%)for significant fibrosis and(100%)for extensive fibrosis while specificity(100%)in all fibrotic stages. the ppv (100%)for significant and extensive fibrosis .npv and accuracy(75%, 80%)respectively for significant fibroses,while it was (100%) for extensive fibrosis respectively.significant correlation between liver biopsy and fibro-test(p0.002)and acti-test(p0.000).significant correlation between liver biopsy hepatitis activity score and apri (p 0.047)and forns score (p0.000). conclusion: forns score wasn't considered since does not discriminate between significant and extensive fibrosis. low sensitivity of apri prohibtes detection of minmal fibrosis and allow undetermined results. fibrosure classified all cases of chronic hcv sufficient to wave liver biopsy pe215 introduction: hcc is the 6 th common cancer. global increase of hepatitis b and c infection, the incidence of hcc steadily increasing. egypt seroprevalence of hcv in nile delta 20 -35%. afp had limited sensitivity 60% and specificity 90% for small hcc. gpc-3 oncofetal protein over expressed in hcc. evaluating validity of glypican-3 as early detector of hcc.:10 healthy controls and 40 hcv positive patients:10 patients chronic hepatitis c virus infection.10 patients compensated cirrhosis [child-pugh class a and b].10 patients decompensated cirrhosis [child-pugh class c]. 10 patients hcc. liver functions: alt, ast, bilirubin(t), albumin, gt.tumor markers: afp and gpc-3.viral markers: hcv antibodies, hbs ag and hbc ab. the median value of gpc-3 in hcc, dc, cc significantly higher than chronic hepatitis and control groups. no significant correlation between afp and gpc-3. auroc of afp 0.85 & auroc of gpc-3 0.84. the diagnostic sensitivity of afp (20 ng/ml) 70% with ppv 53.8%. the specificity85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) 100% with ppv 27%. the specificity 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved specificity to 87.5%. conclusion:gpc-3 although a serological test for early detection of hcc, showed limited specificity, where detected in different stages of chronic liver disease,it is oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 improve prediction accuracy of hcc patients in those showing seronegativity to afp. results: patients with hcv infection (n=388) were significantly older (mean age, 69 years) than patients with dual virus (n=75, 65 years) and hbv infection (n=752; 60 years) (p< 0.0001). the male-to-female ratio for hbv, dual virus and hcv group was 5.2, 3.4 and 1.3, respectively (p< 0.0001). patients in the hbv group more often had higher total tumor volume (mean, 409 cm 3 ) than the dual virus group (244 cm 3 ) and hcv (168 cm 3 ) group (p< 0.0001). no significant differences of the severity of liver cirrhosis, performance status, cancer staging and tumor cell differentiation were noted among the three groups. patients in the hcv group had a significantly poor survival in comparison to the hbv group only in the subset of patients with small tumor volume (< 50 cm 3 ) in the cox proportional hazards model (relative risk: 1.44, p=0.041). conclusions: dual hbv and hcv virus infection does not accelerate the speed of hcc formation in patients with chronic hepatitis b, and appears to have a modified course of carcinogenesis pathway diverted away from the biological behavior of hbv and hcv infection. background: patients presenting with hcc is not infrequent in our clinical practice. the aetiology vary ranging from hbv, hcv, nash and alcohol. the aim of this study was to see the aetiology of hcc in bangladeshi patients. methods: in this retrospective study, records of 976 patients who attended our opd between july 2004 to august 2008 were reviewed. patients having hepatic sol and/or heterogeneous echotexture of liver on usg and/or ct scan were included. diagnosis of hcc was confirmed at usg guided fine needle aspiration cytology with or without elevated serum afp (>500 ng/ml). results: of the 976 patients, 75% (732/976) had hbv infection. hcv infection was diagnosed in 17% (165/976). nash was responsible for 5% (49/976) cases, alcohol in 1% (10/976), while in the rest 2% (20/976) cases no specific aetiology could be established. conclusion: the study shows that hbv is the commonest cause for hcc in bangladesh followed by hcv. background: the aim of this study was to determine whether the hepatitis b virus (hbv) dna viral load and antiviral therapy is associated with hepatocellular carcinoma (hcc) recurrence. methods: this retrospective study involved 93 patients who underwent hepatic resection or radiofrequency ablation for initial hcc curative treatment. the patients were divided into four groups. fifteen patients with low serum hbv dna levels ( 4 log10 copies/ml) at the time of initial hcc treatment received antiviral therapy (lamivudine, adefovir, dipivoxil, entecavir) before hcc appeared (pre antiviral therapy group; pre-tg). thirty-four had low serum hbv dna levels without antiviral therapy (low virus group; lvg). fourteen had high serum hbv dna levels and received antiviral therapy after hcc appeared (post antiviral therapy group; post-tg). thirty patients had high serum hbv dna levels without antiviral therapy (high virus group; hvg). results: the cumulative hcc recurrence rates at 3 years in the hvg, lvg, pre-tg, and post-tg groups were 68.9%, 42.2%, 44.3%, and 57.1%, respectively. there were significant differences in the hcc recurrence rates between the hvg and lvg groups (p = 0.016), and between the hvg and pre-tg groups (p = 0.013). the recurrence rate was lower, though not significantly, in the post-tg group than in the hvg group (p = 0.18). conclusions: not only hbv dna viral load but also antiviral therapy is associated with hcc recurrence. antiviral therapy before hcc appears is important for patients with high serum hbv dna levels to prevent hcc recurrence. background/aims: few reports have described methods for predicting prognosis in unresectable hepatocellular carcinoma (hcc) patients, especially those treated by repeated transcatheter arterial chemoembolization (tae). to determine risk factors for death and determine prognosis in patients treated with repeated-tae, we evaluated clinical data. methodology: we retrospectively analyzed clinical parameters of 224 unresectable hcc patients treated with repeated-tae from january 1997 to december 2007. tae was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. factors affecting survival were evaluated using multivariate analysis after univariate analysis. next, we combined the score for each significant factor into a single prognostic score, after which the results were compared with jis and clip score methods. results: multivariate analysis revealed that bilobular hcc, alpha-fetoprotein ( 400 ng/ml), tumor invasion of the portal vein, tumor size ( 10 cm), and albumin (<2.8 g/dl) were related to poor prognosis, using those 5 factors, we developed a new prognostic scoring system. the 50% survival period was 29.2 months for all subjects, while it was 54.6, 29.2, 15.0, 5.6, and 1.0 months for those with scores of 0, 1, 2, 3, and 4 or over, respectively (p<0.0001), using our new system. clip score was not useful to predict prognosis, while jis score was better. however, subjects with jis scores of 1 and 2 were difficult to differentiate. conclusion: our scoring system was easy to perform and the results showed that repeated-tae was effective for unresectable hcc with a score of 2 or less. local ablative therapies and intrahepatic pressure c. kawamoto 1 , a. yamauchi 1 , k. kaneko 1 , n. miyagi 1 , k. kani 1 , t. aoyama 1 , k. yakabi 1 1 saitama medical center, saitama medical university, japan background: some of the unexpected recurrence observed after radiofrequency ablation (rfa) might be caused by increased intratumoral pressure. the present study examined the relationship between local ablative therapies and intrahepatic pressure. methods: a. basic study: under general anesthesia, laparotomy was performed on 19 pigs. a leveen needle and a percutaneous ethanol injection (pei) needle were inserted into the liver and intrahepatic pressure was monitored using an invasive blood pressure monitor. ablation was performed as follows: 1. rfa. 1) single-step method: after fully deploying the electrode, the power was initially applied at 30 w, then increased in increments of 10 w/min until power roll-off. 2) multi-step method: the array was deployed in 8 steps. at each step, the power was fixed at 30 w until power roll-off. 2. pei. injection of ethanol (2 ml). b. clinical study: we examined the multi-step rfa and pei for hcc. under local anesthesia, intratumoral pressure was monitored. 1. rfa. 39 patients with a mean tumor size of 15.3±4.9 mm were studied. 2. pei. in 10 patients with a mean tumor size of 21.4±8.8 mm, 5 to 10 ml of ethanol was injected per session. results : a. basic study: the intrahepatic pressures were: single-step method, 154.5±30.9 mmhg; multi-step method, 24.1±18.2 mmhg; and pei, 12.0±8.5 mmhg. b. clinical study: intratumoral pressure was 39.5±27.9 mmhg for rfa and 12.2±9.0 mmhg for pei. conclusion: these results suggest that consideration of intrahepatic pressure is crucial in local ablative therapies. background: a late evening snack (les) is recommended for liver cirrhosis. however, no clinical study has evaluated the nutrition status and the effect of les in cirrhotic patients with hepatocellular carcinoma (hcc). we investigated the effect of les undergoing hepatic arterial infusion chemotherapy (haic) in patients with hcc. method: nineteen patients with hcc were enrolled. ten patients were les group, and nine were control group. in the les group, the patients received les supplementation with a branched-chain amino acid (bcaa)-enriched nutrient mixture. in the control group, the patients received ordinary food. there were no significant differences in relation to age, gender, etiology, child-pugh scores, tumor stage, clinical responses to haic between two groups. blood biochemical data, nutrition status using an indirect calorimeter were evaluated at before and at the end of chemotherapy. results: the non-protein respiratory quotient (nprq) and molar ratio of branched-chain amino acid to tyrosine (btr) were significantly improved in the les group but not in the control group. there were no significant differences in the area under the concentration curve for glucose between before and the end of chemotherapy in two groups. background & aims: hepatocellular carcinomas (hccs) often show hypoor mixed vascularity, and the prognosis of these relatively hypovascular hccs is not fully elucidated. cytokeratin (ck) expression profiles may also be useful prognostic indicators, and specifically ck19 may reflect metastastic potency in hccs. this study was to assess the prognostic implication of tumor vascularity and its relation to ck19 expression in hcc patients. methods: a total of 150 patients who underwent surgical resection for hcc were enrolled. tumor vascularity was evaluated according to arterial enhancement pattern on ct scans and ck19 expression was evaluated using tissue microarray methods. clinicopathologic data were analyzed using kaplan-meier and cox proportional hazard model. results: during follow-up period, 91 (60.7%) patients experienced tumor recurrence. forty-five patients (30%) had hypovascluar tumor at the time of diagnosis, and they showed significantly higher positivity for ck19 expression (p=0.001) and shorter disease-free survival (p=0.023) than patients with hypervascular hccs. in addition, recurred tumors in these patients showed more frequently hypovascular pattern than in patients with hypervascular hccs (p=0.001). hypovascularity at initial diagnosis and microvascualr invasion were independent poor prognostic factors predicting survival. following treatment of recurred hccs, hypovascular tumors showed poor response to transarterial chemoembolization (tace), which resulted in shorter overall survival than hypervascular tumors (p=0.057). conclusions: these results demonstrate that tumor hypovascularity in hccs is associated with positive ck19 expression, early tumor recurrence, poor tace response and poor survival. therefore, tumor vascularity may also be a prognostic indicator in hcc patients. background: hepatic stellate cells (hscs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma(hcc). in this study, we determine gene expression changes in two different models of hscs activation and investigate whether induction-activated hscs(ihscs) gene expression changes are different from culture-activated hscs(ahscs). methods: hscs were isolated by density centrifugation and exposed to conditioned medium from rat hcc cell lines c5f. twenty-seven thousands and one hundred gene expression between quiescent hscs(qhscs), ahscs and ihscs was analyzed by microarray and confirmed by real-time rt-pcr and western blot. results: sixteen hundreds and seventy-one probe sets were differentially expressed in ahscs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors. seven hundreds and eleven probe sets were differentially expressed in ihscs. induction-activated hscs showed specific gene expression patterns including raf1, rac2, adam17, wnt6, mmp-9 and tnf, suggesting that hcc cells can specifically induce hscs activation. induction-activated hscs might play a important role in invasion and metastasis of hcc. conclusions: induction-activated hscs gene expression patterns are different from ahscs. culture-activated hscs does not properly regulate gene expression in hscs, suggesting that ihscs may be considered the model for the study of hscs biology in hcc. background: hepatocellular carcinoma (hcc) is a hypervascular tumor, and angiogenesis is important for tumor growth. ephrin receptors are related with vascular system development and the polymorphism of ephb1 in the carcinogenesis of digestive tract has been reported. our aim was to examine the polymorphsims of ephb 1 with the occurrence of hepatocelluar carcinoma in korean population. methods: genomic dna was extracted from 182 patients with hepatocellular carcinoma (hcc), 266 healthy subjects. ephb 1 polymorphism was determined by polymerase-chain reaction-based assays, and the association with hcc was investigated. results: with regard to ephb 1 polymorphism, a/a genotype at rs11926992, t/t genotype at rs7644369, a/a genotype at rs6776570, t/t genotype at rs3821502 and g/g genotype at rs6766459 were significantly associated with hcc but these were not associated with clinical characteristics of hcc. conclusions: five out of seven polymorphisms on ephb1 gene were statistically associated with hcc, in the korean population. therefore, more studies of ephb1 gene polymorphisms including various risk factors should be performed to use as genetic markers of hcc occurrence. background: we aimed to compare the results of hepatectomy for hcc in patients older than 70 years old with those for younger patients. methods: clinicopathological data and outcomes for 155 elderly patients and 333 younger patients with hcc who underwent hepatectomy between 1992 and 2007 were retrospectively compared. results: although postoperative delirium was more common in the elderly group, there were no significant differences between the 2 groups with regard to operative morbidity, hospital death, disease-free survival, and overall survival. the overall recurrence rate was significantly higher in the elderly patients with alcohol abuse than in younger patients with alcohol abuse. multivariate analysis revealed that preoperative alcohol abuse was a prognostic factor for elderly patients. conclusions: elderly patients with preoperative alcohol abuse should be followed closely, even after r0 surgery, because alcohol abuse is strongly correlated with postoperative recurrence and worse survival. background: little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. methods: among 410 patients who underwent curative resection between 1992 and 2005, 180 patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group a), while 46 patients were only transfused with packed red cells (group b), 43 patients were only transfused with fresh frozen plasma (group c), and 141 patients had no transfusion (group d). results: group c had significantly fewer postoperative complications and a shorter hospital stay than group a. preoperative coagulation was significantly worse in group c. survival was significantly better in groups c and d than in group a. conclusions: perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma. background: this study investigated risk factors for postoperative liver failure after resection of hepatocellular carcinoma to detect markers that could identify candidates for hepatectomy. methods: perioperative risk factors for liver failure after hepatectomy were analyzed in 191 patients with hepatocellular carcinoma. results: liver failure occurred postoperatively in 16 patients, 3 of whom died. the hyaluronate/gsa-rmax ratio was a risk factor for postoperative liver failure by univariate analysis and was the only risk factor according to multivariate analysis. all 3 patients who died had a hyaluronic acid/gsa-rmax ratio 500 mg min/dl. conclusions: to reduce postoperative liver failure, preoperative planning should employ various measures of the hepatic functional reserve, including tests of both parenchymal and nonparenchymal liver function. the hyaluronate/gsa-rmax ratio can predict liver failure after hepatectomy, and a ratio greater than 500 mg min/dl is a relative contraindication to liver resection. the patient was a 73-year old japanese man with chronic hepatitis c(ch-c) who achieved a sustained virological response(svr) to interferon(ifn) therapy. as a result the liver functions were normalized and the histological findings of the liver also improved. however, 13 years after svr, mild liver dysfunction was noticed along with a marked increase of tumor markers. several modalities revealed huge liver tumors about 13 cm in greatest diameter in the left lobe invading the bile ducts and another tumor about 3 cm diameter in segment v. we performed liver biopsy and confirmed that this tumor was well-differentiated hepatocellular carcinoma (hcc). only mild fibrosis development could be observed in the adjacent non-cancerous lesions. we successfully treated these tumors with transcatheter arterial chemoembolization and stereotactic radiosurgery. recent studies revealed that the risk of developing hcc still exists even after svr. since most of hcc that develop in patients with svr are usually detected within 5 years, several investigators speculate that hcc is already present but too small to be detected at the time of completion of ifn therapy. this speculation is not the case in our patient, since svr was achieved 13 years ago and no hcv-rna could be detected when hcc appeared. therefore, another possible mechanism should be considered. an annual follow-up with strict surveillance program for hcc should be performed for more than 10 years after the completion of ifn therapy. background/aims: in order to investigate the role and importance of oxidative stress as to carcinogenecity of hepatocellular carcinoma (hcc) we analyze the expression of 8-hydroxydeoxyguanosine (8-ohdg) in the liver tissue of the hcc patients with and without hepatitis viral marker. methods: patients undergoing hepatic resection for the first hcc from 1995 to 2004 were enrolled into the study. only the cases that took no alcohol or small amount of alcohol were enrolled. 24 cases were negative for hepatitis b surface antigen (hbsag) and antibody to hepatitis c virus (hcvab) (nbnc group). 24 were positive for hbsag and negative for hcvab (b group). 21 were positive for hcvab and negative for hbsag and antibody to hepatitis b core antigen (c group). staining with hematoxylin and eosin (h&e) and berlin-blue, and immunohistochemical staining for 8-ohdg were performed using the non cancerous liver regions. the degree of 8-ohdg immunostaining was expressed as the labeling index, which means the percentage of positive hepatocytes per 1000 hepatocytes. results: the labeling index of 8-ohdg for nbnc group is 19.3(±55.3), significantly lower (p=0.014) than that for b group 49.7(±89.5), and also lower (p=0.016) than that for viral group (b group and c group)(35.4±71.8). the labeling index of 8-ohdg had no correlation with grading, staging, fatty and iron deposit among all cases. conclusions: there is possibility that oxidative stress might not associate with the carcinogenesis of hcc in some cases without hepatitis viral infection. background: no effective chemopreventive agent has been approved against hepatocellular carcinoma (hcc) yet. since neovascularization plays a pivotal role in hcc, an angiostatic agent is considered as one of the promising approaches. recently, it has reported that vitamin k2 (vk) and angiotensin-converting enzyme inhibitor (ace-i) exert anti-angiogenic activity. the aim of the current study was to elucidate the combination effect of the clinically used vk and ace-i on cumulative recurrence after curative treatment, especially in consideration of neovascularization. methods: vk (menatetrenone; 45 mg/day) and/or ace-i (perindopril; 4 mg/day) were administered for 36 to 48 months after the curative therapy for hcc. the cumulative recurrence and several indices were analyzed. results: a 48-month follow-up revealed that the combination treatment with vk and ace-i markedly inhibited the cumulative recurrence of hcc in association with suppression of the serum level of vascular endothelial growth factor (vegf); a central angiogenic factor. the serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with vegf. these beneficial effects were not observed with single treatment of vk or ace-i for 36 months. conclusions: the combination treatment of vk and ace-i may suppress the cumulative recurrence of hcc after the curative therapy, at least partly through suppression of the vegf-mediated neovascularization. aim: the aim of this study was to clarify the cilnicopathologic features and management of hepatocellular carcinoma (hcc) patients surviving more than 5 years after hepatectomy. materials & methods: retrospective study was carried out on 823 hcc patients who underwent curative hepatectomy between 1973 and 2002. clinicopathologic factors in 5-year survivors and patients who died within 5 years were compared. the prognostic factors affecting survival were examined among the 5-year survivors. results: there were 290 patients who survived for more than 5 years after initial hepatectomy, and 83 of those patients survived for more than 5 years after hcc recurrence. the overall 3-, 5-, 10-and 20-year survival rates were 57.4%, 40.9%, 17.2%, and 8.5% respectively. in multivariate analysis, absence of underlying cirrhosis, solitary tumor, alfa-fetoprotein less than 1000 ng/ml, and absence of microscopic vascular invasion were favorable independent factors associated with 5-year survival. negative hepatitis c virus antibody status was favorable independent factor associated with longer disease-free interval and survival after tumor recurrence. multimodal treatments such as repeat hepatectomy or percutaneous ablation led to improved survival after recurrence, compared with the survival after transarterial chemoembolization (p<.05). conclusions: the results suggest that patients without underlying cirrhosis who have a solitary hcc that does not demonstrate vascular invasion or high afp levels might survive for longer than 5 years after the initial hepatectomy. close follow-up and multimodal treatment could contribute to prolongation of survival in such patients, even if cancer recurrence occurs. the history of the use of carbon ion radiotherapy (cirt) for treating hepatocellular carcinoma (hcc) goes back to 1995, when clinical trials were initiated at the national institute of radiological sciences. we have already reported that cirt used for the treatment of hcc is safe and effective, and that it causes only minor liver damage. in a phase ii clinical trial, the local control and cumulative overall survival rates were 94 % and 35 % at 5 years, respectively. however, the patients with tumor adjacent to the gastrointestinal tract are thought to be ineligible for cirt because of the high risk of radiation injury of the digestive organs. in order to extend the indication of cirt, we have challenged the cirt for such patients under the use of spacers. a case was a 67-year-old female with 8cm tumor in segment 4. in radiological findings, the tumor revealed typical enhancement pattern for hcc, and was near the ec junction. she had been judged ineligible for hepatectomy because of the high retention rate of indocyanine green. she could undergo the 42.8 gye/2-fraction cirt after the placement of gore-tex soft tissue patch under the laparoscopic procedure. up to the present date, no adverse effect due to the spacer has been occurred, and an apparent anti-tumor effect has been observed. this method seems to have a promising efficacy for extension of the indication of cirt to the patients with tumors adjacent to the gastrointestinal tract. background: previously we reported that high ubiquitination was marker of human hepatocellular carcinoma. on the basis of these finding, we firstly analyzed the effect of bortezomib(proteasome inhibitor) on human hcc cell line. we also reported that hhm/dip1/gcip1 was early marker for human hepatocarcinogenesis. hhm was suggested to be a new tumor suppression gene, but the mechanism was not well confirmed. we analyzed change of hhm signal by bortemib. method and result: we used hcc cell line (huh7, hlf, hepg2) . the inhibitory effect of bortezomib was evaluated using mtt assay. 20nm bortezomib significantly inhibited proliferation of hcc cell line. the inhibitory effect by 20nm bortezomib was similar with 10 m cisplatin. on the other hand, bortezomib has no inhibit effect in isolated hepatocyte from rat. in this condition, we analyzed the expression of cyclin d1, phospho-rb and hhm in hcc cell line by western blot analysis. expression of cyclin d1, phospho-rb decreased, but hhm was increased with time. next we analyzed cell cycle by facs. bortezomib induced hcc cell line into cell cycle arrest in g2/m. the transcriptional activity of hhm was also activated by bortezomib administration using ptimer-promoter-hhm plasmid. conclusion: bortezomib has specific anti-proliferative effect on hepatocellular carcinoma. the induction of hhm by bortezomib might be related with cell cycle arrest. bortezomib will be a useful drug for hcc. neovascularization is required for carcinogenesis of non-alcoholic steatohepatitis: experimental and clinical study m. kitade 1 , h. yoshiji 1 , r. noguchi 1 , k. kaji 1 , t. namisaki 1 , y. aihara 1 , h. background/aim: non-alcoholic steatohepatitis (nash) may progress to liver cirrhosis, and finally hepatocellular carcinoma. recent study suggested that development of hepatic angiogenesis correlates the risk for hepatocarcinogenesis in liver cirrhosis patient. we therefore examined the role of angiogenesis in the hepatocarcinogenesis of nash in both experimental and human study. methods: as an experimental nash model, zucker (z) rats, which naturally develop leptin receptor mutations, and their lean littermate (l) rats were fed a choline-deficient, amino acid-defined (cdaa) diet. in human study, 11 patients with nash-related cirrhosis or pre-cirrhosis, regarded as high risk group of hepatocarcinogenesis, and 13 with simple fatty liver (fl) were enrolled and underwent clinico-pathological examinations. immunohistochemical analysis of 4-hydroxy-2-noneal (4-hne) and cd34 were employed for detection of reacrive oxidative stress (ros) and angiogenesis in the liver tissues, respectively. results: in experimental nash model, both groups showed marked steatohepatitis by feeding cdaa diet. in sharp contrast, the development of glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions and hcc could be observed only in the l-rats. the hepatic neovascularization was also significantly increased only in the l-rats. in human study, both nash and fl exerted a marked elevation of ros. in sharp contrast, significant development of hepatic neovascularization was observed only in nash, whereas almost no neovascularization could be observed in fl. conclusion: in conclusion, these results suggested that neovascularization might play a important role in hepatocarcinogenesis in nash. background: paternally expressed gene 10 (peg10), which was an imprinted gene with an active paternal allele but silent maternal allele, was highly expressed in a great majority of hepatocellular carcinoma(hcc). the aim of this study was to generate transgene mice expressing peg10 in the liver under the control of mouse albumin (alb) promoter and study the integration, transcription, expression of peg10 gene in the transgenic mice methods: the linearized 1716bp transgene fragments, which contained alb promoter and structural gene of peg10, were microinjected into fertilized eggs of mice. then manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. all the newborn mice were screened and identified by pcr detecting genomic dna in tail tissue. as the transgene was driven by the alb promoter, we examined its expression in the liver of transgenic mice by rt-pcr and western blotting. results: the transgene fragment was microinjected into the male pronucleus of 3741 fertilized oocytes. the injected eggs were implanted into oviducts of 94 pseudo-pregnant foster mothers, of which 22 mice became pregnant and give birth to 108 offspring. 65 of them died from unknown reason. among the 43 offspring, 3 were identified to carry peg10 cdna as demonstrated by pcr, and peg10 transgene could be expressed successfully in the liver of the established transgenic mice. the ratio of transgene integration were 6.97% (3/43) by pcr. conclusions: the peg10 transgenic mouse model should be valuable for studying the in viro function of this imprinted gene in hcc. background/aims: brivanib alaninate is the l-alanine ester prodrug of bms-540215, an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. it is being developed in treating hepatocellular carcinoma (hcc), a disease highly prevalent in asia-pacific region. this analysis investigated whether bms-540215 exposure was different between asian and non-asian subjects. methods: a population pharmacokinetic (ppk) model was developed with data collected in 125 subjects (78 non-asian, 47 asian) with advanced and metastatic solid tumors (including hcc) from 2 clinical studies. potential effects of the following covariates on model parameters were examined: age, gender, race, and baseline body weight. model-based simulation was performed to examine bms-540125 exposure in asian and non-asian patients following brivanib doses of 800 mg qd (phase iii dose). results: the ppk of bms-540215 was characterized by a 2-compartment model with first-order absorption and elimination. clearance was found to slightly increase with body weight (p<0.01). however, effects of age, gender and race on clearance were not statistically significant. the median of apparent clearance in asian was 9.5% lower than that of non-asians, which was adequately explained by 16% lower body weight in asians. there was substantial overlap in steady-state bms-540215 auc of asian and non-asian patients, simulated based on their observed body weight distributions in these patient groups. conclusions: bms-540215 pk can be adequately described by a linear 2-compartment model; exposures in asian and non-asian subjects are similar following brivanib doses of 800 mg qd. background/aims: hepatic resection is the standard treatment for hepatocellular carcinoma. in some patients with multiple hcc, one-block resection can not be feasible due to either the tumor location or the reserved liver function. in this study, we attempted to analyze the outcome of multiple-site resection or combined resection and rfa in patients with multiple hcc. the prognostic factors for postoperative survival were also investigated. methods: among 507 patients who received resection from january 1996 to august 2006, 58 patients had a radiologically detected multiple hcc. patients with multiple hcc were divided into: group a, patients treated with one-block resection (n=40) and group b, patients with multiple-site resection or combined resection and rfa (n=18). results: in group b, 6 received multiple-site resection and 12 underwent combined resection and rfa. the clinicopathological variables and postoperative complication rate were not significantly different between the two groups. the 5-year disease-free survival rates for group a and b were 24.1% and 18.3%, respectively (p=0.386). the overall survival rates were also not significantly different (36.9% vs. 39.4%, p=0.528). the multivariate analysis revealed that radiological tumor number 3, edmondsons-steiner grade (iii-iv) and indocyanine green retention rate at 15 minutes> 10% were adverse prognostic factors for overall survival. conclusions: active treatments including multiple-site resection and combined resection and rfa showed similar treatment outcomes compared with one-block resection in patients with multiple hcc. the prognosis after treatment was associated with tumor number, tumor grade and icg r15. background: nasopharyngeal carcinoma (npc) is endemic to southern china. mortalities are mostly associated with secondary metastases. novel treatments for npc metastases are thus urgently needed. we aim to test the efficacy of a physiologically stable gold compound, gold (iii) meso-tetraarylporphyrin 1a (gold-1a), in treating intrahepatic npc metastasis in athymic mice. methods: twenty million of c666-1 human npc cells were injected into the livers of athymic mice to induce primary tumors. gold-1a was administrated by intraperitoneal injection. survival times, tumor volumes and degrees of metastasis of the animals were evaluated. intratumoral microvessel density was determined by immunohistochemical staining for cd34. tube formation by ms1 mouse endothelial cells were conducted with an in vitro angiogenesis assay kit. gene expression level was determined by semi-quantitative reverse transcription-polymerase chain reaction. cell proliferation was performed by methylthiazolyldiphenyl-tetrazolium bromide assay. result: gold-1a prolonged the survival and inhibited intrahepatic and lung metastasis of the tumor-bearing animals. the compound induced tumor tissue necrosis and reduced tumor microvessel formation. in in vitro studies, gold-1a inhibited tube formation and proliferation of ms1 cells, and downregulated the expression of stanniocalcin 1 (stc1), which plays roles in angiogenesis. furthermore, our preliminary data showed that overexpression of stc1 in ms1 cells rescued cells from gold-1a-induced death. conclusion: gold-1a is a novel anticancer agent that prolongs survival of the npc metastases-bearing mice. it inhibits intrahepatic and lung metastasis in vivo and inhibits angiogenesis in vitro, in part via downregulation of stc1. tbx3 is a transcriptional repressor that is important for embryonic development. overexpression of tbx3 was found in a large variety of cancers, including breast cancer, ovary cancer, cervical cancer, lung cancer, bladder cancer and liver cancer. tbx3 promote carcinogenesis by bypass cellular senescence via suppression of p14 arf . our resent studies revealed that two key motifs composed of 7 + 3 residues are essential for its transcriptional repression. based on this finding, we designed a set of peptides to block its transcriptional repression activity and tested their antiviral effects. we found that tat-tagged peptides (taps) effectively transduced hepatoma hepg2 and bel7404 cells at almost 100% efficiency and inhibited cell growth in a dose dependent manner. further studies revealed that the tap treated cells underwent up-regulate apoptosis via suppression of p14 arf both at mrna and protein levels, demonstrating the potential of novel taps for anti-hcc treatment in the future. safety and long-term outcomes of radiofrequency ablation therapy in elderly and cirrhotic patients with hepatocellular carcinoma k. kakisaka 1 , h. kuroda 1 , k. kasai 1 , y. takikawa 1 , k. suzuki 1 1 iwate medical university background and purpose: a tendency of the aging in patients with hepatocellular carcinoma (hcc) is predominantly seen in japan. in fact, the mean age of patients with hcc in our institute in 1990 was 60.1 years old, while that in 1999 was 67.8 years old. it is not still remained whether the percutaneous radiofrequency ablation (rfa) therapy in elder patients with hcc is safety and equal in therapeutic usefulness compared to the non-elder patients with hcc. subjects and methods: two hundred six cirrhotic patients with hcc (376 tumor nodules) received rfa therapy curative intent since august, 2006 were enrolled. we divided all patients into two groups: over 65 years (elder group: n=135) and under 65 years (non-elder group: n=71), and compared the patient's characteristics, tumor factors and survival rate and causes of death in two groups. results: the characteristics of patients, tumor factors, cumulative survival rate and recurrence rate were not revealed in two groups. although in elder group two patients complicated aspiration pneumonia and respiratory depression due to sedation under rfa respectively, total occurrence rate of complications did not differ between two groups. conclusion: rfa therapy is safety and effective even in elder patients with hcc, although their care is necessary to prevent any complications which are often occurred during the rfa therapy. background and purpose: the aim of this study is to evaluate whether administration of the branched-chain amino acid (bcaa) enriched nutrient (namely, aminoleban en, ostuka pharmaceutical company, japan) might improve protein-energy malnutrition (pem) status and quality of life (qol) in cirrhotic patients with hcc receiving rfa therapy. subjects and methods: thirty-five cirrhotic patients with hcc who had received rfa therapy from october 2005 to october 2006 in our institute were randomized into two groups: diet with supplementation of aminoleban en (en group: 20 patients, 420 kcal/day) and diet only (control group; 15 patients). the total intakes of calories (30-35 kcal/kg) and protein (1.0-1.5 g/kg) were equal between tow groups. the primary end point was event-free survival rate (development of liver cancer, rupture of esophageal varices, or progression of hepatic failure) and second end points were serum albumin levels and the health-related qol by shortform-8 questionnaire (sf-8). results: total intakes of calories and protein were similar during the one year after rfa. no significant differences in event-free survival rate were seen between two groups. however, decreased serum albumin levels and one (general health perception) of domains in sf-8 were significantly improved in en group compared to the control group. conclusion: supplementation of bcaa-enriched nutrient may improve the impaired liver function and qol after rfa therapy. large scale prospective study should conduct to confirm these results near the future. backgrounds and aims to investigate the effects of selective cox-1 and cox-2 inhibitor on proliferation and apoptosis of hcc cell. methods hep3b and snu 387 cells were treated with ns-398 and sc-560. mtt assay, caspase 3/7 activity assay and tunel assay were performed. cox protein and mrna expression were measured by western blot and real time rt-pcr. results in hep3b cell line, cox-1, cox-2 (50, 100, 200 um) and combination (25+25, 50+50, 100+100 um) treatment after 24hr showed a significant dose dependent inhibitory effect on cell growth (p<0.05). cox-1, cox-2 (100 um) and combination (50+50, 100+100 um) treatment after 24hr significantly increased caspase 3/7 activity (p<0.05) and induced apoptosis (p <0.05). however, the combination treatment could not showed a additive effect to cox-1 or cox-2 inhibitor (p>0.05). in snu 387 cell line, cox-1 inhibitor and combination treatment showed a inhibitory effect on cell growth (p <0.05) similar to hep3b cell line but any of treatment could not induce apoptosis significantly (p >0.05). in cox protein and mrna expression, snu 387 cell line showed significant cox-1 predominency (p=0.037) but hep3b cell line showed cox-2 predominency (p=0.032). conclusions in hcc cells, no additive effect of the combination treatment of cox-1 and cox-2 inhibitors could be anticipated. the apoptosis inducing effect of cox inhibitor could be different between hcc cell lines. more studies for the mechanism of different response to cox inhibitor between cell lines is needed. background: the aim of this study was to determine the maximum tolerated dose and recommended dose of combination chemotherapy with mitoxantrone and uracil/tegafur (uft) (phase i part), and to clarify its efficacy (tumor response, overall survival, and progression free survival) and safety in patients with advanced hepatocellular carcinoma (hcc) at the recommended dose (phase ii part). methods: patients eligible for study had histologically confirmed, chemo-naïve advanced hcc, who were unsuitable for resection, local ablation therapy or transcatheter arterial chemoembolization. the therapy consisted of mitoxantrone dosages (6, 8 and 10 mg/m 2 /day) intravenously on day1 and oral administration of uft 300 mg/m 2 on day 1 through day 21. the treatment was repeated every four weeks if there was no evidence of tumor progression or unacceptable toxicity. results: a total of 25 patients were entered into the study. all had a good ecog performance status score of 0-1. in phase i part, dose limiting toxicities occurred in all three patients (two patients: grade 4 neutropenia, one patient: grade 3 creatinine elevation) given mitoxantrone at dosage of 10 mg/m 2 /day, and the recommended mitoxantrone dosage was 8 mg/m 2 /day. among 19 patients administered at the recommended dosage, one patient (5.3%) achieved a partial response, 8 patients (42.1%) had stable disease and 10 patients (52.6%) had progressive disease. one-year survival proportion, median survival and median progression free survival were 26.3%, 8.4 months and 2.5 months, respectively. the most common toxicities were grade 3-4 leucopenia (15.4%) and neutropenia(10.3%). conclusion: mitoxantrone 8 mg/m 2 with uft 300 mg/m 2 /day is recommended dose. this regimen is generally well tolerated, but appears to have little activity for advanced hcc. these findings do not support its use in practice, and further trials with this regimen in patients with advanced hcc are not recommended. the study assessed the benefits of 3-d reconstruction of spiral ct scans for the diagnosis of and surgical guidance to large liver tumors or tumors at the hepatic hilum. we retrospectively analyzed 33 cases of children with such tumors treated in past 5 years.the patients were examined by 3-d reconstruction using 64 slice spiral ct. in 28 cases, the volume of tissue removed exceeded 1/3 the entire volume of the liver. in 5 cases, the excised tissue represented less than 1/3 of the total liver volume, but the location of the tumor was adjacent to major hepatic vessels. pathological diagnoses included hepatoblastoma (n = 20), hepatocellular carcinoma (n = 4), mesenchymal hamartoma (n = 4), teratoma (n = 1) and adenoma (n = 4). all children had curative resections with tumor-free microscopic margins. 3-d ct imaging can provide high quality images and accurate location of the tumors. it could help the surgeon identify the tumor borders accurately and devise a safe surgical strategy. with its help the surgeon could identify vital hepatic blood vessels before operation, and can avoid massive hemorrhaging during operation. background: to investigate the association between c-509t polymorphism of transforming growth factor (tgf)-1 gene and hbv-related hepatocellular carcinoma (hcc). methods: patients with hbv infection (196 cases were hbv carriers, 379 cases were hcc) and 299 healthy volunteers were enrolled. the polymorphism of tgf-1 gene c-509t was identified by polymerase chain reaction-restriction fragment length polymorphism method. the concentrations of plasma tgf-1 were measured by enzyme linked immunosorbent assay (elisa). tgf-1 mrna expression was quantified by real-time pcr. a recombinant construct containing -509c>t variant as promoter and cat as reported gene was transfected into hepg2 cells. the reporter gene cat was detected with elisa. results: the ct genotype at position -509 of tgf-1 gene prevailed in all three groups, the frequency of genotype cc and allele c at -509 in hcc were significantly higher than those of the hbv carriers and controls. the plasma tgf-1 concentration among the three genotypes did not show any significant difference in three groups. however, both the tgf-1concentration and liver mrna levels were statistically higher in patients with cc genotype than in those with tt genotype in the hcc group. reporter gene cat was elevated when hepg2 were transfected with -509c-cat recombinant construct compared to that with -509t-cat one (p<0.05) conclusion: the presence of c allele at position -509 may play an important role in the development of hbv-related hcc through influencing tgf-1 expression both at mrna level and protein level. background: to assess diagnostic value of n-glycan markers in identifying hepatocelluar carcinoma (hcc) from liver fibrosis after hbv infection. methods: a total of 273 cases of hbv related liver fibrosis (n=128) and hcc (n=145) patients as well as matched healthy controls (n=120) were recruited. routine liver function and tumor markers were detected by automatic biochemistry or immunological analyzer. n-glycome of serum protein was profiled by dna sequencer-assisted fluorophore-assisted carbohydrate electrophoresis with a capillary electrophoresis-based abi3130 sequencer. results: the abuncance of a single agalacto biantennary glycan (ng1a2f, peak 4) was increased in liver fibrosis and decreased in hcc, while that of a branching triantennary glycan (na3fb, peak 9) was decreased in fibrosis and increased in hcc. the efficacy of the log ratio of above two n-glycan abundance [log (p9/4)] was similar to afp in differentiation hcc from fibrotic patients. with logistic regression analysis, the accuracy and sensitivity of the diagnostic model combining afp with n-glycan analysis(cscore b) were increased 7-10% compared to afp. log(p9/4) was even more powerful in monitoring the progresison of hcc with the specificity improved 16% and accuracy improved 8% compared to that of afp. besides, log(peak 9/4) was correlated well with other tumor markers and tnm stages. conclusions: the log ration of the abundance of a branching triantennary glycan (na3fb, peak 9) to a single agalacto biantennary glycan (ng1a2f, peak 4) and the model combining afp with n-glycome markers are promising in hcc diagnosis and progression monitoring. the low incidence of tumor seeding and post-procedure bleeding after radiofrequency ablation (rfa) of hepatic tumors has been attributed to the use of thermocoagulation of the tract, which results in necrosis, upon electrode withdrawal. however, different investigators use different techniques with no experimental evidence of the effectiveness of a particular technique. objective: we aimed to compare the necrotic zone produced using different electrode withdrawal techniques. methods: eighteen tract ablation zones were created in ex vivo porcine livers by withdrawing an internally-cooled rfa electrode (cool-tip radiofrequency system, valleylab) 1-2 mm/second using energy-dependent (20 vs.40 vs.60 vs.80 watts) and temperature-dependent (80 vs. 90 0 c) techniques. horizontal mathematical modeling suggests an impractical number of radiofrequency ablation (rfa) zones needed in order to ablate a medium-large hepatic tumor. however, overlapping rfa zones may increase the necrotic diameter disproportionately to that deduced from single ablation alone. objectives: to compare the necrotic diameter in single (group1), dual overlapping (group2) and dual non-overlapping (group3) ablation. methods: single (n=5) and dual (overlapping n=18; non-overlapping n=6) ablation zones were created in ex vivo porcine livers using cool-tip rfa electrodes. necrotic diameter was measured at the midpoint (maxd) of the single and the two distinct rfa zones of the dual ablation groups and compared with the necrotic diameter at the tip of the second ablation (maxd-o), corresponding to the point of overlap in group2. the rfa electrode was withdrawn 2.5 and 4.1 cm before re-ablating for group2 and group3, respectively. results: despite no difference in end-rfa temperature between 3 groups (group1=75.6+7.4cvs.group2=73.9+7.3cvs.group3=74.8+4.8c; p=0.873), maxd was significantly greater (p=0.011) in group2 (4.1+0.7cm) as compared to group1 (3.3+0.6cm) and group3 (3.3+0.4cm), with no difference between group1 and group3(p=1.00). further proof of synergism between two overlapping ablations is that the maxd-o in group2(4.3+0.6cm) was larger than maxd of group1 (p=0.042) and group3 (p=0.028), and was similar to maxd of group2 (p=1.00). conclusions: overlapping two rfa zones results in incremental increase in necrotic diameter compared to single and dual non-overlapping ablation. this may explain the discrepancy in the number of ablation zones needed between clinical and mathematical modeling studies. background: hepatocellular carcinoma (hcc) is the fourth most common cancer worldwide, main etiological factors being chronic infections with hepatitis b and c viruses. the present study was undertaken to evaluate the association of glutathione-s-transferase (gst) t1 and m1 null genotypes and microsomal epoxide hydrolas e(mephx) polymorphisms with hepatitis virus related hcc risk in indian population. subjects and methods: three groups of subjects were considered viz. control (n=169), chronic viral hepatitis (n=174) and hcc (n=63). pcr-rflp was used for this polymorphic study. genotype distributions between categories were compared using the 2 test; odds ratios (ors) and 95% ci were calculated to express the relative risk. results: presence of gstm1 null genotype significantly (p<0.05) decreased the risk for hcc development among chronic viral hepatitis subjects. however, gstt1 null genotype was associated with an increased risk for hcc by 2.23 and 1.42 times among control and hepatitis subjects respectively. in case of mephx, tyr113his and his113his genotypes significantly (p< 0.05) reduced the risk of hcc development in both viral hepatitis and control subjects. in case of mephx exon 4 genotypes, arg139arg imposed an approximate 2 fold risk for hcc development in the two groups. combination of heterozygous mutant genotypes at mephx exons 3 & 4 also imposed around 2 fold risk (non-significant) for hcc. conclusions: polymorphic forms of gst and mephx share an association with viral related hcc risk in indian population and should be further evaluated as the candidate genes to determine individual susceptibility for viral related hcc. background : the association between type 2 diabetes mellitus (dm2) and hepatocarcinoma (hcc) has been identified in the last ten years. methods: to clarify the temporal relationship between dm2 and hcc and the possible effects of antidiabetic therapy on hcc risk, we recruited 465 patients with hcc compared with 490 control subjects without liver diseases and 618 cirrhotic patients. results: prevalence of dm2 was 31.2% in hcc, 23.3% in cirrhotic and 12.7% in control group. in univariate and multivariate analysis, the odds ratio (or) for hcc in diabetic patients were respectively 3.12 (ci 2.2-4.4; p <0.001) and 2.2 (ci 1.2-4.4; p= 0.01). or in univariate analysis were higher in male than in female patients. in 84.9% of the patients dm2 pre-exists the diagnosis of hcc from a mean time of 141.5 months. moreover, the insulin treatment was more frequent in diabetic hcc patients than controls and we report an or for hcc of 2.99 (ci 1.34-6.65; p= 0.007) in patients treated with insulin or sulfonylureas, and an or of 0.33 (ci 0.1-0.7; p= 0.006) in patients treated with metformin. conclusion: our study confirms that male patients with type 2 diabetes mellitus have a significantly increased risk of hcc independently of other cofactor such as hbv, hcv and alcoholic abuse. dm2 is a pre-existing disease in most hcc patients and suggests that insulin and sulphonylurea treatments in dm2 are associated with an increased risk of hcc development, while metformin may have a protective effect. background & aims: over the last few years, techniques that allow systematic analysis of chromosome aberrations at a genome-wide level were applied to hcc. the purpose of this study is to apply gene loss expression profiling in the attempt to discover new related genomic regions not revealed by loh or cgh, and search the new tumor suppression genes for hcc. methods: primary hcc and corresponding non-tumor liver tissues were obtained from surgery. serologically, 13 cases were with hepatitis b virus infection and 12 cases were with hepatitis c virus infection. four non-viral infected tissues from four patients receiving surgical resection for hepatic adenoma or focal nodular hyperplasia.affymetrix genechip, u133a, was used to compare the loss and gained gene expression in liver needle biopsy samples (n=54). results: after adjusting by chromosome arm length, 16p, 17p, 19p, 19q and 22q showed higher gene loss-expression ratio (>= 10 loss / cm) in the comparison between normal samples and tumor samples; 1q, 2p and 4q showed higher gene loss-expression ratio in the comparison between tumor and non-tumor tissues. more than 50 genes showed different loss expression level in this study. for example, cd10 was loss expression in all non-tumor samples comparing to four normal samples. ficolin 3 and ficolin 2 were loss expression in hcc samples with hbv infection and with hcv infection, respectively. conclusion: our results revealed the potential tumor suppression genes and the genomic region they harbored. further study is needed to validate the observation. background/aims: hepatocellular carcinoma is common malignancy in human, accounting for 1 million deaths in the world annually. caspase 8, as an initiator caspase, is involved in the induction of apoptosis. survivin, a novel inhibitor of apoptosis is related to the ability to inhibit caspases and involved in critical steps of onset and progression of hcc with unfavorable prognosis. methods: to explore the possibility that the epigenetic alteration of caspase 8 and survivin genes is implicated in the development and progression of hcc, promoter methylation of two genes was analyzed in 73 cases of primary hcc by methylation specific pcr. the relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathologic parameters was also investigated. results: the methylation of caspase 8 (34.2%, 25/73) and survivin (32.9%, 24/73) demonstrated a negative correlation with immunohistochemical expression of capsase 8 (47.9%, 35/73) and survivin (43.8%, 32/73) (p=0.042 and p=0.001 respectively). methylation of caspase 8 and immunohistochemical expression of its gene product was significantly correlated with apoptosis (p=0.026 and p=0.032). survivin nuclear immunoreativity revealed significantly correlated with proliferative activity of tumor cells (p=0.001). by survivial analysis, the negative caspase 8 expression and positive survivin expression showed worse prognosis in hcc, that was statistically insignificant (p>0.05). conclusion: in conclusion, caspase 8 and survivin may contribute an important regulatory mechanism for tumor cell proliferation and apoptosis, and may be prognostic predictors in hcc. injection was recently reported to be effective against hcc with pvi, though the therapy is not always applicable for the patients with arterial abnormality. therefore we tried combination therapy of transcatheter arterial cisplatin embolization and radiation, and will report the effectiveness and toxicity of the therapy. methods: the combined therapy was conducted in 7 hcc patients with pvi. transcatheter arterial embolization with 1mg/kg cisplatin powder (ia call) was performed against intralobar lesions, followed by external radiation targeted for pvi (60gy in 2 gy fractions). the following variables were evaluated with the survival rate: gender, age, viral etiology, child's class, performance status, and location of pvi. results: one (16%) patient showed complete response and another two (33%) partial response. two (33%) showed no change, and one (16%) showed progress of disease. the survival rates at six months among overall patients were 85.7%. adverse events were limited to nausea and appetite loss. one of the patients with partial response underwent curative resection, and is still alive without any recurrence for 530 days. conclusions: the combination therapy of cisplatin embolization and radiation is safe, effective and also feasible to the patients with arterial abnormality. this therapy is suggested to be a useful alternative therapy for the patients with extensive pvi. recently, the injection port has been used for hepatic arterial infusion chemotherapy (hai) in japan. hai is usually used for the treatment of multifocal bilobar tumors of the liver or hccs combined with portal vein tumor thrombosis (pvtt), not amenable to tace. this study examined the efficacy and toxicity of repeated hepatic hai using lipiodol suspension mixed with cisplatin powder. methods: from april 2005 to september 2008, 20 patients with inoperable advanced hcc were enrolled in this study. all received cisplatin powder (50mg) and lipiodol (2ml) suspension, with an intervening 4 weeks interval. the drugs were delivered from an injection port. 13 patients had hcc with pvtt, and 7 had hcc without pvtt. 11 patients with liver function of child grade a, 7 of grade b, and 2 of grade c were enrolled. result: the mean number of hai given during the follow-up period was 4.1 times. we found complete response in 1 case, partial responses in 6, no change in 6, and progressive disease in 7. the overall response rate was 35.0%. the 1-year survival rate was 36.7% and the 2-year survival rate was 12.1%. although 3 patients had cisplatin-induced anaphylaxis, no severe adverse events (hepatic failure and renal failure) were observed. conclusion: chemo-lipiodolization using cisplatin powder delivered via an injection port provides some clinical benefits without severe adverse events in patients with far advanced hcc. background: recently, the antitumor efficacy of angiogenesis inhibitors is expected in the treatment to hepatocellular carcinoma. the gene expression relevant to the vascularization, which is a target of these inhibitors, has a difference according to each case and it is thought that it influences the therapeutic effect of them. however, there are still few reports of mrna expression of vascular endothelial growth factor (vegf) receptors in hcc. methods: the relative mrna level of vegf and its receptors (kdr and flt-1) was analyzed using quantitative rt-pcr in 51 patients with hcc. matched samples of hcc (t) and non-tumor liver tissue (nt) were obtained by fine needle (21gauge) biopsy. results: gene expression level of vegf and flt-1 was significantly higher in hcc than nt (vegf; p<0.001, flt-1; p<0.001). according to the clinicopathological findings, gene expression level of vegf and kdr in hcc was significantly high in hypervascular hcc compared to hypovascular hcc (vegf; p=0.002, kdr; p=0.042). additionally, flt-1 tended to be expressed higher in hypervascular hcc than hypovascular hcc (p=0.09). moreover, gene expression level of vegf, kdr and flt-1 tended to be higher in advanced-stage hcc than early-stage hcc. conclusion: not only vegf but kdr and flt-1 were highly expressed in hypervascular and advanced hcc. aims: fibrinogen-like protein 2/fibroleukin (fgl2) has been reported to play a vital role in the pathogenesis in mhv-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo-and xeno-graft rejection by mediating "immune coagulation". fgl2 functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. therefore, this study was designed to examine the role of fgl2 in tumor development. methods: tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (hcc) model on nude mice (established from high metastasis hcc cell line mhcc97lm6) were obtained. results: hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human hcc nude mice. hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, nk cells, and cd8 + t lymphocytes and vascular endothelial cells. hfgl2 mrna was localized in cells that expressed hfgl2 protein. fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. in vitro, il-2 and ifn-increased hfgl2 mrna by 10-100 folds and protein expression in both thp-1 and huvec cell lines. one-stage clotting assays demonstrated thp-1 and huvec cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. conclusion: the hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction. . the therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least 2 cycles, when responses to treatment were evaluated by eastern cooperative oncology group criteria. results: of 146 patients treated (male, 79%; median age, 64 years), 64% had child-pugh a, and 31% had b. 90% had either metastasis or vascular invasion. 71% had metastasis and 49% had vascular invasion. on the basis of independent assessment, three (2.1%) patients achieved a complete response, thirteen (8.9%) had a partial response, and 42 (28.8%) had stable disease. there was no grade 3/4 drug related toxicities. median overall survival was 6.9 months. conclusion: combination therapy of ifn +5-fu has modest efficacy in hcc. background: amt is a mixture of approved pharmaceuticals in low therapeutic doses (human insulin and chlorpheniramine) and herbal components (aqueous camomile extract). preclinical and phase i data in healthy volunteers showed a favourable safety profile for amt. this pilot study should examine efficacy and safety of amt in the patients with advanced hepatocellular carcinoma (hcc). methods: thirteen patients with advanced hcc (tnm stage iii-iv), who did not respond to existing therapy, were treated with i.m. amt at 0.066 ml/kg up to a maximum volume of 5 ml twice daily for 1-10 months. primary study objectives: clinical benefit response (cbr). secondary objectives: safety of amt, tumor response according to who-recist criteria, quality of life (qol) and iimmunomodulatory effects. the effects were evaluated by cytokine production of pbmcs before and after the treatment. results: there were no significant safety issues. four and 3 patients showed positive and stable responses for cbr, respectively. tumor response was 1 pr, 6 sd and 6 pd. even in the patients with pd, 2 and 1 patients showed positive and stable responses for cbr. qol data showed clear improvement. immune monitoring demonstrated effects of amt on the functional immune parameters in about half of patients. in the patients with pr, histological examination showed tumor necrosis and many lymphocytes including plasmacytes infiltrating in the tumor. conclusion: these results suggest that a promising rate of patients with advanced hcc respond clinically to the amt treatment without significant safety issue and amt has some immuno modulatory capacities. background/aims: dysplastic nodules are important due to premalignant potential. the aim of this study was to evaluate the electron microscopic findings of liver dysplastic nodule in patients with liver cirrhosis. methods: a total of 5 patients (mean age: 64±9 years old, male 4) with dysplastic nodules which suspected as malignant nodule (mean size 1.38±0.22 cm) was enrolled from 48 cases of liver cirrhosis undergone ultrasonography-guided biopsy from december 2006 to january 2008. the etiologies of liver cirrhosis were as follows; alcohol (1 patient), hepatitis b virus (2), and hepatitis c virus (2). results: hepatocytes showed rosette formation of regenerative hepatocyte or degeneration. the nucleus was round or oval shaped and the nucleus membrane was irregular. the nucleolus was prominent and clear, the mitochondria were crowded to one side in the cytoplasm with megamitochondria. glycogen granules and lipofuscin pigments were abundant. sinusoid formation was poorly developed and collagen fiber bundles were increased. the hepatocytes of rosette formation and bile ductules cell made of canal of hering, which was dilated and microvilli was decreased. the number of canal of hering was 7, which was composed of 1.7±0.8 with hepatocyte and 2.1±0.7 with bile ductule cell, respectively. there was no oval cell in the canal of hering, which was relatively well developed. schwann cells were clustered together in nerve plexus. therefore, these electron microscopic findings showed that dysplastic nodule was similar to early hepatocellular carcinoma. conclusions: this study showed that dysplastic nodule in liver cirrhosis is nearly identified to early hepatocellular carcinoma. dcp is an important risk factor for recurrence after radiofrequency ablation of single hepatocellular carcinoma -< 3cm in diameter r. kuromatsu 1 , a. takata 1 , n. fukushima 1 , s. sumie 1 , m. nakano 1 1 division of gastroenterology, department of medicine, kurume university school of medicine background and aims: the aim is to analyze the risk factors for local recurrence + intrahepatic metastasis after radiofrequency ablation (rfa) and hepatic resection (hr) for single hepatocellular carcinoma (hcc) < 3cm in diameter. methods: between 1999 and 2007, 219 patients with single nodule < 3cm in diameter and child-pugh grade a were treated by hr and rfa, and recurrence rate and survival rate using kaplan-meier method, and important risk factors for recurrence using cox's proportional-hazards regression model were analyzed. factors used for multivariate analyses were age, gender, viral marker, tumor diameter, afp, afp-l3, dcp, and platelet count. results: mean age was 66 years old, m/f ratio was 136/83, hr/rfa was 59/160, and mean observation period was 1194 days. five-year survival rates, and 2-year local recurrence-free + intrahepatic metastasis-free rates were not significant between hr group and rfa group (82/72%, 92/89%). in rfa group, the only independent risk factor for local recurrence-free + intrahepatic metastasis-free survival was dcp (p=0.0034). tumor diameter was not significant for recurrence. in hr group, there was no risk factor for recurrence. in pathological analyses of hr group, dcp had a tendency to associate with microvascular invasion (p=0.065). conclusions: rfa was effective for hcc < 3cm in diameter and dcp < 40 mau/ml. hepatic resection should be selected for single hcc with dcp > 40 mau/ml even though hcc <3cm in diameter. background: radiofrequency ablation (rfa) is now a common treatment for small hepatocellular carcinoma (hcc). however, critical complications after rfa such as rapid intrahepatic dissemination have been reported. in this study, we investigated the method how to estimate the malignant potential of small hcc by dynamic ct before rfa. methods and results: firstly, 61 hccs less than 3cm in diameter were analyzed. those tissues were classified into 4 groups as followed, small nodular type with indistinct margin (type e), simple nodular type (type 1), simple nodular type with extranodular growth (type 2), confluent multinodular type (type 3). in the type 2 and 3 groups, portal invasion over vp1 were observed more frequently than those in the type 1 and e groups. at the next step, these 61 hccs were classified into above-mentioned 4 types by two radiologists according to the shape of early stain or defect of delay phase of dynamic ct before operation. the accorded rate was 87% between those classifications. next, 45 patients, which had solitary hcc less than 3cm in diameter and treated with rfa, were classified into those 4 types by dynamic ct before rfa. the recurrence rate and prognosis of those patients were examined. in the type 2 and 3 groups, the recurrence rate was higher and significant worse prognosis was showed than those in type 1 and e group . conclusion: it was suggested that hcc with type 2 and 3 might process higher malignant potential and rfa should be carefully performed on those types of hcc. background/aims: hypoxia-inducible factor-1 (hif-1 ) is the central transcriptional factor in the cellular response related to various aspects of cancer biology, including proliferation, survival, angiogenesis, and extracellular matrix metabolism to hypoxia. il-8 became known to replace hif-1 functions in the other cancer cell lines. the aim of this study was to evaluate whether il-8 may induce angiogenic factors without hif-1 by inflammation signal of hypoxic condition. methods: hif-1 knockdown cell lines of hcc (huh7 and hepg2) were constructed by rna interference tools, and cultured under normoxia (21%o2, 24 hours) and hypoxia (1%o2, 24 hours) conditions. following transfection, the amounts of hif-1 , il-8, angiogenic factors and matrix metalloproteinase (mmp) were examined using rt-pcr and western blotting, respectively. results: the expression of hif-1 , angiogenic factors, mmp, il-8 was markedly enhanced in wild types that were cultured under hypoxia, and the hypoxic induction of angiogenic factors and mmp was partially blocked in hif-1 knockdown hcc cell lines. nf-b inhibitor suppressed angiogenic effects by blocking il-8 activity. conclusion: these data suggest that il-8 induced tumor angiogenic factors in hif-1 knockdown hcc cell lines. background there were some reports that liver caner related to the levele of sera hbvdna our research focused on the relationship between the quantity of hepatocellular hbv cccdna , tdna and liver cancer. methods the samples included the liver tissue of 24 chb patients (chb group) and the para-liver cancer tissue of 26 primary liver cancer patients (phc group) the quantity of hepatocellular hbvcccdna, tdna were assayed by fq-pcr in both groups. result: the quantity of hepatocellular hbv cccdna in chb group was 11.56±16.14 copys/cell higher than phc group(3.96±9.27 copys/cell), p=0.011; the quantity of hepatocellular hbv tdna in chb group was 57.38±91.64 copys/cell higher than that of phc group(35.07±93.03 copys/cell),p=0.13.; conclusion: the quantity of hepatocellular hbvcccdna, tdna can not be used as predictors of liver cancer for hepatitis b patients. hepatic cancer predominantly occurs in males. this is almost a commonsense to most of us. but the detailed mechanisms underlying such phenomenon are still not well-known. the average age of liver cancer patients are about 30-40 years old. so most female patients have udergone pregnancy at least one time. pregnancy is a very important event before or during the development of liver cancer in females. in this special period, not only sex hormones secrete in a strange manner, but also immune system functions in a special module which is very different from normal. so it is urgent to investigate the impact of process of pregancy on the development of hepatic cancer. in this study, 100 female sd rats are randomly divided into two groups: pregance group and controll group. rats in both groups are injected iv diethylnitrosomine(a chemical carcinogen). in pregnance group, rats are raised together with male rats in 3:1 ratio(3 female, 1 male) to make every rats undergo pregnancy. while in controll group, rats are coupled with spermaduct-ligated male rats. the size and amount of hepatic cancers in pregancy group are smaller and less than those in controll group. the survial rate is also significantly higer than that in controll group. we conclude that the process of pregnancy exerts an inhibitory role in the development of chemical induced hepatic cancer in rats. acknowledgement: this project was sponsed by the national natural science foundation of china. the number of the grant is 30600727: the use of alpha-fetoprotein measurement in detection of recurrent hepatocellular carcinoma after living donor liver transplantation n. yamashiki 1,2 , y. sugawara 1,3 , s. tamura 3 , r. tateishi 2 , h. yoshida 2 , j. kaneko 3 , y. matsui 3 , n. kokudo 1,3 , m. omata 2 1 organ transplantation service, 2 department of gastroenterology, university of tokyo, 3 department of surgery, university of tokyo background: the recurrent hepatocellular carcinoma (rhcc) after liver transplantation (lt) can occur in 10 to 20 % of transplant recipients despite with a careful patient selection. for the surveillance of rhcc, frequent measurement of alpha-fetoprotein (afp) and annual ct scan is commonly used. however, the usefulness of afp is not clear. we report the update of our experience using our surveillance protocol. methods: between 1996 and march 2008, 330 adult living donor lt were performed at the university of tokyo. among them, 100 recipients with hcc in their explanted liver were subjected to analysis. we used monthly measurement of afp and des-gamma carboxy prothrombin (dcp) with annual dynamic ct scan. results: 83 met milan criteria pre-operatively and 12 did not. 5 were incidental hcc. rhcc was experienced in 9 patients at 10 (2-24) months after lt. recurrence sites were graft (1), lung (2), bone (3), and multiple organs (3). rhcc was first suspected from elevation of tumor markers in 8; afp in 4, dcp in 2, and both afp and dcp in 2. rhcc was confirmed with ct scan (7) or mri (2) in 4 (0-6) months after the first sign of rhcc. when the cutoff level of afp>20ng/ml was used, the sensitivity and specificity for rhcc were 66% and 100%. six cases were treated surgically of which two achieving prolonged survival. conclusions: although the confirmation of the rhcc sites required multiple imaging studies, afp measurement was useful as for the first sign of rhcc. purpose: to evaluate the therapeutic effect of heated (60 c) lipiodol via hepatic artery administration in vx2 rabbit liver cancer model. materials and methods: thirty male new zealand white rabbits were randomly divided into 3 groups with 10 rabbits for each group. vx2 carcinoma cells were surgically implanted into the left liver lobe. the tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of tumors detected by ultrasonograph reaching 2 to 3 cm. under the anaesthesia, transcatheter hepatic arterial embolization was performed and doxorubicin-lipiodol (37ºc) (1 ml), lipiodol (60ºc) (1 ml) and control (physiological saline (37ºc) (1 ml)) were injected into hepatic artery of the 3 different groups. one week later, the volume of tumor was measured by ultrasonograph again. the serum of all rabbits was collected before injection and at 4 and 7 days after injection and the level of aspartate aminotransferase (ast) was checked. the survival period of 3 groups of rabbits after treatment was also recorded. during the last course of their disease, the rabbits were given some analgetics to relieve suffering. results: the tumors' growth rate in lipiodol (60ºc) background/aims: hepatocellular carcinoma (hcc) is one of the male-dominant cancers, and hepatitis c virus (hcv) is one of the causes of hcc. it was reported that androgen receptor (ar) is expressed in hcc and its surrounding tissues. androgen signaling and ar may be involved in hepatocarcinogenesis. in this study, we investigated whether hcv interacts with androgen signaling in human hepatocytes. methods: hcv protein expression vectors were co-transfected with ar-expression vectors and ar-responsive element-driven reporter vector into immortalized human hepatocytes (ihhs) and human hepatoma cell lines. kinase inhibitors were used to examine the activation of the akt, mapk, and jak/stat3 pathways. real-time pcr and western blotting were performed. cell culture grown hcv (hcvcc) were also used, and angiogenesis was evaluated by tubule formation assays in human coronary microvascular endothelial cells in the presence of 5 -androgen-1 -ol-3-one. results: hcv enhances ar-responsive gene expression in the presence of androgen. hcv core protein has the strongest effects and induced ar activation associated with jak/stat signaling. hcvcc enhances vegf mrna expression and angiogenesis. conclusions: hcv core protein is an enhancer in androgen signaling and can be expected to play an important role in hcv-related hepatocarcinogenesis. background: to evaluate the therapeutic outcomes and the toxicity of the combination of arsenic trioxide and the chinese traditional jianpiliqi (jplq) formula in the treatment of advanced hepatocellular carcinoma (hcc). methods: patients with advanced hcc, not suitable for resection but with normal major organ functions, were enrolled to receive a therapeutic regimen consisting of intravenous arsenic trioxide (6 mg / m 2 ) administration from days 1-14, and an oral administration of jplq formula twice daily from days 1-28. each cycle was composed of 28 days and treatment could expand up to 4 cycles before evidences of intolerable toxicity or disease progression. result: one patient had partial response, one had minor response, 15 showed stable disease and 15 (46.9%) had disease progression. total disease control rate was 53.1%, median survival time was 5.8 months (2-23.9 ms), and time to progression was 4.2 months (1-16.9 months). the incidences of grade 1-3 abdominal distention and nausea/vomiting were 65.6% and 37.5%, respectively. increases in ggt occurred in 4 patients (2 grade 2, 1 grade 3, and 1 grade 4) and increases in serum creatinine in 2 patients (1 grade 3 and 1 grade 4), respectively. conclusion: compared with the single arsenic trioxide treatment reported in past literature, treatment by arsenic trioxide combined with jplq showed modestly higher anti-tumor activity and tolerable toxicity in patients with advanced hcc; its manageable toxicity and increased tumor response rate may offer a better treatment regimen, and deserve further investigation. aim: to investigate the effect of osteopontin (opn) expressions down-regulated by rna interference (rnai) on the invasion and metastasis of human hepatocelluar carcinoma (hcc). methods: hcc cell line (hcc-lm3) was transfected with the chemically synthesized small interfering rna (sirna) in study arm and with non-specific sirna in control arm. real-time pcr and western blotting were used to quantify the mrna and opn protein levels. the malignant phenotypes including cellular growth rates, colony formation and matrigel invasion activities of the hcc cell line were analyzed. results: in study arm opn mrna expressions decreased 75% and opn protein decreased 80% compared to those of blank arm. the number of formed colonies and migrating numbers of the cells in vitro decreased significantly (64.6% and 59.6% respectively) in study arm compared to these of blank controls (p<0.05). the parameters in the control arm did not differ from those of the blank arm (p>0.05). conclusion: the specific sirna was able to reduce opn expressions at both the mrna and protein levels and significantly diminished the invasiveness of hcc cells. methods: the expressions of mif and vegf in hcc and adjacent tissues were detected from 4 patients. specific sirna targeting mif gene was synthesized, and transfected into the hcc cell lines (plc and hepg2) in study group and non-specific sirna was used in controls. the mrna and protein expressions of mif and vegf were examined by pcr and western blot. results: mif and vegf mrnas were overexpressed in the hcc tissues compared with adjacent tissues (rq=8.91±1.85 and 3.00±0.86, p 0.01). the mrna and protein expressions of mif and vegf of hcc cell lines significantly decreased in study group compared with controls (p 0.01). vegf mrna levels decreased 75.6%±2.6%; 79.8%±1.2% in plc, and 73.6%±4.6%; 80.7%±2.2% in hepg2 cells when disposed with sirna 50nm and 100nm. vegf protein levels also significantly reduced in study group p 0.01 . conclusions mif and vegf mrnas were overexpressed in the hcc tissues in vivo, and mif sirna was able to knock down the expressions of mif and vegf in hcc cell lines in vitro. y.y. li, y.c. zhang, y.j. zhou, y.m. wei aim: to identify tumor-associated genes by constructing transcription profiles of pure hepatocellular carcinoma (hcc) tissues and normal liver tissues with the combination of laser capture microdissection and microarray. methods: hcc cells and normal liver cells from resection samples of 4 patients were laser capture microdissected. micro-rna was isolated from them for linear amplification then crna was tested with whole genome microarray. differentially expressed genes were screened. results: the quality control of this technique was satisfactory with rna integrity number>7, a260/a280 ratio for crna measurement=2.0~2.1 and good pictures for microarray. compared with normal liver tissues, hcc had 1361 differentially expressed genes, with 607 being up-regulated and 754 being down-regulated genes respectively. among the top ten ranked up-and down-regulated genes (total 20), 5 genes were known as hcc differentially expressed genes, 11, known as other tumors expressed genes previously. four unknown tumor related genes (depdc1b, aspm, fcn2 and bbox1) were detected in this study. conclusion: the combination with laser capture microdissection and microarray was effective in screening the differentially expressed genes of hcc. background/objective: young patients present with large hcc on initial presentation are not uncommon. our aim is to study the computed tomography(ct) imaging of hcc and the clinical features of this special group of patients. methods: 521 hcc patients had ct imaging of liver peformed in a three year period, 385 patients had ct imaging peformed at the time of initial hcc diagnosis in our centre and were selected. they were divided into three age groups: young patients with age </= 40 year-old(group 1), age 41 to 60(group 2), age >60(group 3) to study imaging and clinical factors. univariate and multivariate analysis by cox regression model done to look for prognostic predictive factors. results: infiltrative tumour in ct scans, symptomatic presentation, child's and tm staging are prognostic factors in hcc. conclusion: young hcc patients have larger infiltrative tumour in initial ct scans and more being symptomatic. age is not an independent prognostic factor. aim: to investigate the expression change of nk cells receptor nkg2d from human peripheral blood in patients with primary carcinoma of liver and study the relationship between nkg2d expression and cytotoxicity of nk cells. background/aims: lens culinaris agglutinin-reactive alpha-fetoprotein (afp-l3) is a specific protein produced by hepatocellular carcinoma(hcc), which is more valuable than afp in the diagnosis of hcc. aptamers are oligonucleotide ligands binding to target molecules sensitively and specifically, which are screened from a great capacity of synthetically oligonucleotide library by systematic evolution of ligands by exponential enrichment (selex). our aims were to select the aptamers against afp-l3 from a self-designed ssdna library for potential application in diagnosis of hcc. methods: a random ssdna library and its corresponding primers were designed and synthesized. aptamers against afp-l3 were selected by selex. individual aptamers were separated by polymerase chain reaction-single strand conformation polymorphism (pcr-sscp) analysis and characterized. results: a ssdna library of 78 nucleotides with 40 random nucleotides in middle were designed and used for the selection. the binding rate of library against afp-l3 was increased from 5.1% to 48.2% after 13 round selection. seven aptamers (s1 to s7) were isolated, and their sequences in random region and secondary structures were different from each other. all aptamers could bind afp-l3 in a different extent, and the dissociation constants of s4 and s7 are 650nmol/l and 132nmol/l. conclusions: aptamers for afp-l3 are successfully screened out and could bind afp-l3 specifically. methods: flow cytometry was used to determine the number of nk cells and the expression of nk cells receptor nkg2d from human peripheral blood in patients with 20 case primary carcinoma of liver 23 case hepatitis b cirrhosis 20 case hepatitis b and 20 healthy cases and enzyme mark instrument was used to detect cytotoxicity of nk cells in all cases. results: killing rate of nk cell for k562 cell,nkg2d expression level of nk cells, and the number of nk cells in the patients with primary carcinoma of liver decreased significantly p<0.05 compared with those in the healthy subjects and hepatitis b group ,and decreased a little compared with those in the hepatitis b cirrhosis (p>0.05).the activity of nk cells showed a obvious positive-correlation with the number of nk cell and expression level of nk cell receptor nkg2d. conclusion: the cytotoxicity of nk and the nkg2d expression of nk cells decreased significantly from human peripheral blood in patients with primary carcinoma of liver .the activity of nk cells is closely related to the nkg2d expression level of nk cells. enhancing the nkg2d expression level of nk cell may provide a new idea for adoptive immunotherapy of primary carcinoma of liver. and alpha-fetoprotein afp in serum and tissues for primary hepatic cancer(phc). methods: sixty-six phc and 32 cirrhotic patients were enrolled. in phc patients,male /female was 48:18, age was 55.4 ± 9.91.of them, 16 patients were defined as stage a-a. in cirrhotic patients, male /female was24:8, age was 53.3 ± 5.81. serumgpc3 was detected using elisa. serum afp was detected using electrochemiluminescence. the hepatic expressions of gpc3 and afp were measured using immunohistochemistry in 21 phc and 6 cirrhotic patients. results: the cutoff value of afp diagnosis for phc was 400 g / l or more, afp positive in phc patients was 28.8% (19/66); the cutoff value of gpc3 diagnosis for phc was 300ng / l or more, gpc3 positive was in 47.0 % (31/66), p = 0.031. in a-a stage phc patients,the positive of gpc3 and afp was 62.5% (10/16), 0(0/16), respectively,p = 0.000. in serum afp negative or positive patients, the positive of gpc3 was 46.7% (14/30) , 47.2% (17/36), respectively,p = 0.964. the relationship between gpc3 with age, sex, child-pugh grade, hbv infection, tumor size and metastasis were not observed.the positive expression of gpc3 and afp in hepatocellular carcinoma tissue was 85.7% (18/21), 4.8% (1 / 21), respectively, p = 0.000. neither gpc3 ,nor afp in the paracarcinomatous and cirrhotic tissue, was expressed. conclusions: diagnosis of glypican-3 protein for primary hepatic cancer is superior to afp.gpc3 can be regared as a early marker to diagnosis phc. objective: to investigate the effects and the possible mechanism of curcumin on the proliferation and the invasion of human hepatocellular carcinoma in vitro and in vivo. methods: hcclm3-rfp cell lines were maintained in dmem medium supplemented with 10% fetal bovine serum. the fluorescent areas of hcclm3-rfp were photographed daily and repeated in 4 consecutive days after curcumin treatment for obtaining cell growth curves. the cell morphologic changes were also observed. cell invasion experiment was performed with boyden chamber array. the rfp-expressing human hcc xenograft model in nude mice was established to study the anti-tumor effects of curcumin. the ctc was detected by facs. the expression of cyclind1 and mmp-2 was detected by sybr green real-time pcr. results: after incubation with 20 m, 10 m and 5 m curcumin respectively for 24, 48 and 72 hours, the growth of hcclm3-rfp was significantly inhibited and some morphologic changes were observed. the mean tumor size in nude mice treated with curcumin since day 21were significantly less than those of the control group(p 0.01). the mean metastasis area of lung and the number of ctc in curcumin group on day 35 were remarkably less than in the control group(p 0.01). the mrna levels of cyclin d1 p 0.01 and mmp-2 p 0.05 in curcumin group on day 35 were significantly lower than in the control group. conclusion: curcumin can inhibit the proliferation and invasion of hcclm3 cell line not only in vitro but also in vivo mainly by down-regulating the expression of cyclin d1 and mmp-2 in mrna levels. phosphorylated erk is a potential predictor of sensitivity to therapy with sorafenib in hepatocellular carcinoma -evidence from in vitro study z. zhang 1 , y.h. wang 1 1 background: sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (hcc) and thus sets the new standard for the first-line treatment of advanced hcc. however, it remains unresolved to predict the drug sensitivity in treating hcc with sorafenib. pretreatment perk level has been shown to be associated with favorable response to such therapy in a phase 2 preclinical study, indicating that perk may be a potential biomarker for treatment of hcc with sorafenib. methods: the effects of sorafenib and 5-fluorouracil on cell proliferation were evaluated by cell viability assay in four types of hcc cell line (smmc-7721, mhcc97-l, mhcc97-h and hcclm6), with different metastatic potential and basal perk expression. levels of perk expression were determined by immunocytochemical analysis and quantification, along with western blot analysis. correlation analysis was carried out between the ic50 values of drugs and mean optical density values of perk. results: the basal perk levels increased stepwise in cell lines in accordance with their metastatic potential. sorafenib inhibited erk phosphorylation at a concentration between 5 and 20 m dose-dependently, while no changes were observed after 5-fu treatment. correlation analysis between the ic50 values and mod values of perk revealed that the effects of sorafenib were significantly correlated with basal perk levels (spearman r=-0.8671, p=0.0003). on the other hand, the resistance to 5-fu were significantly associated with basal perk expression in these hcc cell lines (spearman r=0.7846, p=0.0009). conclusions: in this vitro study, perk was confirmed to be a useful biomarker predictive of sensitivity in treating hcc with sorafenib. the raf/mek/erk pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in hcc. background: to investigate the dynamic expression of igf-ii and igfbp-3 and its alteration of bcl-2 in hcc. methods: hcc models were induced with 2-faa on male sd rats. morphological changes of livers were observed and the dynamic changes of liver or serum igf-ii, igfbp-3, and bcl-2 were quantitatively analyzed by elisa. the expression and distribution of liver igf-ii were observed by immunohistochemistry. result: hepatocytes from granule-like degeneration to a typical hyperplasia to hcc and the progressing increasing of the levels of hepatic igf-ii after rats induced by 2-faa. the levels of igf-ii in hepatoma and sera were significantly higher than any of other groups. the positive relationship of igf-ii was found between liver and sera (p<0.01). the igfbp-3 levels in hepatoma were significantly lower than that in other groups (p<0.01) and the progressing increasing of the levels of hepatic bcl-2 expression during the course. the levels of bcl-2 in hepatoma tissues were significantly higher than those in normal and degeneration ones. the immunohistochemistry evidences indicated the positive expression and hepatocyte distribution of bcl-2 in rat hepatoma. conclusion: hepatic igf-ii, igfbp-3 and bcl-2 may participate in hepatocyte canceration and accelerate the occurrence and development of hcc. the expression of igf-ii and igfbp-3 could be useful molecular markers for early diagnosis and prognosis of hcc. background: this study was done to assess the etiological role of hepatitis b virus (hbv), hepatitis c virus (hcv) and aflatoxin b1(afb1) in development of hepatocellular carcinoma (hcc) in bangladesh. it was also investigated whether alpha-feto protein (afp) and protein induced by vitamin k absence or antagonist ii (pivka-ii) has any diagnostic advantage over each other methods: fifty five histologically proven hcc patients were tested for serological markers of hepatitis b and hepatitis c, and afb1-dna adduct. during the diagnosis, they were also investigated for liver function tests, afp pivka-ii. results: out of fifty five hcc patients, 41(74.5%) were found positive for serological markers of hbv, 21(38%) for hcv and 15(27%) for both. eight cases (14.5%) were negative for the markers of hbv and hcv. however, none had afb1-dna adduct above normal range. both pivka-ii and afp is strong marker for hcc with satisfactory level of sensitivity and specificity; but pivka-ii is more sensitive (92.7%) and afp is more specific (96%). conclusions: hbv and hcv is the major etiological agent responsible for the development of hcc in bangladesh. background: to investigate the influences on the malignant transformation of hepatocytes through the intervention of nf-b activation pathway. method: hcc models were induced with 2-faa on sd rats, thalidomide was administered intragastrically and rats were sacrificed fortnightly interval to the twelfth week. morphological changes were observed by he staining. nf-b expressions were detected by ihc. the relationship between nf-b expression and pathological characteristics in hcc and non-hcc were analyzed. results: rat hepatocytes showed vacuole-like denaturations at the early stages, then dysplastic nodules appeared at middle stage, and finally progressed to tubercles of cancerous nest, all of which were highly differentiated hcc. thalidomine can repress the morphologic change of liver cells. there were only punctiform denaturations at the early and middle stage; nodosity hyperplasy and minority atypical hyperplasia were found at the finally stage. the ihc results demonstrated that nf-b level was significantly higher than those in normal ones, and the nf-b level of livers in hcc was higher than those in thalidomide group. an increasing tendency of nf-b was found from normal to hcc. nf-b in hcc were significantly higher than those in nc. the nf-b levels with thalidomide intervence raised first and decreased later. nf-b expressions in hcc were higher than that in their non-cancerous tissues. no positive relationship presented between nf-b expression and histological differentiation grade or the number of tumor, and size of tumor. conclusion: decrease nf-b expression can inhibit hcc development and nf-b is expected to be a new molecular target of hcc therapy. method: the cellular distributions of vegf expression in hcc tissues were investigated by immunohistochemistry. the levels of total rna and vegf were quantitatively detected in hcc, their paracancerous, and distal cancerous tissues, respectively. simultaneitily, serum vegf were analyzed in patients with chronic liver diseases for clinical values. results: the positive expression showed palm-yellow or palm-brown granules and distributed in hepatocyte plasma of hccs. the incidence of vegf was 63.9% in hcc tissues, 78.3% in non-encapsulated hccs, and 90.9% in hccs with extrahepatic metastasis, respectively. no significant difference was found between hepatic vegf and hcc diameter or differentiation degree. the specific concentration (pg/mg liver) of vegf expression was significantly higher (p<0.01) in hcc than their paracancerous or distal cancerous tissues, respectively. the circulating vegf was abnormally elevated in hcc. if the cut off values was more than 280 pg/ml, the incidence of serum vegf was 88.4% in hcc, 14.3% in chronic hepatitis, and 10% in liver cirrhosis, respectively. the combined vegf and afp can increase positive rate up to 94.2% for hcc. conclusion: the vegf overexpression is a useful marker for vascular invasion and metastasis of liver tumors. background: hepatocellular carcinoma (hcc) represents a major health problem world wide. it accounts for 90% of all primary liver cancers and is the fifth most common malignancy (1). objectives: evaluation of radiofrequency thermal ablation versus transarterial hepatic chemoembolization with the effect of viscum (fraxini 2) on tumour recurrence. methods: 60 patients with hcc were enrolled in the study. group 1 include 30 patients and were treated with radiofrequency thermal ablation (15 patients of them received viscum 2 by subcutaneous route for 2 years). group 11 included 30 patients with hcc and were treated by tace (15 patients of them received viscum 2 subcutaneously for 2 years). results: group 1 patients showed total ablation in 70% with persistant inactivity during 2 years follow up. group 11 did not show significant difference from group 1 as regards relapse rate nor the performance status. complications as nausea, vomiting, fever, jaundice, and elevation of transaminases were significantly more encountered with tace. viscum 2 did not significantly arrest tumour recurrence. conclusion: non surgical patients with hcc can achieve curative treatment with radiofrequency with minmal side effects. tace is a palliative treatment option for large hcc. a new technique had been attempted to increase the field of radiofrequency ablation of expandable electrode needles in the treatment of hepatic neoplasms much larger than the routinely covered size of 5-7 cm according to the needle size overcoming the technical difficulties usually met with in the overlapping balls technique due to the hyperechoic focus that develops at the needle tip making reinsertion difficult and inaccurate. in this technique, two or three needles were inserted from the start into the mass with accurate estimation of the exact field of ablation of each needle trying to cover the whole extent of the mass before application of radiofrequency waves. 40 patients were included in the study, all presented with hepatic neoplastic mass lesion that range in size between 7 and 10 cm in its maximum diameter. all had a pretreatment helical (triphasic) ct study for accurate delineation of the whole extent and vascularity of the mass. two needles were sufficient to cover the whole extent of the mass in 23 patients (57%) while in the remaining 17 patients (43%) three needles were necessary. the procedure was done under general anathesia and ultra sound guidance, patients tolerated procedure well with smooth recovery. no major complications. follow up spiral (triphasic) ct was done 2 weeks after ablation revealed percentage of tumour necrosis of 90% or more in 30 patients (75%), 70-90% in 6 patients (15%) while in the remaining four patients (10%) the percentage was 50-70% necrosis. in conclusion this technique should be considered in the treatment of hepatic masses larger than the usual field of the needle. results: the median value of gpc-3 in hcc, dc, cc was significantly higher than chronic hepatitis and control groups. no significant correlation found between afp and gpc-3. auroc of afp was 0.85 & auroc of gpc-3 was 0.84. the diagnostic sensitivity of afp (20 ng/ml) was 70% with ppv 53.8%. the specificity was 85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) was 100% with ppv 27%. the specificity was 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved the specificity to 87.5%. conclusion:gpc-3 although it is a serological test for early detection of hcc, it showed limited specificity, where it is detected in different stages of chronic liver disease, as it is an oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 may improve the prediction accuracy of hcc patients in those showing seronegativity to afp. outcome of inoperable hepatocellular carcinoma patients receiving transarterial chemoembolization: retrospective analysis in an asian regional hospital w.m. yip 1 , k.f. li 1 , k.k. li 1 , m.l. szeto 1 1 background: hepatocellular carcinoma (hcc) is a common cancer worldwide causing substantial mortality. although surgical resection is a form of curative treatment in hcc, only a minority of patients is suitable for this treatment and the postoperative recurrence remains high. transarterial chemoembolization (tace) is a treatment option for inoperable hcc and it was proven by randomized control trials that tace can prolong survival in selected patients. the aim of this study is to evaluate the survival and the prognostic factors in patients with advanced hcc treated by tace. methods: seventy four patients with inoperable hcc diagnosed from january 1998 to december 2003 were analyzed retrospectively in this study. only patients with unresectable hcc or who refused operation were included. patients with advanced cirrhosis, extrahepatic metastasis or previously treated hcc were excluded. multiple host, tumor and treatment variables were analyzed in order to evaluate the predictive factors of favorable response to treatment and better survival. results: the median survival of the study patients was 213.5 days. the cumulative survival rates at 1 year, 2 year and 3 year were 28.4%, 12.2% and 6.8% respectively. by multivariate analysis, superselective cannulation performed in tace (hazard ratio: 0.47, 95% ci: 0.23-0.95, p=0.034), embolization with gelfoam (hazard ratio: 0.30, 95% ci: 0.11-0.80, p=0.017), treatment interval more than 45days (hazard ratio: 0.33, 95% ci: 0.15-0.72, p=0.006), child-pugh grade b (hazard ratio: 5.62, 95% ci: 2.11-14.97, p=0.001), and pre-treatment serum fp level (hazard ratio: 2.93, 95% ci: 1.50-5.73, p=0.002) were independent predictors of survival. conclusions: survival of patients with inoperable hcc is still grave despite treatment. this study provided information in predicting the survival of patients with inoperable hepatocellular carcinoma treated by transarterial chemoembolization. result: age <65, total bilirubin (tb) <2.0mg/dl, albumin (alb) 3.5g/dl, prothrombin time (pt) 70%, platelet counts (plt) 10.0 10 4 /mm 3 , single nodule, and type of treatment (surgery or local ablation therapy) were linked to increased survival at univariate analysis of clip 0-1 hcc patients. of clip 2-6 hcc patients, tb <2.0mg/dl, alb 3.5g/dl, des-gamma-carboxy prothrombin (dcp) <100mau/ml, absence of vascular invasion, and type of treatment were correlated with survival. the following factors were related to survival by multivariate analysis: clip 0-1 hcc patients; age, alb, single nodule, and absence of vascular invasion, clip 2-6 hcc patients; age, tb, alb, alpha-fetoprotein (afp) <100ng/ml, dcp, absence of vascular invasion, and type of treatment. conclusion: age, albumin, vascular invasion were significant predictors of survival both clip 0-1 and clip 2-6 hcc patients. clip 0-1 hcc patients: single nodule; clip 2-6 hcc patients: lower levels of tumor markers and patients receiving promising treatment had a better chance of prolonged survival. the role of gross classification as the predictor of microvascular invasion in hepatocellular carcinoma. s. sumie 1 , r. kuromatsu 1 , k. okuda 1 , e. ando 1 , a. takata 1 , n. fukushima 1 , m. sata 1 1 background; the presence of microvascular invasion (mvi) as the risk factor in hepatocellular carcinoma (hcc) is controversial. the aim of this study was to determine the outcomes and predictive factors after hepatic resection for hcc with mvi. methods; one hundred and ten patients who underwent curative resection for hcc were included in this retrospective study. the risk factors of these patients for recurrence-free and disease-specific survival were investigated, and the clinicopathological factors predicting the presence of mvi were also evaluated. result; multivariate analysis showed that cirrhosis and mvi were identified as independent risk factors for recurrence-free survival. the 2-year recurrence-free survival rates for patients with and without mvi were 44.6% and 76.7%, respectively. multivariate analysis showed that the number of tumors, presence of mvi, and im were identified as independent predictors of disease-specific survival. the 5-year disease-specific survival rates for patients with and without mvi were 59.3% and 92.0%, respectively. by univariate analysis, mvi was significantly associated with greater tumor size, gross classification, histological grade, and intrahepatic micrometastasis (im). gross classification proved to be the only independent predictive factor for mvi by multiple logistic regression analysis. the gross classification could be evaluated by preoperative imaging diagnosis. conclusion; mvi is strongly associated with recurrence and survival in hcc patients after curative resection. furthermore, gross classification of hcc can be helpful in predicting the presence of mvi. background: hcc is a common cause of cancer morbidity and mortality. pxd101 is a novel, low molecular weight, histone deacetylase inhibitor. this phase i study aims to determine dose limiting toxicity (dlt) and maximum tolerated dose (mtd). methods: patient eligibilities include unresectable disease, ecog 2, adequate organ functions. pxd101 was given intravenously on day 1-5 every 3 weeks; dose levels were: 600 (level 1), 900 (level 2), 1200 (level 3) and 1400 mg/m 2 /day (level 4). dlts are defined as grade 4 hematological toxicity or grade 3/4 non-haematological toxicity during cycle 1 (according to nci ctc v3), or treatment delay >2 weeks. the mtd is defined as the dose below which > 2 of 3 or > 2 of 6 patients experiencing dlt. results: 18 patients were entered; level 1 (3), level 2 (3), level 3 (6) and level 4 (6). grade 3/4/5 toxicities in cycle 1 included: raised alt 1/0/0, diarrhea 1/0/0, abdominal distension 1/0/0, anaemia 1/0/0. a total of 56 cycles were administered; overall grade 3/4/5 toxicities: raised alt 1/0/0, bilirubinaemia 1/0/0; cardiac ischaemia 1/0/0; diarrhoea 1/0/0, abdominal distension 2/0/0, anaemia 2/0/0; variceal haemorrhage 1/0/0; hypercalcaemia 1/0/0; hyperkalaemia 0/1/0; hyponatraemia 1/1/0; infection 2/0/0; liver dysfunction 0/2/0; muscle weakness 1/0/0; abdominal pain 1/0/0; prolonged qtc 1/0/0; syncope 1/0/0; seizure 1/0/0. there were 3 sd and 15 pd. conclusion: at the maximum dose of 1400 mg/m 2 /day, mtd has not been reached. pxd101 is very well tolerated. sponsor: the division of cancer treatment and diagnosis, national cancer institute, usa. tumor thrombus (pvtt) is prone to be produced in the portal vein near the main tumor nodule for hepatocellular carcinoma (hcc) patients and its molecular mechanism is still unclear. in this study, we first established a hcc cell line named csqt-01 from resected tumor thrombus in portal vein in a patient with histopathologically proved to be a moderately differentiated hepatocellular carcinoma . this cell line was composed of polygonal shaped cells and its peaks of the chromosome number was 69 and 77. study on stem cell biology in this cell line suggests that cd133 cells represent about one fourth of the tumor cell population and cd133(+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. with this cell line model and resected tumor thrombi specimen, we also studied the different expression of proteins in primary tumor and tumor thrombus and found 20 proteins expressed differentially between primary tumor and the pvtt. from these proteins, annexinv, prx , cycb were selected for further analysis to find potential biomarkers of pvtt in hepatocarcinogenesis. for clinical study, we recommended a new tumor thrombus type system ( type i iv) according to anatomic features of portal vein and tumor thrombus of hcc developing modes, then evaluate this type system to predict prognosis of hcc patients. the retrospective data of 406 hcc patients with pvtt underwent resection shows that the 1y, 2y, 3y overall survival rates were 44.7 , 26.3 and 19.7 for type i, 29.9 , 20.6 and 12.4 for type ii, 25.0 , 12.5 and 3.6 for type iii, 27.3 , 0 and 0 for type iv, respectively, suggests tumor thrombus type system may be helpful to determine treatments and prognosis of hcc patients with pvtt. polyprenol could decrease the risk of hepatocarcinogenesis in hbv g. kuznecova 1,2 , s. kuznecovs 1,2 , i. kuznecovs 1,2 background: over-expression of p-glycoprotein (pgp) is associated with liver cancer development from hbv . glycoprotein synthesis in malignant tissues is limited by dolichyl phosphate (dolp). the aim of the present study was to investigate the effect of polyprenol (pp) which provides a dolp substitute in regulation of n-glycosylation on pgp over-expression in the development of liver cancer in hbv infection. methods human hepatocytes, infected with hbv and human hepatocarcinoma hep 3b cell line were used. pgp was assessed by an immunohistochemical technique. dolp fractions were analysed by hplc methods. results it is confirmed that plasmatic membrans of hepatocytes cells contain 7,9 -9,4% of pgp (the total protein amount) as a resistance marker. hbv infected cells differ from normal hepatocytes in pgp content by 4-5 times and hep 3b cells differ by 10-12 times the study showed 5-fold dolp decrease in hbv infected cells and 10-fold dolp decrease in hep3b cells. the investigations demonstrate that the situation can be changed by treatment with dolp and pp. the dolp concentration in hbv infected hepatocytes was returned to the normal level. it is established that dolp in the concentration 10 -6 m aid 6-8-fold reducing pgp in membranes of hbv infected cells. background: metastasis is one of the most complicated and major pathological processes responsible for poor prognosis of hepatocellular carcinoma. snail was recently highlighted as a critical transcriptional factor for tumor metastasis. method & result: real time rt/pcr and western blot analysis demonstrated that snail mrna and protein, respectively, were induced by 12-otetradecanoylphorbol-13-acetate (tpa) in hepatoma cell hepg2. blockade of gene expression of snail by antisense oligodeoxynucleotide and/or sirna technique can prevent not only the tpa-triggered emt/cell migration and growth inhibition of hepg2 but also tpa-induced down-regulation of e-cadherin and up-regulation of p15ink4b. moreover, the tpa-triggered promoter activation of p15ink4b was also prevented. on the other hand, two of the hepg2 clone overexpressing snail, namely s7 and s15, had a scattered fibroblastic morphology and acquired higher motility than parental hepg2. also, the proportion of g0/g1 phase of s7 and s15 was higher than that of parental hepg2, consistent with the longer doubling time of both cells. semiquantitative rt/pcr analysis demonstrated a greatly elevated gene expression of snail accompanied with decreased e-cadherin and increased p15ink4b in both snail-overexpressing cells. on the transcriptional level, p15ink4b promoter activity was 2.6-fold higher in s7 as compared with parental hepg2. furthermore, electrophoretic mobility of dna fragments encompassing proximal p15ink4b promoter can be retarded by incubation of nuclear extract of s7. conclusion: our results demonstrated that snail play diverse trans-regulatory roles in hepg2. notably, we suggested that snail may upregulate p15ink4b gene expression by directly activating its promoter. a. schmitt-graeff 1 , r. fischer 1 , m. grosse-perdekamp 1 , o. skalli 2 1 universityhospital freiburg , 2 louisiana state university health sciences center, shreveport background/aims: synemin is an intermediate filaments (if) protein which affects the motility of several cell types by modulating the dynamic properties of alpha-actinin and f-actin. we have previously shown that synemin is expressed in resident hepatic stellate cells (hsc) and myofibroblasts (mf) in hepatic inflammation and fibrosis. in the present study we evaluated systematically the expression of synemin in a large cohort of western european hepatocellular carcinoma (hcc). methods: single and double immunolabelin for alpha-smooth muscle actin (sma), vimentin, cd31, cd34, cd68, cea, cd10, cellular retinol-binding protein1 (crbp-1) and synemin were performed on 75 paraffin-embedded hccs and 10 controls. results: synemin-positive hscs/ mfs were a hallmark of non-neoplastic fibrotic liver tissue at the border to the neoplastic lesion but were absent from normal controls. tumour cell plates of the trabecular and pseudoglandular types of hcc were covered by scattered synemin-positive cells outlining sinusoidal structures. a subpopulation of these cells showed features of pericytes while others resembled endothelium. this pattern correlated with the degree of differentiation and was not observed in poorly differentiated hccs which generally contained rare intratumoral mfs. conclusion: the presence of synemin-positive hscs/mfs in the vicinity of hccs suggests a possible contribution of mesenchymal cells to the promotion of liver carcinogenesis. since synemin expression is linked to motility, a migration of this cell type into the tumour and a differentiation in vascular mural cells may be implicated in sinusoidal remodeling and the expansion of the neoplastic population. a.s. butt 1 , a. ahmed 1 , s. hamid 1 , w. jafri 1 , h. ali shah 1 1 aim: to estimate the prevalence of viral marker negative hcc and to compare the clinical, biochemical, histological, radiological characteristics and initial treatment response among patients with viral marker negative and viral marker positive hcc. methods: medical records of patients diagnosed to have hcc visiting aga khan university hospital, karachi during january 1998 to december 2007 were reviewed. patients were divided in to nbnc-hcc(those who have negative hbsag and anti-hcv antibody)and viral hcc(those who have positive hbsag and anti-hcv antibody)group. results: out of 433 patients 68(15.7%) had nbnc-hcc. over all mean age was 57.48± 10.9 years and 69.5% were males. the proportion of hcc detected under surveillance was significantly smaller in nbnc-hcc group(p0.02). there was no difference in distribution of age, gender, bmi, child score, bilirubin, serum albumin, prothrombin time and alfa feto protein in both groups. however, patients with viral-hcc were found to be more thrombocytopenic(52.67±86.7vs.226.15±153.9,p<0.001) and had hepatopulmonary syndrome. on liver biopsy greater proportion of moderate to poorly differentiated hcc was observed in nbnc group(19.2%vs.7.9%,p<0.001). hcc measuring 5cm in diameter(60.3%vs.41.9%, p 0.01), non -solitary hcc(p0.038) and portal thromboses(p0.01) were strongly demonstrated in nbnc-hcc group. involvement of right hepatic lobe and extra hepatic tumor spread was greater in nbnc-hcc group but that difference was not statistically significant. out of 178 patients who underwent for liver transplantation(0.5%),tace(36.7%),resection(1%),ethanol ablation(2%) and chemotherapy(2%), poorer responses were observed in nbnc-hcc group (p 0.04). conclusion: hcc secondary to nbnc-cirrhosis is not uncommon. patients with nbnc-hcc tended not to be under surveillance that leads to diagnoses at more advanced stage and poor prognosis. background: hepatocellular carcinoma is a common malignancy in asia and is related to the high prevalence of chronic viral hepatitis. we examined the clinical features, treatments and survival rates in asian americans with hcc. methods: retrospective cohort study of 278 hcc patients who presented to the ucla liver cancer center in los angeles, california, usa from september 2000 to december 2007. results: two hundred and seventeen of 278 (78%) hcc patients were male, 58% and 30% had hbv and hcv infection respectively, and 73% had cirrhosis. hcc patients detected by surveillance had smaller tumor sizes, more within the milan and ucsf criteria, lower hcc tokyo system scores and had improved 1,3,5 year overall patient and disease free survival rates compared to hcc patients who presented with symptoms (p<0.01 to p<0.0001). by multivariate analysis, independent predictors of patient survival were tumor volumes greater than 5cm (hr1.48, p=0.04), afp per unit log10 increase (hr 1.12, p=0.02), hcc tokyo score per unit increase (hr 1.3, p<0.0001), liver transplantation (hr 0.14, p<0.0001), hepatic resection (hr 0.18, p<0.0001), rfa (hr 0.17, p<0.0001), tace (hr 0.44, p=0.0006), and hepatitis b infection (hr 0.71, p=0.03). factors associated with disease free survival were age per year increase (hr 1.01, p=0.03), meld per unit increase (hr 0.97, p=0.0049), liver transplantation (hr 0.23, p<0.0001), and hepatic resection (hr 0.39, p<0.0001). conclusion: hbv and hcv infection accounts for the majority of hcc in asian americans. hcc detected by surveillance resulted in treatments which improved overall patient and disease free survival. treatment of small ( 2cm) hcc tumours can be achieved by surgical resection and complete eradication always correlates with good patient's outcome, with low local recurrence and high survival rates. indeed, surveillance program for the early detection of small hcc tumour is imperative to facilitate curative treatment, and hence better survival. discovery of new blood-based biomarkers is obligatory and vimentin is a distinct novel small hcc tumour marker herein identified using proteomics. experimental design: a total of 76 liver tissues were evaluated by 2-de analysis. differentially expressed proteins were unequivocally identified by maldi-tof/tof and validation of the best candidate from protein to gene levels. indirect elisa assay was developed to detect soluble vimentin from 152 serum samples. results: vimentin was significantly over-expressed in small hcc tumours compared to non-malignant controls and maintained expression in >2cm tumours using 2-de analysis. blind verification displayed over-expression of vimentin in both transcripts and proteins levels. soluble vimentin was significantly detected at high level in small hcc as well as in overt hcc tumours. receiver operating characteristic analysis showed vimentin exhibited 40.91% sensitivity and 87.50% specificity in detecting small hcc at a cutoff of 245ng/ml. combined diagnostic performance of soluble vimentin and serum afp increases the detection sensitivity and specificity to 50.53% and 98.15%, respectively. conclusion: in this context, over-expression of vimentin is associated with the favourable 2cm sub-class of hcc thus may potentially be used as an effective serum-based diagnostic marker for cancer surveillance in high-risk cirrhotic patients. purpose: our recent comparative oncogenomic analysis in mouse model has identified yap (yes associated protein) as a novel oncogene in hcc. however, its clinical significance is unknown. in this study, we aimed to investigate the clinical values of yap as an independent prognostic marker in hcc. experimental design: a total of 177 hcc cases with retrospective clinicopathologic and follow-up data were recruited in this study. both tumor and adjacent non-tumor tissues were examined for immunoreactivity of yap expression by immunohistochemistry. clinicopathologic features and yap expression were investigated with pearson 2 test. hcc-specific disease free survival and overall survival with yap expression were analyzed by kaplan-meier curves and log-rank test. cox regression was used to test the independence and magnitude of the effects. results: yap was found over-expressed in hcc (62.1%) with nuclear expression pattern. positive yap immunoreactivity was significantly correlated with worse tumor differentiation grade (p=0.021) and high serum alpha-fetoprotein (afp) level >400 ng/ml (p<0.001). kaplan-meier plot and cox regression showed that yap was an independent predictor for hcc-specific disease free survival (hazard ratio, 1.653; 95% ci, 1.084-2.522; p=0.02) and overall survival (hazard ratio, 2.226; 95% ci, 1.312-3.778; p=0.003). conclusions: yap expression in hcc is correlated with tumor differentiation and serum afp level. it served as an independent prognostic marker for hcc. background: integrative analysis of global protein and mrna expression patterns could help researchers to understand cancer cell physiology without the need of any prior hypothesis. methods: we used a 2d-page approach to profile and compared the global protein expression profiles of hepatitis b virus-related hcc tissues, adjacent non-tumor liver tissues, normal liver tissues and hcc cell lines. subsequently, we established the bioinformatic tools for integrative analysis of gene expression and protein expression data. we compared the dysregulated protein list and the dysregulated gene lists obtained by meta-analysis of microarray gene expression data from 6 research centers in different countries. results: we identified 66 proteins dysregulated in hcc. hierarchical clustering analysis revealed that there was a progressive change of protein expression patterns from normal liver, adjacent non-tumor liver tissues, hcc tissue, then to hcc cell lines. according to the biological functions, the differential proteins could be classified into various groups, including heat shock protein, chaperone, kinase substrate, cell signaling, apoptosis regulation, transcription regulation, free-radical scavenger and metabolic enzyme. ontology analysis of the genes with consistent dysregulations at both mrna and protein levels identified specific pathways down-regulated during the progression of hcc. the inhibition of those pathways provides new insights in the hepatcarcinogensis and treatment strategies. results: in pre-s/surface regions, hcc patients had higher frequencies of pre-s deletions, amino acid substitutions at codon 4, 7, and 81 in pre-s1 genes, at the start codon in pre-s2 genes, and at codon 68 in surface genes. but they had a lower frequency of amino acid substitution at codon 2 in pre-s2 genes than those without hcc. in bcp/precore regions, hcc patients had higher frequencies of c or g1753, a1762/t1764, t1846, and a1899 than those without hcc. multivariate analysis showed that pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. the hbv with a complex mutation pattern (pre-s deletion, t1762/a1764, and a1899) rather than a single mutation was associated with hcc. patients with combined mutations of t1762/a1764 and pre-s deletion, t1762/a1764 and a1899, pre-s deletions and a1899, and t1762/a1764, pre-s deletions and a1899 had a 7.81, 7.7, 7.0, and 16.88 fold increased risk of hcc, respectively, compared to patients with wild-type at both or three genomic regions. conclusions: pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. combination of these viral mutations appeared increasing hcc risks. high peritumoral expression of placental growth factor in hepatocellular carcinoma is a poor factor for survival after curative resection h.x. xu 1 , x.d. zhu 1 , p.y. zhuang 1 , w.zhang 1 , h.chuan sun 1 1 background/aims: angiogenesis plays a significant role in the metastasis and recurrence of hepatocellular carcinoma (hcc). placental growth factor (plgf), which is one member of the vascular endothelial growth factor family, may have prognostic values in patients after curative resection of hcc. methods: expression of plgf was assessed by immunohistochemistry in tissue microarray containing paired peritumoral liver tissue and tumor from 105 patients underwent hepatectomy for histologically proved hcc. prognostic values of plgf and clinicopathological factors were evaluated. result: plgf staining was mainly on the cytoplasm of tumor cells or hepatocytes. the mean integrated optical densities of peritumoral and intratumoral density of plgf were 0.018±0.018 and 0.006±0.007 respectively. peritumoral plgf density was significantly higher than that in tumor (p<0.001), and this result was also validated in another cohort of 37 patients by quantitative real-time reverse transcription-pcr (p=0.007). intratumoral density of plgf was not correlated with common clinicopathological factors (eg, tnm stage, tumor size, microvascular invasion, intra-hepatic metastasis) or overall survival (os) (p=0.425) and time to recurrence (ttr) (p=0.419). however, peritumoral density of plgf, which was correlated with tumor size (p=0.003) and intrahepatic metastasis (p=0.004), was a prognostic factor for both os (p=0.015) and ttr (p=0.018). in multivariate analysis, peritumoral expression of plgf was also an independent prognostic factor for os (p=0.015, rr: 2.500 95% ci: 1.191-5.253 ) and ttr (p=0.045, rr: 2.013 95% ci: 1.014-3.996). conclusion: peritumoral expression of plgf in hcc patients is an independent risk factor for survival and recurrence, and may be a target of anti-angiogenic therapy in preventing post-operative recurrence. purpose: to further research rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection for treatment of advanced hepatocelluar carcinoma (hcc). methods: 25 cases were treated with transhepatic artery chemoembolization (tace) + radiofrequency ablation (rfa) + introportal vein chemotherapy (pvc) + percutaneous ethanol injection (pei) (four combined group) and this method was compared with 23 cases that were performed tace + pei (two combined group) . the serum level of afp was measured respectively after 2 and 6 months, ct scan and color doppler ultrasound were measured after treatment for six months. results: the serum level of afp declined in two groups after 2 months. for treatment after six months, afp in four combined groups was rose lower than two combined group (x 2 =5.06, p<0.05). ct and doppler ultrasound examination, four combined groups was superior than two combined groups to the control in tumor shrinkage (x 2 =8.29, p<0.01) and blood supply x 2 =6.81, p<0.01), relapse and mortality are also less. conclusions: rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection is a safe, effective combined method and has less complication in treatment of advanced hcc. poster exhibition -hcv poster session, hall 5b on the average, hepatitis c virus infects 1.2% of the population worldwide. in egypt, the prevalence rates reach 20% in some areas. ability of the virus to persist in about 75-85% of infected individuals is related to the virus higher mutation rate. six major hcv genotypes have been identified. genotype 4 seems to be confined to the middle east and central africa. extra hepatic syndromes have been reported in up to 1/3 of hcv patients. we aim in this study to determine the relationship between viral genotypes and specific extra hepatic haematological disease in patients with chronic hepatitis c. the study group included 60 selected patients with chronic hepatitis c having various haematological problems. we studied hepatitis c virus genotypes using rt-pcr. we found among 60 patients , 57 genotype 4 (95%) and 3 patients genotype 1a (5%).28 patients (46.66%) were diagnosed as chronic hepatitis c with associated thrombocytopenia, 16 patients (26.66%) were diagnosed mixed essential cryoglobulinemia(mec), 13 patients (21.66%) were diagnosed non-hodgkin's lymphoma, and 3 patients (5%) were aplastic anemia. positive serum cryoglobulins level was found in 20 patients (33.3%).no significant correlation was found between the level of viraemia and specific haematologic disease, biochemical liver markers or liver enzymes (p>0. 05). we did not find correlation between hcv genotype and specific extrahepatic haemological disorder in hcv infected patients. several environmental, genetic and immunological factors may contribute in disease progression. results: in the targeted area 111 shops of barbers were successfully interviewed and total 715 questionnaires were filled by both groups. the mean age were found in both groups of barbers (n=186) and clients (n=529), 28.47 years. the both groups showed that there are no any drugs which can protect us from diseases. both of the groups were not vaccinated for hepatitis b diseases. regarding the care providers the barbers replied that they prefer registered medical practitioners and the clients generally prefer the hakeems. those who knew hepatitis as liver disease, were 73 (39.2%), out of 186 barbers only 68 ( 36.6%) were knowing about hepatitis-b&c, when we enquired about routes of hbv& hcv transmission only (37%) replied correct routes of transmission in both groups. about hbv vaccination (49.7%) were aware, only (14.8%) were vaccinated against hbv. 60% barbers claimed for disinfection of instruments before shaving (88.9%) claimed for use of new blades. in the sero-surveillance the hbv found was very low and hcv became epidemic (5.7% -14.4 %) respectively. conclusion: the both groups need awareness for transmission. the use of new blade for the clients reduces the burden of hbv and hcv. the study highlights the roles of male sex, older age, and genotype 1b in the progression from chc infection to hcc. patients with higher hcv viral load potentially tend to develop hcc; however, hcc occurrence could be prevented using antiviral treatment. these two points need to be clarified further by a larger study population with longer follow-up period. an approach have recently been described that retroviral vectors encoding t cell receptor (tcr) genes are used to redirect the specificity of normal peripheral blood lymphocyte (pbl)-derived t cells to recognize the tumor antigens. the therapy in which t cells have been genetically modified with tcr genes to recognize hcv would represent a novel approach for the treatment of hcv infections and hcv-related malignancies. we have previously shown that hcv+ liver transplant patients that have received hla disparate liver allografts have hcv reactive t cells of host origin in their peripheral blood that are restricted by the donor hla molecules. initial studies indicate that the tcrs expressed by hcv reactive t cell clones from these patients have relatively high affinity for their ligands. we have cloned and expressed two tcrs which mediate recognition of the 1406-1415 and 1073-1081 epitopes from the hcv ns3 protein. the results indicate that these tcr transduced t cells can recognize the wild type epitopes, as well naturally occurring mutant variants of these epitopes. most importantly, the tcr transduced t cells could also recognize hcv+ hepatocellular carcinoma cells. these data suggest this high affinity hcv-specific tcr might have potential new immunotherapeutic implications. background: factors associated with svr in patients without an rvr remains unclear. methods: 200 hcv-1 (100 for 24 and 48 weeks, separately) and 150 hcv-2 (100 for 24 weeks, 50 for 16 weeks) patients were randomized to peginterferon-alpha-2a and ribavirin for analysis. results: multivariate analysis showed that treatment duration and a complete evr were the strongest independent factors associated with an svr. a higher svr rate and a lower relapse rate were observed in the standard regimen group than in the abbreviated group in patients who had a cevr (table 1). the best levels of viral loads in predicting cevr at week 4 were < 10 4 iu/ml (table 2) . conclusion: it was crucial to achieve a cevr with adequate treatment duration in patients who failed to achieve an rvr. our aim was to evaluate the impact of some biochemical, histological and viral factors on both evr and svr in patients with genotype 1 chronic hepatitis c (chc) treated with peginterferon plus ribavirin. patients and methods: we evaluated retrospectively 188 naïve patients with chc treated with peginterferon plus ribavirin at standard weight-based doses for 48 weeks. biopsies were assessed for inflammatory activity and fibrosis. steatosis was categorized by the proportion of hepatocytes per low-power field with fatty changes: >5%, >5-33%, 34-66%, >66%. biopsies were also assessed for stainable iron using the brissot scoring system. all patients were evaluated for metabolic syndrome (ms) using the ncep-atp iii criteria. results: evr was achieved in 115/188 pts (61.17%) while svr occurred in 82/115 (71.3%). after adjusting for sex and age, independent factors that negatively interfered with both evr and svr were: fibrosis score, steatosis, iron score, homa-ir index and viral load. after excluding the patients with ms criteria (n=32), evr was observed in 102/156 (65.4%) and svr in 82/102 (80.4%). factors that independently influenced both evr and svr were: fibrosis score, steatosis, iron score and viral load. conclusion: fibrosis, steatosis and iron scores, as well as viral load are independent parameters that can affect both evr and svr in genotype 1 chc patients, regardless the presence of ms. if ms is present, high homa-ir index can also additionally impair viral response. issue/argument: asia has rising cases of hepatitisb/c. alcohol/food-habits cause high prevalence in rural/tribal areas. lack of monitoring/follow-up complicates management. vaccines emerge as hope. clinical-trials of vaccines debated-issue. design of hepatitis-vaccine-trials in developing-countries complex ethical-issue. we focus on controversies identified in international/regional/local cme/pharma programs as vulnerability of volunteers to exploitation by foreign/local research-groups/funding-agencies. critical task is protect interests of vaccine-subjects in face of substantial-risks. determine if hepatitis-vaccine-volunteers will have access to treatment during trial. access to vaccine-trial-outcome. interaction with seniors 19 th apasl-congress from developed-countries will give voice to such burning-issues. methodology: researchers/pharmaceuticals/govt-policy planners need to develop forum to solve these problems. ngo's can play pivotal role. obligation on part of researchers to create mechanisms to offset anticipated risks of participation in controversial, risky vaccine-development. conclusion: counselling/right to withdraw from trial be made basic guideline. apart from monetary aspects unsuspected adverse reactions/deaths be properly evaluated/monitored. researchers need to evolve policy-guidelines to overcome barriers as variation in interpretation of essential ethical ideas, legal-system-differences, educational/economic-status. need to develop common consensus between research-community/pharma sector to reduce suffering of hepatitis-affected patients community. recommendations: researchers/ngos should come together at 19 th apasl-congress platform to form workgroup to settle these issues. we shall raise our this burning issue & present hepatitis-prevention-advocacy plan of our ngo graphically to apasl-2009 participants. results: 53% patients expressed that alternative-medicines-rx most important factor to cope with hepatitis. higher scores of qol (anova p < 0.001) correlated with alternative-medicines-rx. our ngo-initiative suggests that over 70% patients will need well trained specialist for home-based-care unit. conclusions: life-span/qol of hepatitis-sufferers depends on appropriate-palliative-care. ngo-personals should be trained in palliative-care-services. our data is being used for palliative care advocacy. field of spiritual/psycho-social/community support is fertile ground for further investigations. such use of complimentary indian medicinal plant extracts needs further evaluation in a large group in multicentre trial. treatment with adacolumn in patients with hepatitis c related who have undergone kidney transplantation: preliminary study g. novelli 1 1 la sapienza university introduction: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. background: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: the treatment was found to be safe without hemodynamic or infective complications. considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. m. sharaf-eldin 1 , h. el batae 1 , n. abd el-ghaffar 2 , w. rasheed 2 1 tanta faculty of medicine , egypt., 2 national research centre, cairo, egypt aim: we aimed to characterize serum cytokine levels of interleukin-1 beta (il-1 ) and interleukin -6 (il-6) in hcv infected patients & in patients with hepatocellular carcinoma (hcc) in comparison to control group and their possible use as markers of disease progression. patients and methods: sixty patients were divided into three groups: group i: twenty hcv infected patients without cirrhotic changes. group ii: twenty hcv infected patients with liver cirrhosis (lc). group iii: twenty hcv infected patients with hcc and 20 healthy subjects as control group. all patients and control group were subjected to biochemical and serological tests, anti hcv, hcv (rt-pcr) and cytokines measurements of serum il-1 & serum il-6 levels. results: showed a high statistically significant elevated serum il-6 and il-1 levels in patients with chronic hcv infection in comparison to control group. highly statistically elevated levels of il-6 and il-1 in liver cirrhosis and higher levels were found in hcc group in comparison to control group. the levels of il-6 and il-1 increased significantly in hcv infected patients as the disease progress. conclusion: serum il-1 , and il-6 levels are elevated in patients with hepatitis c-related liver diseases, especially in lc and hcc patients. their levels reflect hepatic dysfunction better than liver inflammation parameters; accordingly, we may use serum il-1 and il-6 as markers for liver disease progression in hcv-infected patients instead of invasive techniques. atsushi tanaka 1 , naoko hanawa 1 , mitsuhiko aiso 1 , yoriyuki takamori 1 , hajime takikawa 1 1 teikyo university school of medicine background and aim: pegylated interferon (peg-ifn) therapy is not indicated for many cases with hcv-related cirrhosis due to various adverse effects. however, patients with hcv cirrhosis are at high risk for development of hepatocellular carcinoma (hcc). thus we have introduced low-dose peg-ifn treatment for patients for compensated hcv cirrhosis. patients and methods: selection criteria for low dose peg-ifn is 1) compensated hcv-related cirrhosis, and 2) either the elderly (>65) or presence of thrombocytopenia (<8.0x10 6 / l). we have treated patients who met these criteria with low-dose peg-ifn, consisting of either peg-2a 90 g/1 -2w or peg-2b 0.5 g/kg/w+ribavirin 200mg/d. [results] twenty patients with compensated hcv cirrhosis (all patients genotype 1b) have been treated with low-dose peg-ifn (peg-2a:9, peg -2b+rib:11 . the age, platelet counts (x10 6 / l), and alt (iu/l) of 20 patients at baseline were 64.1±8.3, 7.8±4.6, and 90.4±68.6 respectively. all patients were well tolerated. low-dose peg-ifn has been continued 59.6±43.8 weeks on average. although viral response was not detected, biological response (br), defined as maintenance of alt within normal range, was obtained in 12 patients (12/20=60%). of note, neither development of hcc nor decompensated cirrhosis was observed in these 12 br cases. by contrast, hcc and decompensation developed in 5 and 1 patients respectively among 8 patients who failed to achieve br. conclusion: low-dose peg-ifn treatment was safe and well tolerable, and could potentially prevent hcc or decompensation in patients with liver cirrhosis when br was obtained. aims: to study the efficacy of peginterferon and ribavirin in treating chronic hepatitis c (chc) with genotype 6a in hong kong chinese. methods: to assess sustained virological response (svr) (serum hcvrna< 500iu/ml) at 6-months follow-up. results: nine patients with genotype 6a chc (included from jan2003 to dec2007) received peginterferon and ribavirin. mean age: 50 (range 28-64). mean alt before treatment: 96 iu/l (range 29-173 iu/l). seven patients had liver biopsy performed, only one showed stage 3-4 fibrosis and others showed active hepatitis without advanced fibrosis. mean serum hcv-rna: 9.17x10 5 iu/ml(range 9.0x10 3 -3.3x10 6 iu/ml). six patients had received peginterferon alfa-2b (1.5mcg/kg/week), other 3 received peginterferon alfa-2a (135mcg/week). ribavirin dosage ranged from 600mg-1000mg/day depending on body weight and baseline haemoglobin. treatment durations were 48-52 weeks in 7 patients, 24-26 weeks in 2 patients as one showed rapid virologic response at week 4 and the other was intolerant to side effect of peginterferon. eight patients had early virologic response at week 12 and one had >2log drop of hcvrna. eight patients had end-of-treatment response. eight patients (88.9%) achieved svr at end of follow-up. two patients who received only 24-26 weeks of combination therapy also achieved svr. the one who failed to achieve svr was at older age of 60 and had advanced fibrosis. conclusions: the efficacy of pegylated interferon and ribavirin in treating chinese patients with chronic hepatitis c genotype 6a can achieve high sustained virologic response rate of 88.9%. t. bharati 1,2 , p. kar 2 , a. mohammad 2 , k. mariappan 1 , j. annamalai 1 , r. introduction: hcv is a recognized cause of hcc. information on hcv genotypes in hcc are scanty in india. methods: a total of 154 hcc cases from delhi, 96 hcc cases from madurai and 246 cases of chronic hepatitis without hcc were controls in the study. rt-pcr for hcv rna and genotyping were carried out in all the cases results: in group-i, hcv rna was positive in 26.58% hcc cases in which genotype 3 was found in 57.1% genotype 1 was observed in 26.2% hcc cases. whereas 16.6% cases remained nontypable. in group-ii, hcv rna was positive in 23.95% hcc cases, with genotype 3 in 30.4% cases, genotype 1 in 52.2% cases and genotype 4 in 4.34% cases. however, 13.04% cases remained nontypable. out of the 246 control cases, 187 were ch and 59 were cirrhosis. in ch group, hcv rna was positive in 22.45% cases in which, genotype 3 was detected in 71.4% cases whereas genotype 1 was observed in 21.4% cases . however 7.1% cases remained nontypable. in cirrhosis group, hcv rna was positive in 37.2%cases. genotype 3 was found in 59.1% cases. while genotype 1 was present in 31.8% cases and 9.1% cases remained nontypable. conclusion: genotype 3 in delhi and genotype 1 in madurai were predominant. in hcc cases. our study demonstrates that no particular hcv genotypes were associated with hcc and genotype did not appear to influence the development of hcv-associated hcc. background/aims: the standard treatment for chronic hepatitis c infected with hcv genotype-1 is a combination of pegylated interferon alfa and ribavirin for a 48weeks. it is unclear if 24weeks treatment is possible for patients showing a rapid virologic response (rvr) without compromising the sustained virologic response (svr) in korea. method: between june 2005 and july 2008, among patients chronically infected with the hcv genotype-1 (hcv-1) who were treated with pegylated interferon alfa subcutaneously once weekly plus ribavirin (weight-based), 20 consecutive paients who had low pretreatment viral load ( 1.5x10 6 copies/ml) and rvr were treated for 24weeks and then followed up for 24weeks. the hcv rna was quantitatively assessed pretreatment, at 12weeks of treatment and was qualitatively assessed at 4weeks of treatment, the end of treatment (24weeks), 24weeks after end of treatment. rvr was defined as undetectable hcv rna at the 4weeks. results: baseline characteristics of patients was as followed; age (30-65years:mean 45years), bmi (21-27kg/m²:mean 23.5kg/m²), hcv rna titer (0.3-1.4×10 6 copies/ml:mean 0.5×10 6 copies/ml), alt (5-751iu/l:median 77iu/l). among the 20 patients, all patients (100%) had sustained virologic response (svr). conclusions: hcv-1 infected patients with a low baseline hcv rna concentration ( 1.5×10 6 copies/ml) who had hcv rna negative at week 4 of treatment may be treatment for 24weeks without compromising sustained virlolgic response. however, an additional trial will be needed to optimize the treatment duration. background/aims: acute hepatitis c (ahc) has a high chronicity rate of up to 50~84% if it is not treated. although the good treatment response to pegylated interferon (peg-interferon) therapy has reported, there is not definite guideline to treat of ahc in korea yet. the aim of our study was to investigate the clinical course and treatment outcome of ahc in single center of korea. methods: we performed a retrospective analysis of 35 patients who were diagnosed with ahc during the period from may 1996 to december 2007. the diagnosis of ahc was based on seroconversion to anti-hcv antibody or the clinical and biochemical diagnostic criteria satisfactory to ahc and on the presence of hcv rna in first serum sample. the spontaneous resolution was defined as loss of hcv rna in serum for 6-month in untreated group, and in treatment group, the sustained virological response (svr) was defined as a index of treatment success. results : thirteen of thirty-five patients were treated, six of thirty-five were untreated and observed clinical course, and sixteen patients were not followed up after diagnosis. in treatment group, nine of thirteen (69%) acquired svr, and two of six (33%) showed spontaneous resolution in untreated group. ten of thirteen treatment patients used conventional interferon, and another three patients used peg-interferon. conclusion : compared with untreated group, there was higher svr rate in treatment group (33% vs. 69%). so early interferon treatment in acute hepatitis c should be considered. background: this study was conducted to identify predictors of thyroid dysfunction and to determine whether virologic factors or treatment response affect thyroid dysfunction development during peginterferon (pegifn) therapy in chronic hepatitis c patients. methods: sixty chronic hepatitis c patients treated with pegifn -2a or -2b in combination with ribavirin from 1st july 2004 to 30th july 2008 were included in this study. treatment responses were evaluated and thyroid functions were assessed every 4 weeks. results: seventeen patients (28.3%) experienced thyroid dysfunction during treatment, and that occurred more frequently in women and in patients with a lower body mass index (bmi). the proportion of patients with a high viral load (a serum hcv rna titer >750,000 iu/ml) was significantly higher in the thyroid dysfunction group rather than in the euthyroid group(88.2% vs. 48.8%, p=0.005). among patients with hcv genotype 1, the rate of sustained virologic response was lower, and relapse occurred more frequently in the thyroid dysfunction group than in the euthyroid function group during pegifn-based therapy(svr, p=0.024; relapse, p=0.017). the female gender and the high viral load were independent predictors of thyroid dysfunction in multivariable analyses (female, or 9.44, p=0.009; high hcv rna titer, or 8.40, p=0.030) . conclusion: the risk of thyroid dysfunction during pegifn therapy for chronic hepatitis c was found to be higher for women and for those with a low bmi and a high viral load. background: in peginterferon alpha2b (peg-ifn 2b) and ribavirin (rbv) combination therapy for 48weeks for patients with chronic hepatitis c, it is still difficult to predict which patients will achieve sustained viral response (svr) at the completion of this therapy. aim: to predict svr and non-svr (relapse) at the end of this combination therapy by determining changes of serum hyaluronic acid (ha) levels. methods: eighteen patients were enrolled and their serum ha levels were measured before therapy, and after the 1st, 2nd, 3rd, and last trimesters during therapy. results: eleven patients achieved svr and 7 became relapsers. all patients showed higher ha levels in the 1st trimester than the pretreatment levels. in the svr group, 3 of 11 (27.2%) patients in the 2nd, 5 of 10 (50.0%) in the 3rd, and 9 of 11 (81.8%) in the last trimester showed lower ha levels than the pretreatment levels. by contrast, in the relapser group, none in the 2nd, 1 of 6 (16.7%) in the 3rd, and 1 of 7 (14.3%) (p<0.05) in the last trimester showed lower ha levels than the pretreatment levels. this study revealed that as the 48-week therapy went on, ha levels were more likely to fall below the pretreatment levels by the last trimester in patients achieving svr. however, ha levels of relapsers tended to continuously be above the pretreatment levels. conclusion: determination of changes of serum ha levels during peg-ifn 2b and rbv therapy predicts svr and non-svr at the completion of this therapy. results: the most frequent lymphomas were with high malignancy (40%), intermediate (32.5%) and low degree (27.5%). cryopathy was negative (87.5%). the presence of viral markers was performed soon as possible after the nhl diagnosis, at the same time (52.5%) or during the first year of evolution (32.5%). the prevalence of the hcv infection was 15%, comparable to the one in the control patients group (22.68%), admitted in a gastroenterological clinic. on the other hand, this prevalence is significantly increased compared to the one in the general romanian population (4.9%). the patients with nhl and hcv infection belonged especially to the low and intermediate malignancy degrees; the survival was influenced by the malignancy degree and not by the presence of hcv infection. the prevalence of hbv infection in the tested patients was 2.5%, being lower than that of hcv infection (2.5% vs. 15%, p = 0.113) but comparable to the one in the general population (2.5% vs. 6.3%, p = 0.525). conclusions: the prevalence of hcv infection in the patients having nhl was 15%, comparable to the one in the control group, but significantly increased compared to the one in the general population, leaving open the issue of a causal relationship between hcv infection and nhl. iron hepatic overload and hepatitis c d. damian 1 , m. grigorescu 1 , m.d. grigorescu 1 , t. zaharie 1 1 third medical clinic, cluj napoca, romania aim: evaluation of the prevalence and the degree of iron loading and the relationships with the clinical, biological and morphological changes. method: 212 patients with chronic hepatitis c were included, to whom we tested the blood iron level. in order to evaluate the hepatic iron accumulation we performed the perls staining, using a qualitative analysis and a semiquantitative scoring system (deugnier). results: from a total of 212 patients, 16.5% presented increased blood iron level (p = 0.000). the evaluation of the liver iron loading was performed in 54 patients, some having normal blood iron level (n = 34) and others (n= 20) increased (p = 0.007). the stainable iron was observed in 27 patients. the iron loading was usually low, the deposits were observed mostly at the sinusoid cells and the hepatocyte and less in the portal spaces, usually as a pale staining or of small, nonmerging granules. the total iron score deugnier was low. the increased blood iron correlated with the alt and ggt levels, the necroinflamatory activity and fibrosis. no correlationships between stainable iron and increased blood iron. the presence of liver iron accumulation only correlated with the fibrosis degree. conclusions: of the 212 patients to whom we tested the blood iron, 16.5% had increased levels. the perls staining was positive in 50% of the patients. the iron loading was mainly low, with a more frequent distribution in the sinusoid cells and in the hepatocytes and correlated only with the stage of fibrosis. response patients whose hcv rna became negative at 12 -16 weeks t. ide 1 , t. arinaga 1 , k. ogata 1 , i. miyajima 1 , k. kuhara 1 , r. kuwahara 1 , m. background/aim: chronic hepatitis patients whose hcv rna became negative at 4 weeks of peg-interferon/ribavirin treatment achieved excellent svr(sustained viral response) rate of almost 90%. however, in patients whose rna became negative after 20 weeks, the svr rate is very low. since many patients became rna negative at 12-16 weeks, it is important to clarify the characteristics of the patients. material and method: among 234 patients, 41 became rna negative at 12-16 weeks and the therapy completed (total 48-60 weeks). the characteristics were analyzed by using sex, age, weight, bmi, alt, gtp, hemoglobin, platelet counts, ccr, hyaluronic acid, the mutation of hcv core region (aa70, 91) and interferon sensitivity determining region, adiponectine, home-ir, rna dynamics, dose and the treatment period. result: the svr rate was 75.6%(31/41). because all 10 patients of non svr were female, we compared these 10 patients and 15 female svr. the platelets counts were low in non svr (non svr 15.0 ± 2.8 (x10 4 /mm 3 ) vs svr 20.2 ± 4.8 (p<0.05)). the mean dose of ribavirin was lower (447 ± 126 mg/day) in non svr (p<0.05) than in svr (625 ± 127). conclusion: as for the characteristics of the patients whose hcv rna became negative at 12-16 weeks but became non svr, female, low platelet count and low dose of ribavirin were important factors. in the patients who received reduced ribavirin doses, the idea to increase the ribavirin dose and to maintain it are necessary. (ex, use 400mg and 600mg alternately) pe329 background: chronic hepatitis c virus (hcv) infection poses a challenge for a growing number of infected patients who exhibit disease complications, including cirrhosis, hepatocellular carcinoma, and liver failure in china. the combination treatment of peginterferon alpha (peg-ifn alpha) plus ribavirin (rbv) is recommended as a standard care for hcv infections, which can improves hepatic markers and eradicates the virus in about 50% of patients. however, a significant number of patients do not respond to therapy or relapse following treatment discontinuation. several viral, hepatic, and patient-related factors influence response to therapy. methods: in our clinical practice, a total of 77 interferon-naïve patients (61% male; median age 47 years) with chronic hepatitis c include 11 cirrhotic patients (no genotyping) received peg-ifn alpha-2a 180 mcg/week plus rbv 900-1200mg/day for 48 weeks and follow up 24 weeks. results: show that the patients have more rvr and evr rate (54% and 90.9% respectively). while the svr (undetectable hcv-rna 24 weeks after treatment completion) rate is only 51.5% in conclusion: comparing with the data of clinical trail, the rvr, evr and eotr were higher, while svr was the same in chinese patient with chronic hepatitis c patients received the combination therapy of peg-ifn plus rbv. the reason of high relapse was still unknow. although optimal duration of retreatment and benefits and safety of maintenance therapy have not been determined, an extended duration is likely needed, even for the patients who achieved evr. s. nakamoto 1 , f. imazeki 1 , k. background/aims: recently amino acid (aa) substitutions in hepatitis c virus (hcv) core region (double wild (dw); arginine at aa 70, leucine at aa 91) were reported to be associated with sustained virological response (svr) in a combination therapy of peginterferon and ribavirin. we evaluated the viral factors influencing treatment response. methods: nucleotide sequences of core region were determined directly in 104 patients with genotype 1 and high viral load ( 100 kiu/ml) treated with peginterferon-alpha 2b and ribavirin for 48 weeks. rapid virologcal response (rvr) was defined as more than 2 log decrease of hcv-rna during the first four weeks of therapy and early virological response (evr) as that during the first 12 weeks. svr was defined as negative hcv-rna 6 months after the end of treatment and non-virological response (nvr) as less than 2 log decrease of hcv-rna during the treatment. results: dw at aa 70 and 91 was shown in 17/44 (35%) patients with rvr and in 5/44 (11%) with non-rvr (p=0.003), in 25/72 (35%) with evr and in 1/28 (3.6%) with non-evr (p=0.001), in 16/37 (43%) with svr and in 6/44 (14%) with non-svr (p=0.003), and in 1/24 (4%) with nvr and in 25/79 (32%) with non-nvr (p= 0.005). in multiple logistic regression analysis, dw was significantly associated with rvr, evr, svr and nvr. conclusions: dw at aa 70 and 91 in hcv core region was closely associated with virological response in a combination therapy of peginterferon and ribavirin. medicine and hepatology, henry dunant hospital, athens, greece, 3 biometrics, ist gmbh, mannheim, germany, 4 background: among patients with chronic hcv treated with pegylated interferon and ribavirin, the highest sustained virologic response (svr) rates are achieved in patients with a rapid virological response (rvr). here we investigate how the time taken to become hcv rna undetectable influences the probability of relapse during untreated follow-up. methods: data from 569 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180µg/week plus ribavirin 1000/1200mg/day were included in the intent-to-treat analysis. response was classified as rvr, complete early virological response (cevr) slow responder and non-evr. results: there was a correlation between the time required to become hcv rna undetectable and the relapse rate after stopping treatment. patients with an rvr had the lowest relapse rate (4%); this increased among patients with slower responses. conclusion: there was an inverse correlation between the time taken to achieve a virologic response and the probability of relapse. background: rapid virologic response (rvr; hcv rna <50iu/ml) at week 4 of treatment with pegylated interferon plus ribavirin can be used to predict the probability of achieving an svr. patients with detectable hcv rna at week 4 have a lower probability of achieving an svr than those with an rvr; further subdivision of these patients may be useful in predicting outcomes. methods: we conducted a retrospective analysis including 569 genotype 1 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180 g/week and ribavirin 1000/1200 mg/day. patients were categorized as rvr and non-rvr. those without an rvr were further subdivided into detectable but unquantifiable, 3, 2, 1 or <1 log10 drop in hcv rna. the proportion of patients with undetectable hcv-rna at week 12 and achieving an svr was calculated within each category. results: rvr and non-rvr patients had an 88% and 43% rate of svr respectively. among non-rvr patients, rates of svr depended on the categorical response at week 4: detectable but unquantifiable hcv rna, 77%; 3 log10 drop in hcv rna, 61%; 2 log10 drop, 43%; 1 log10 drop, 27%; and <1 log10drop, 13%. independent of week 4 response, undetectable hcv rna at week 12 was also highly predictive of svr. conclusions: patients achieving an rvr have high rates of svr. among patients who do not achieve an rvr a more precise prediction of svr can be achieved by considering the extent of viral load reduction at week 4 and week 12. retrospective japanese validation study of fibrotest and actitest in patients with chronic hepatitis c. n. nagata 1 , t. mine 1 1 background: fibrotest (ft) and actitest (at) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis c virus. the aim of this study was to perform a validation study the discordances between ft and at(ft/at) and liver biopsy in patients with chronic hepatitis c in japan. methods: 117 serum samples of chronic hepatitis c patients sended at -80°c at the biochemistry department of pitié salpetrière hospital were analysed between july and august 2007. ft/at components were assessed on thawed sera for 110 patients. 96 from 110 patients had liver biopsy at the moment of serum analysis. liver biopsy fibrosis and activity scores were assessed by a pathologist in japan according to metavir scoring system. for each individual test-ft/at the following statistical analysis were performed result: ft observed auroc for the diagnosis of advanced fibrosis was 0.81 and after adjustment according to the prevalence of different stages of fibrosis the auroc was 0.89. this difference could be explained by the non-homogenous distribution of different stages of fibrosis (low prevalence of extremes stages of fibrosis -f0 and f4-and high prevalence of adjacent intermediate stages -f1 and f2). the observed auroc of ft for the diagnosis of precirrhosis and cirrhosis was 0.86 and the observed auroc of at for the diagnosis of moderate to severe activity was 0.74 . conclusion: these results are similar to those observed in all independent validations worldwide. background: accurate monitoring of hcv-rna level throughout anti-hcv therapy is key factor for predicting sustained virological response (svr). real-time detection polymerase chain reaction (rtd-pcr) based methods are sensitive, have wide dynamic range of quantification and carryover contamination caused by classical pcr. aim: to compare rtd-pcr based assays; cobas ampliprep/cobas taqman (cap/ctm) and recently developed abbott realtime hcv for hcv rna quantification and measurements differences by 2 assays in different genotypes. methods: in total, 253 serum samples were used including, 135, 39, 24, 15, and 40 with genotypes 1b, 2a, 2b, 3a and 4 respectively were tested quantatively for hcv-rna by cap/ctm and abott realtime. results: good correlation between two assays as overall (r=0.96) with correlation coefficient (r) in genotypes 1b, 2a, 2b, 3a ranged between 0.99 to 0.98 and least in genotype 4 (r=0.78). mean differences between cap/ctm and abott realtime was significnat in genotypes 1b and 4. significantly hcv-rna genotype 4 underestimation by cap/ctm (4.3+0.9 log iu/ml) than abbott realtime (4.8+0.9 log iu/ml; p=0.01). in genotype 1b, significantly higher hcv-rna measurement by cap/ctm (5.7+1.7log iu/ml) than abbott realtime (5.0+1.4log iu/ml, p=0.001). two hcv genotype 4 samples showed measurement differences (cap/ctm minus abbott realtime) of -3.75 and -1.68 log iu/ml. studying genotype 4 sequences within 5 utr , target for cap/ctm rt-pcr amplification, revealed nucleotide polymorphisms at positions a107, a165, t203, a205, and a243. conclusion: different measurement efficiency by commonly used cap/ctm in different genotypes compared to abbott realtime. 6 new york, new york, usa, 7 vertex pharmaceuticals, 8 cambridge, ma, usa, 9 duke clinical research institute , 10 duke university, durham, nc, usa background: prove1 is a placebo-controlled study of 250 subjects with genotype 1 chronic hepatitis c randomized to 48 weeks of peginterferon-alfa-2a 180 ug/week (p) plus ribavirin 1000-1200 mg/d (r) (pr48, n=75), or 3 regimens of 750 mg q8h telaprevir (tvr) with pr: tvr/pr for 12wks followed by pr for 0wks (t12/pr12, n=17), 12wks (t12/pr24, n=79) or 36wks (t12/pr48, n=79). the impact of african american race (aa) and bridging fibrosis on sustained virologic response (svr) was examined. methods: subjects with cirrhosis were excluded from study. fibrosis was categorized as mild/minimal, portal, or bridging from biopsy within 2 years. itt analysis was performed. results: overall, svr was achieved by 41% of subjects in the pr48 group, 35% in t12/pr12 group, 61% in t12/pr24 group, and 68% in t12/pr48 group. subgroup analyses indicated svr was improved with tvr/pr (tvr/pr arms pooled) vs pr48 alone in aa subjects (44% (8/18) vs 11% (1/9)), and in subjects with bridging fibrosis (69% (22/32) vs. 26% (5/19)). adverse events leading to discontinuations were more frequent in the tvr/pr groups (21% vs. 10%). rashes, gastrointestinal events and anemia were more common in the t/pr arms, and rashes were more frequently severe (7% vs 1%). conclusions: tvr-based treatment for 24 or 48 weeks was associated with an increase in svr rates compared to pr48. subgroups with impaired response to standard peg-ifn/rbv therapy appeared to benefit from the addition of telaprevir. adverse events leading to discontinuation were more frequent in tvr-based regimens. background and aims: induction of type i ifns is a core issue in antiviral responses and must be tightly controlled. the protein kinase tbk1 is critically involved in virus-triggered type i ifn signaling. in previous studies, an alternatively spliced isoform of tbk1, termed tbk1s, was identified to be induced in both human and mouse cells. bound to rig-1, it is able to disrupt the interaction between rig-i and visa. this study was designed to observe the expression of tbk1s in hcv-infected patients. methods: total rna was extracted from samples of peripheral blood mononuclear cells obtained from 11 hcv patients, 9 hcv patients treated with ifn-/ribavirin and 9 healthy controls, and subjected to real -time pcr using the primer-probe sets for human tbk1s, tbk1 and ifn-genes. results: the tbk1s expression was significantly elevated in hcv-infected patients, while treatment of hcv-infected patients with ifn-/ribavirin resulted in down-regulation of tbk1s to the normal level. conclusions: the study strongly supports the idea that expression of tbk1s is correlated with hcv infection, and indicates that tbk1s may play an important role in the regulation of hcv infection.this work was supported by nsfc(30571643, 30672380, 30700702 background: infringement of iron metabolism is one of fibrosis progressing factors during diffuse liver diseases. the interrelation between the syndrome of iron overload (sio) and svr achievement is studied during chronic hcv infection treatment. methods: 68 patients with chronic hcv infection (genotyping: 1-34; 1+3-5; 3-22; 2-6; 4-2) are investigated. sio criteria: iron increase-more than 37 mkmol/l, ferritin -more than 200 mkmol/l, percent of transferriny saturation with iron (%tf) -more than 50%. results: sio revealed in 10 patients (14.7%): 5 patients -1 genotype (2 assotiative with a diabetes) and 5 patients-genotype 3 (3 -in combination with liver steatosis and obesity). venipuncture series were done up to getting ferritin referential parameter values before therapy beginning. rvr: sio -5 patients, normal metabolism -48; evr: 6 and 54, svr: 4 and 54 relatively. 5 nonresponding patients (sio) had steatosis and diabetes, hereditary hemochromatosis (c282y/h63d) is verified in 1 case. increase of ferritini values and %tf during therapy and positive hla-a3 and hla-b7 is registered in nonresponding patients. conclusions: sio in hla-a3, hla-b7 and c282y/h63d positive patients is independent predictor of nonresponse during peginterferon alpha-2a (40 kd)" ribavirin treatment. a.p. srivastava 1 , g. dogra 2 , s. sachdeva 1 , n. nigam 1 , a. chakravarty 2 1 dr. rml hospital, new delhi (india)-110001, 2 maulana azad medical college & associated hospitals, new delhi, india background: hepatitis c virus (hcv) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. genotyping and assessment of viral load in hcv patients are vital for designing therapeutic strategies. we aimed to determine the pattern of hcv genotypes and its association with viral load and biochemical profile. methods: 71 hcv rna positive patients were included in the present study attending the medical-opd and wards of dr rml hospital, a tertiary care hospital in new delhi during 2006-2008. hcv genotyping was carried out by restriction fragment length polymorphism (buoro et al 1999) followed by the type specific primers from the core region (ohno et al 1997) . viral load estimation was carried out by taqman real time pcr system using previously described method (martell et al 2000) . result: 67.6% of cases were having genotype 3 (3a, 3b, 3f & 3i) followed by genotyping 1 (1a & 1b) in 26.8% and genotype 2 in 5.6%. there was no statistical significant difference seen in the biochemical profile between the three groups of genotypes. genotype one was associated with a significantly higher viral load as compared to the genotypes three and two. parentral mode of transmission was accounted for the 68% of all the infected cases. conclusion: hcv genotypes 3 and 1 accounted for 94% of our cases. the genotype 1 is associated with higher degree of disease severity as assessed by viral load. also two unusual subtypes 3i and 3f were identified from this geographical region. a.p. srivastava 1 , g. dogra 2 , s sachdeva 1 , n. nigam 1 background: the development and resolution of an inflammatory process is regulated by a complex interplay among cytokines that have pro and anti-inflammatory effects. regulatory mechanisms that control the production of cytokines include genetic polymorphism in particular promoter/leader region. polymorphisms may directly or indirectly affect the binding of transcriptional factors, consequently increasing or decreasing the production of mrna, thus regulating cytokine production. we aimed to determine the polymorphism of tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) genes in chronic hepatitis c patients. methods: 40 hcv rna positive patients were included in the present study conducted during 2006-2008. 25 healthy controls were also included. genomic dna was extracted by using q1a amp dna blood kit protocol according to manufacture's instruction and desired fragment was amplified by using the primer's of vidigal et al 2002. result: genotyping of -308-promoter variant of tnf-alpha was performed by pcr. polymorphism in the tnf-alpha (g/g, g/a and a/a allele) was different between hcv patients and healthy controls. il-10 variants (c/t, c/c) were more frequent among hcv patients as compared to healthy controls. conclusion: genetic polymorphism analysis on il-10 promoter have indicated that distribution pattern of il-10 polymorphism was significantly different between controls and hcv patients. furthermore, polymorphism in promoter region of tnf-alpha (-308) was found, though the difference was not significant. since this is a preliminary study, we believe that our findings may stimulate further research on larger number of patients. introduction: the assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. although liver biopsy is the gold standard for fibrosis assessment, it is invasive and may have some risks, this has led to the development of non-invasive biochemical markers of liver fibrosis. fibro-test which have five parameters used for the quantitative assessment of liver fibrosis. our aim is to validate the performance of fibro-test in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 50 patients with chronic hepatitis c genotype 4. all biopsies were scored using metavir system by two independent pathologists. fibro-test was done with (biopredictive, houilles, france) for the assessment of liver fibrosis. sensitivity, specificity, ppv and npv were measured for distinguishing between different degrees of severity of fibrosis. results: patients (45 male and 4 female) age ranged 21-56 years, liver biopsy showed 4% (f0), 40% (f1), 20 % (f2), 28% (f3), 8% (f4). the efficacy of fibrotest is 63.26%, sensitivity 83.3%, specificity 53%, positive predictive value 50% and negative predictive value 85%. conclusion: fibrofast has a low performance in assessment in fibrosis in chronic hepatitis c genotype 4. introduction: liver biopsy is the reference method for assessing liver fibrosis. however, it is invasive, costly and has some limitations. european liver fibrosis (elf) markers have shown to be accurate in assessing liver fibrosis in a range of chronic liver disorders. our aim is to test the performance of elf markers in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 199 patients with chronic hepatitis c genotype 4. all biopsies were staged for fibrosis using metavir system by two independent pathologist. elf markers were done by (diagnostic & operations, england) and fibrosis scores were derived using the published elf algorithm. the area under the curve (auc) for receiver operator characteristic curves was measured along with sensitivity and specificity, positive (ppv) and negative (npv) predictive values for distinguishing between different stages. results: patients (179 male and 20 female), age was ranged 25-51 years, liver biopsy showed 2% (f0), 27% (f1), 34% (f2), 26% (f3) and 11% (f4). elf markers had no correlation with fibrosis score where r = -0.003, p = 0.963, aucs: 0.469, specificity 87.9 %, sensitivity only 9.3 %, ppv: only 6.03 %, npv: 61.5 % and efficacy 58.2%. conclusion: the performance of elf marker is low and can not be used for assessment of fibrosis in chronic hepatitis c genotype 4. background: the prove2 trial is a randomized, placebo-controlled study that assessed the safety and efficacy of 750mg q8h telaprevir (tvr) combined with 180 g/week peg -ifn alfa-2a (p) ± 1000-1200mg/day ribavirin (r) in chronic hcv genotype 1-infected treatment-naïve patients without cirrhosis. methods: overall, 323 patients received tvr + pr for 12 weeks (t12/pr12; n=82), tvr + pr for 12 weeks then pr for 12 weeks (t12/pr24; n=81), tvr + p for 12 weeks (t12/p12; n=78), or to pr for 48 weeks (pr48; n=82). primary endpoint: sustained virologic response (svr, undetectable hcv-rna 24 weeks post-treatment). results: baseline characteristics were well balanced across groups. numerically higher svr rates were observed in patients receiving t12/pr24 (69%; p=0.004 for difference vs. pr48) than t12/pr12 (60%), t12/p12 (36%) or pr48 (46%). relapse rates were lower in the t12/pr24 group (14%) than the t12/pr12 (30%), t12/p12 (48%) and pr48 (22%) groups. the relapse rate in patients receiving t12/pr24 with 4-week and 12-week undetectable hcv-rna was 7% (3/45). the aes occurring more frequently with the t/pr regimen were pruritus, rash, asthenia, nausea and anemia. in the t/pr arms, 12 patients discontinued due to rash, 2 discontinued due to pruritus, and 2 patients due to anemia. conclusion: these results showed that a telaprevir-based regimen led to significantly higher svr rates than pr, and indicate that this regimen could shorten the overall treatment duration from 48 weeks to 24 weeks for most patients infected with hcv genotype 1. a.c. cardoso 1 , c. stern 1 , r. moucari 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: this study evaluated the effect of the response (svr) to therapy on fibrosis stage, as assessed by ls, in patients with advanced fibrosis (f3) or cirrhosis (f4). methods: hcv patients with f3 or f4 who received interferon-based treatment were studded. ls was assessed after treatment (median delay of 36 months, 2-206) in patients with or without svr. correlations between ls and clinical and treatment characteristics were analyzed. results: 114 patients were included: male gender (72%), mean age (54±9 years), diabetes (26%), mean bmi (26±6 kg/m2), genotype 1 (59%). 33% had svr. ls was performed 0-3, 3-6, >6 years following treatment. by linear regression, the median of the ls was independently associated with svr (p=0.005) and diabetes (p=0.008). svr patients had lower ls (8.4 kpa; range 3.3-45) than non svr patients (15.7 kpa; range 5.3-75) (p<0.001). among the svr patients the median ls was lower when the delay between ls and the end of treatment was longer (10.9,8.8,6. 3) (p=0.02). on the opposite, among the non-svr patients the median ls was not significantly different (p=0.57). the median of liver stiffness was higher in patients with diabetes (p=0.006). bmi and dyslipidemia did not influence the median of the ls. conclusion: in patients with advanced fibrosis or cirrhosis, ls was lower in patients with svr and decreased with time while it was higher and did not decrease in non-svr patients. ls could be important for assessment of fibrosis stage during the post-treatment follow-up. to study peripheral blood and intrahepatic natural killer (nk) cells in patients with chronic hepatitis c in relation to disease activity and severity of hepatic fibrosis. patients & methods: fifteen untreated patients with histologically-proven chronic hepatitis c, and 12 matched healthy subjects. the nk cells and natural killer t (nkt) cells were identified in fresh whole blood samples using two-color flow cytometric assay as cd3 -cd56 + and cd3 + cd56 + positive cells. immunohistochemical staining of liver biopsies taken from all patients was done using monoclonal antibody against cd56 for detection of nk cells and rabbit polyclonal antibody against smooth muscle actin (sma) for identification of activated hepatic stellate cells (hscs). results: patients with chronic hepatitis c showed significant decreases in the percentages of nk cells and nkt cells in peripheral blood. a negative correlation was found between serum hcv rna levels and the percentages of peripheral blood nk cells and the intensity of intrahepatic nk cells. the percentages of circulating nk cells and nkt cells and the intensity of intrahepatic nk cells were inversely correlated with the metavir fibrosis stage and the steatosis grade, and also with the intensity of intrahepatic activated hscs. conclusion: patients with chronic hepatitis c had significant deficiency in circulating nk and nkt cells as well as in intrahepatic nk cells. this may provide a possible mechanism for the suppression of innate immunity against hcv. background: hcv infection is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. the virus is classified in six genotypes and more 50 subtypes, which are related distinct with antiviral therapies reply. in brazilian amazon, epidemiologist's studies in blood donors had pointed high frequency of genotype 1 (74%) followed by genotypes 3 (25%) and 2 (1%). however, epidemiological research in populations of risk to the infection still is scarce. aim: to determine hcv genotypic frequency in 116 blood donors, 55 patients with blood transfusions multiples, 57 patients in hemodialysis and 52 drugs users in the state of pará, brazilian amazon. methods: using real time pcr and nucleotide sequencing followed phylogenetic analysis had been gotten viral diagnosis and genotyping. results: in blood donors, hcv distribution was constituted by genotypes 1 (93.1%) and 3 (6.9%). in multitransfunded patients occurs maximum prevalence of genotype 1 (100%), probably reflect of genotype 1 specific transmission of blood donors population. on the other hand, in hemodialysis patients had been detected genotypes 1 (85.7%), 2 (3.6%) and 3 (10.7%), result of a bigger diversity of transmission routes (transfusional, interfamilial, nosocomial, etc) . in drug users occurs the biggest frequency of genotype 3 (38.1%) with prevalence of genotype 1 (61.9%), suggesting that the sharing of abuse machinery is allowing strains diffusion of genotype 3. conclusions: the genotype 1 possesses the biggest frequency in different population. moreover, through hcv genotypic frequency if it detached the contribution of transmission distinct routes indicated by previous epidemiologists researches. virol. 2007 ). we established real-time polymerase chain reaction (pcr) assays for the easy detection of these hcv mutations. methods: plasmids p-core-w, including wild type hcv core coding region (70r and 91l), and p-core-m, including mutant type hcv core (70q/h and 91m), were constructed by cloning and pcr-based mutagenesis for control vector of wild type core and that of mutant core, respectively. using serially diluted forms of these vectors, sybr green-based real-time pcr detections with mutation-specific primers were performed. results: analysis of known scalar concentrations of references indicated that the detection limits of these methods were at least 10 copies, 10 copies, 1000 copies, and 10 copies of 70-wild, 70-mutant, 91-wild, and 91-mutant, respectively. each primer could clearly distinguish the difference between p-core-w and p-core-m at the same copy numbers. concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-w versus p-core-m could be distinguished. on the other hand, for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. conclusions: this method could represent a useful alternative for the detection of genotype 1b hcv core amino acid substitutions 70 and 91 and be reliably applied for rapid screening. efficacy and tolerability of hcv treatment in asian patients according to age and genotype at a tertiary centre in western australia n. saroj 1 , n. kontorinis 1 , t. lorenzo 1 , m. marion 1 , s.l. chen 1 , w. cheng 1,2 1 royal perth hospital, 2 centre for international health, curtin introduction: race and ethnicity can influence efficacy and tolerability to treatment in hcv. the higher response rate in asians is thought to be associated with better adherence and tolerability. objectives: (1) to evaluate the adherence according to age and genotype (2) to assess the effect of age on treatment efficacy (3) background: hepatitis c virus (hcv) infection is a major health problem. there is huge regional variation in its prevalence and genotypic distribution. voluntary blood donors are thought to have somewhat lesser prevalence than the rest of the community. reliable statistics are not available for the entire country, particularly for the rural areas. it is important to know local situation and rationalize use of limited resources. methods: retrospective study of the records of patients attending the free liver clinic (flc) of our hospital located in a rural area of pakistan, and those screened for hcv infection prior to voluntary blood donation. results: patients at flc (324 out of 1638 [20%; males 65%] were found to have higher chances of being reactive for hcv antibodies as compared to voluntary blood donors (121/804 [14%]; p = 0.004; or 1.39 -95% ci = 1.11 -1.75). out of a total of 1022 hcv reactive patients, 904 (88%) were found to be positive on hcv rna testing. out of a total of 166 typeable genotypes, 125 (75%; 95% ci = 68.7 -81.9, estimated odds = 3.05) were infected with a single genotype, and only 7 patients (4%) were infected with genotype 1, either alone (n=4) or in combination with 3a. conclusions: one out of every 5 people tested in our flc is seropositive for hcv, and 14% of "healthy" voluntary blood donors have the same results. genotype 1 is very rare in our region. s.a. batool 1 , s.z. abbas 1 1 department of gastroenterology, muhammad hospital, mirpurkhas, pakistan background: hepatitis c viraus (hcv) infection is common in our region. data is not available on success rates of conventional interferon (inf) based products here. we attempted to find out the dominant genotype, and to determine the success rate of conventional inf-based treatment in eradicating hcv. methods: retrospective case series study of hcv infected patients' records treated with 14 different brands of inf. results: 320/1858 (17%) of all patients tested were positive for hcv antibodies. hcv-rna was tested by pcr for 1022 patients, of which 904 (88%) turned out to be positive. genotype type 3 was the dominant genotype -found in 118/168 (70%) patients. 101 men and 57 women were treated with various brands of inf with the same manufacturer's brand of ribavirin. the overall etr achieved was 100/142 (70%) -58/85 (68%) men and 42/57 (74%) women. 41/62 (66%) of genotype 3 achieved etr. there was no significant difference in average ages for those who achieved good etr and those who did not (39 years each). the etr achieved by different brands ranged from 48% to 91%. svr was achieved by 17/30 patients. conclusions: 17% of all people tested positive for hcv antibodies, of which about 88% had evidence of active hcv infection. etr achieved by different brands averaged 70%. this was 74% in female sex, although age did not appear to be a factor in determining a favourable etr. patients & methods: a total of 439 consecutive diabetic patients of either sex were evaluated for hcv and hbv infection by using enzyme linked immunosorbant assay (eliza-3) along with serum alt levels. on the basis of this test, the patients were divided into two groups, sero +ve and sero -ve. different variables were: age, sex, bmi, area of residence (rural or urban), type and duration of dm, smoking, literacy and alt. results: males 50.3% and females 49.7%. age ranged from 18 to 95. majority were married (98.4%), from rural area (70.4%), had type-2 dm (99.8%), normal weight (39.2%), normal alt(60.1%) and non-smokers (78.6%). seroprevalence for hcv, hbv and both were 25.1%, 1.8% and 1.6%. two groups were made, sero +ve and sero -ve. raised alt (59.2%) was significant (p<0.05) factor while all others variables were insignificant (p>0.05). conclusion: hbv and hcv infections are more prevalent in dm with increased alt levels. while hcv infection is more common than hbv in patients with dm. hepatitis c virus (hcv) envelope proteins (e1 and e2) mediate the entry of virus into host cells by binding to its cellular receptors and resulting in the fusion of the viral membrane with host cell membrane. the expression and secretion of biologically active envelope proteins in vitro have proven to be a difficult task due to the high degree of glycosylation and the existence of hydrophobic domains within these sequences. in order to obtain glycosylated, correctly-folded hcv envelop proteins in large quantities, we optimized the dna sequences of hcv envelop proteins by substituting the encoded sequence with human preferable codons and expressed them in human embryonic kidney (hek) 293 cells. both proteins were detected intracellularly, with a small portion secreted into supernatant. in order to enhance secretion, truncated forms of envelop proteins including e2 tm, e2384-661, e2484-661 were also expressed. both full-length and truncated forms of envelop proteins were glycosylated and expressed at high level. in addition, we also expressed the codon-optimized hcv receptors cd81 and claudin-1 in 293 cells. by comparing the expression level of codon-optimized sequences and the sequences that were obtained from cdna library by pcr, we found that codon-optimization enhance protein expression significantly in 293 cells. these results not only lay solid foundation for further research concerning the mechanism of hcv entry, including the optimal ph and right protein conformation for fusion, cell types that permit viral entry; but also potentiate a useful cell model for testing antiviral agents. background: prolactin (prl) is an immunoregulatory hormone secreted from lymphocytes, however, prl induction in relation to hepatitis c virus (hcv) infection has not been elucidated. methods: serum prl levels were measured in both 232 subjects of our hcv cohort study and 31 male patients of the hospital, who were chronically infected with hcv. furthermore, serum prl levels were compared in 27 male patients before and after interferon therapy. we measured expression of prl mrna level in pbmcs in 12 male patients, and also investigated prl mrna of pbmcs collected from 5 healthy men that stimulated by hcv produced by huh7.5 cells in vitro. result: serum prl levels were significantly higher in the hcv-infected subjects than in the controls (p< 0.01). they were significantly higher in hcv-infected male subjects than in the controls (p< 0.001). serum prl levels were significantly higher in male patients than in the controls (p<0.01). serum prl levels decreased significantly after interferon therapy in patients with sustained virological response to therapy (p<0.05). the levels of prl mrna in pbmcs derived from hcv-infected patients were significantly higher in 12 male patients than in the controls (p<0.001). conclusion: the high levels of prl expression are associated with hcv infection in carriers. background and objectives: hepatitis c virus (hcv) is a major cause of chronic liver hepatitis, cirrhosis, and hepatocellular carcinoma.current clinic standard therapy is interferon alpha (ifn-) combination with ribavirin, but this treatment is associated with adverse effects and often fails to induce a sustained response. until recently, development of a hcv cell culture system (hcvcc) provides a suitable tissue culture system to study the complete hcv life cycle. in this study, we tested the effect of ifn omega (ifn-)-a member of type interferon on hcv compared with ifnbased on hcv 1b replicon and hcvcc. methods: we compared ifn-and ifn-2a effects on hcv rna replication and protein expression, as measured by ribozyme protection assay and western blot. we also compared the intracellular protein level of phosphorylated signal transducer and activator of transcription 1 (p-stat1) treated with different interferon type and concentration with western blot analysis. results: hcv rna and protein level were inversely related with ifnconcentration and compared with ifn-2a, at the same concentration, the hcv rna and protein levels treated with ifn-were lower than that treated with ifn-2a p 0.05 .also based on the hcv rna analysis, ec50 of ifn-was 10 folds lower than ifn-2a. ifns increased intracellular p-stat1 level at a dose dependent manner and compared the same concentration of ifn-and ifn-2a, p-stat1 protein level was higher in ifn-treated group p 0.05 . conclusions: these results demonstrate distinct antiviral effect of ifncompared with ifn-2a and this difference maybe partly caused by the stronger stimulation of ifn receptor . outstanding antiviral activity of ifnmay be useful for developing new hcv treatment strategies. background & aim: hepatitis b virus (hbv) infection with undetectable levels of hepatitis b surface antigen (hbsag) is called an occult infection, which although has been described among subjects with chronic hepatitis c liver disease in the western world, it's prevalence and clinical significance are still ambiguous in the indian subcontinent. materials and methods: we investigated hbv-dna pcr in serum samples of 260 hbsag negative subjects with chronic hcv-related liver disease, and 70 apparently healthy volunteers negative for hbsag and anti-hcv as control. results: serum samples found positive by at least two independent pcr assays were considered hbv dna positive. hbv-dna was detected among 19 hcv-related chronic liver disease (cld) patients (7.3%), which was higher (p = 0.2) as compared with the control volunteers (4.3%). it was more frequent (37.5%) in 24 anti-hbs negative/anti-hbc positive patients than in 180 anti-hbs/anti-hbc positive (5 %, p < 0.05). hcv rna by qualitative pcr was significantly (p <0.001) higher in occult hbv compare to non-occult. hcv genotype 1b was predominantly associated with occult hbv (73%), especially among subjects with hepatocellular carcinoma (hcc) (p<0.05) as compared to non-occult hbv cases. though not significant, frequency of occult hbv infection was higher than healthy controls and hcv 1b genotype was significantly associated in patients with hcc. conclusion: this study suggests that in all hbv-endemic areas, the possibility of occult hbv in patients with hcv should be considered and hbv-dna should be performed. j. zhao 1,2 , w.d. cai 2 , l. chen 2 , y.x. gan 2 , m.l. he 1 , x.r. wang 1 1 background: besides hiv and syphilis, hepatitis c virus (hcv) is also rapidly spread among men who have sex with men (msm). this study was designed to identify the prevalence of these 3 sexual transmitted diseases in msms in shenzhen, china. methods: a cross sectional study was conducted by using time location sampling method from april to july, 2008. 831 msm participants (including 454 male sex workers) were recruited and finished guided self-administered questionnaires (or interviews if they have difficulty in reading or understanding) in 37 venue-date-time randomly selected from 48 active venues. results: results were analyzed using spss. 736 blood samples were collected for hiv, syphilis and hcv test. participated msms were between the age of 18 to 51 years (25.5±5.9) with a majority of 20-29 years (73.7%). most of them finished junior high school education (74.9%). 84.2% had high level of knowledge on modes of transmission and prevention. likewise, 56.0% msms have ever sold sex to men, 38.3% of them were self identified as gay, 35.1% as bisexual. 78.3% msms had multiple male sexual partners and 50.3% msms always used condom. 48.6% of them had sex with women in the past 6 month, and the condom use rate decline to 35.1% during both male and female sex. hiv positive rate is of 6.7% and syphilis for 18.3%, hcv is only found in 3 cases (0.4%). conclusions: a greater number of the participants have both male and female sex partners. this survey shows that hcv infection rate is still low among msms in shenzhen, although the hiv and syphilis rate is high and continuing increased in the past few years. the change of insulin sensitivity in hepatitis c patients with normal insulin sensitivity s.g. park 1 , y.k. cho 1 , j.w. lee 1 , j.w. yun 1 , h.j. kim 1 , w.k. jeon 1 , b.i. background: hepatitis c virus (hcv) infection is associated with a high prevalence of diabetes mellitus (dm). insulin resistance (ir) is known to play a crucial role in the development of dm in chronic hepatitis c (chc) patients. we prospectively investigated the change of insulin sensitivity in chc patients during 5-year period, and analyzed factors significantly associated with ir. methods: subjects consisted of 62 non-cirrhotic chc patients with normal alanine aminotransferase (alt) and normal insulin sensitivity (chc group), and healthy control group of 172 subjects matched by age, sex, body mass index and life styles. we compared initial baseline insulin sensitivity, metabolic parameters and incidence rate of ir at the end of follow up period in both groups. the change of insulin sensitivity and metabolic parameters and development of ir was analyzed, and factors associated with development of ir were evaluated. results: ir developed in 22.5% of 62 chc patients and 5.2% of 172 normal individuals (p<0.001). hcv infection per se and genotype 1 were independent risk factors of ir. initial fasting glucose 90-100 mg/dl, fasting insulin 10 uiu/ml, homa-ir 2.3-2.7 were significantly associated with development of ir in chc group. conclusions: hcv infection is independent risk factor of ir. even if chc patients with normal insulin sensitivity, careful monitoring for ir is necessary. prevalence of viral hepatitis c in latvia i. tolmane 1,2 , b. rozentale 1,2 , j. keiss 1 , f. arsa 1 1 sa infectology center of latvia, 2 riga stradin's university background and aim: viral hepatitis c (vhc) because of its prevalence and clinical course has become one of the most actual infectious diseases in the world. to date chronic hepatitis c affects over 170 million individuals worldwide. chronic vhc is a leading cause of cirrhosis and hepatocellular carcinoma. the aim of this study was to investigate how many residents of latvia, that are over 18 years of age have been exposed to vhc (anti-hcv prevalence) and how many are infected at the moment (hcv-rna prevalence). until now such research has not been performed in latvia. methods: from the register of general practitioners there were randomly selected 26 gp's from different regions of latvia, 60 persons over 18 years of age were selected out of each gp register and tested for anti-hcv with screening test (elisa). in case of positive result antibodies were confirmed with western-blot reaction and person was tested for hcv-rna (pcr). results: in total 1591 person was invited by general practitioners for the test and 1442 persons responded (response rate 90.6%). confirming test (western-blot) was positive in 34 participants and out of which hcv rna test was positive in 25 patients. conclusions: there are 2.4% of people exposed to hepatitis c virus in latvia and 1.73% are infected with hepatitis c virus, respectively, 1734 infected persons per 100 thousand individuals. genetic variation in the ikk/nf-b pathway and the live fibrosis progression in chronic hepatitis c r. sho 1 , k. ishii 1 , r. ishii 2 , h. watanabe 2 , k. sugahara 2 , y. nishise 2 , k. okumoto 2 , t. saito 2 , s. kawata 2 , a. fukao 1 1 department of public health, 2 department of gastroenterology, yamagata university faculty of medicine background/aims: i b kinase/nf-b (ikk/nf-b) signaling pathway is thought to play critical roles in liver inflammation and fibrogenesis. we carried out a haplotype-based association study to examine the contribution of common genetic variations in the genes encoding nf b inhibitor kinase alpha and beta (ikbka and ikbkb; the major components of ikk/nf-b pathway) to the progression of live fibrosis in chronic hepatitis c. methods: based upon the common single nucleotide polymorphisms (snps; minor allele frequency(maf) 0.05) and linkage disequilibrium (ld) information derived from the hapmap, we selected 5 and 3 tag snps from ikbka, and ikbkb, respectively, for genotyping. by using melting curve analysis, snps were genotyped in 217 chronic hepatitis c patients, including 80 patients with hepatocellular carcinoma. association between common genetic variations in ikbka/ikbka and platelet count (plt) was tested by both genotype-and haplotype-based approaches. results: we succeeded in genotyping a total of 8 tag snps that efficiently capture common variation across the 32 kb-block of ikbka and the 25 kb-block of ikbkb. for each of genes tested, 5 haplotypes were found in population studied. all snps were in hardy-weinberg equilibrium, but no significant association was observed between any single tag snp or haplotype and decreased plt in patients analyzed. conclusions: our data suggest that it is unlikely that polymorphisms within the ikbka and ikbkb genes are involved in the progression of live fibrosis in chronic hepatitis c. further studies on genetic variations in other nf-b-related genes in chronic hepatitis c are needed. background: hepatitis c virus infection is a major burden after liver transplantation. the effective treatment for patients who underwent liver transplantation has not been well established. management of these patients is the most challenging task. cyclophilins are essential host factors for hcv replication. we report here the efficacy of divided administration of ifn plus cyclosporine a in the treatment of chronic hepatitis c patients who failed peg-ifn or ifn combined ribavirin. patients and method: we prospectively included 59 patients (median age, 63) with genotype 1b and, failures to combination ifn plus ribavirin or combination pegylated ifn plus ribavirin. the present treatments consisted of an induction therapy, an intensified therapy and a maintenance therapy. the induction therapy comprised intravenous 1 mu ifn every 4 hours for the first 3 days, 1.5 mu ifn every 6 hours for the next 4 days and 2 mu ifn every 8 hours for the following 3 weeks, totaling 168 mu of ifn . the intensified therapy was induction therapy shortened to 2 weeks. the maintenance therapy comprised of pegylated ifn 2b and ribavirin. csa was given 4 times daily during the induction and the intensified therapies. ribavirin was given twice daily during the maintenance therapy. results: the end treatment response and sustained virological response rate of the present study were 73 % (43/59) and 59% (35/59), respectively. the relapse rate was 19 %(8/43). non-responders was 16 % (3/19). all adverse effects were completely reversible. the treatment protocol was well tolerable. conclusion: we concluded that our protocol should be effective in failures to the previous combination therapies. host factor targeting treatment will become a promising treatment option. cyclophilin targeting treatment is a promising new anti-hcv treatment k. inoue 1 , t. watanabe 1 , s. yoshiba 1 1 background: hepatitis c virus (hcv) is the most common cause of chronic liver disease. however, the efficacy of currently available treatments is limited. we recently reported the effects of combined interferon-/cyclosporin a treatment. cyclophilins are associated with hcv replication and bind cyclosporin a. which cyclophilins are closely associated with hcv replication remains controversial. in this study, several cyclophilins were found to be essential host factors for hcv replication and hcv replication was rescued by overexpression of cyclophilin a in the presence of cyclosporin a. methods: we evaluated the effect of cyclosporin a and its analogues on the replication of hcv in vitro using several types of hcv replicon. the gene expression of representative cyclophilins and pin-1 was knocked down using small interfering rna2 (sirna) to identify cyclophilins associated with hcv replication. the specificity of the effect of sirna was confirmed by western blot analysis. the effect of overexpression of cyclophilins on hcv replication in the presence of cycloporin a was also studied. results: cyclosporin a and its analogues suppressed hcv replication in a dose dependent manner. cyclophilin f, cyclophilin lc1 and cyclophilin lc2 as host factors which are closely associated with hcv replication, in addition to the previously reported cyclophilin a. knockdown of chclophilin b showed little effect on hcv rna replication. cyclophiln-dependent hcv replication varied among the three hcv replicon cell-lines used. overexpression of cyclophilin a rescued hcv replication in the presence of cyclosporin a. conclusions: these findings suggest several cyclophilins are essential host factors for hcv rna replication. thus potent cyclophilin inhibitors have the potential to be anti-hcv drugs. background/aims: hepatitis c virus (hcv) genotypes 1-6 have a worldwide distribution. types 1a and 1b are predominant in northern europe and north america, and in southern and eastern europe and japan, respectively. type 3 is endemic in south asia and is variably distributed in different countries. genotype 4 in egypt, genotype 5 in central and south america and genotype 6 is common in china, japan and south east asia. in pakistan 3a is the commonest genotype, which is associated with the most favorable outcome regarding end treatment response and sustained virological response after 24 weeks of therapy. the aim of this study is to find out hcv genotypes in newly diagnosed chronic hepatitis c patients. methods: this observational study was conducted in chronic hepatitis c patients. all patients had raised alt levels for last 06 months, had positive polymerase chain reaction (pcr) for hcv rna by real time method and liver biopsy was done in all patients under national program for prevention and control of hepatitis during year 2006 -2007. genotyping was done on roche genotyping kit. data was analyzed by spss 13.0 results: out of 164 patients, 85.9% (n=141) were genotype 3a. 6.1% (n=10) were genotype 3b. 3.0% (n=5) were genotype 1a. n= 01 had genotype 1b. 4.2% (n= 7) had mixed genotype (3a,3b/1a,1b,3a,3b). conclusion: majority (85.9%) of chronic hepatitis c patients were genotype 3a which is associated with favorable outcome after 24 weeks of interferon and ribavirin therapy and only 3.0% had genotype 1a in this cohort. s.t. zhou 1 , y. zhao 1 , f.j. zhang 1 background/aims: as human immunodeficiency virus (hiv) infected children who are receiving antiretroviral therapy (art) are living longer in china, comorbidities of hepatitis b virus (hbv) and hepatitis c virus (hcv) coinfection should be carefully considered when making management decisions. however, the coinfection rate of either hbv or hcv is unknown in hiv-infected children in china. we evaluated the seroprevalence of hbv and hcv in the china national pediatric art cohort of hiv-infected patients. methods: patients were selected from hiv infected children medically eligible for art who were enrolled into the china national pediatric art cohort since 2004. interviews, medical assessment, serology for hbsag, anti-hcv antibody, transaminase levels, and hiv serostatus and cd4 counts at baseline of patients were obtained. results: 42 of 763 hiv-infected children were hbsag seropositive (5.50%; 95%ci: 3.88%-7.12%), and 69 of 662 children were anti-hcv antibody seropositive (10.42%; 95%ci: 8.09%-12.75%). only age was associated with hbv coinfection. multivariate analysis revealed that children infected with hiv through contaminated blood or transfusion of blood products were 6.35 times more likely to be anti-hcv antibody positive than those infected with hiv through other routes. and children from central china provinces, henan, anhui, shanxi, and hubei were 2.5 times more likely to be hcv seropositive. conclusion: the high seroprevalence of hbv and hcv coinfection in hiv-infected children attending china national pediatric art cohort calls for routine screening for hepatitis viral coinfection and modification of the management of hiv-infected children in china. background: bms-790052 is a first-in class and highly selective hepatitis c virus (hcv) ns5a inhibitor with picomolar in vitro potency against genotypes 1a and 1b. in a sad study with healthy subjects, bms-790052 was safe, well-tolerated, and had a pharmacokinetic profile suggestive of once-daily dosing. methods: the objectives of this randomized, double blind, placebo-controlled, sad study were to evaluate the safety, tolerability, antiviral effect and pharmacokinetics of bms-790052 in patients with genotype 1 chronic hepatitis c (chc). treatment naïve or experienced patients were randomized to receive 1, 10, or 100 mg of bms-790052 or placebo. results: all bms-790052 single doses were well tolerated and had a safety profile similar to that of placebo. following oral administration, bms-790052 was readily absorbed with dose proportional exposures over the studied dose range. the mean terminal half-life of bms-790052 was approximately 12 hours. mean decline in hcv rna 24 hours after a single 1, 10 and 100 mg dose of bms-790052 was 1.8 log10 (range 0.18 to 3.0 log10), 3.2 log10 (range 2.9 to 4.0 log10) and 3.3 log10 (range 2.7 to 3.6 log10), respectively. the 100 mg dose resulted in a mean decline of 3.6 log10 (range 3.0 to 4.1 log10) 48 hours after dosing, which was maintained at 144 hours. conclusions: single doses of up to 100 mg of bms-790052 were safe and well tolerated in patients chronically infected with hcv genotype 1. bms-790052 produced a robust decline in hcv rna and has a pharmacokinetic profile that potentially supports once-daily dosing. background: the global infection rate of hcv is approximately 3%, and nearly 3.2% in china. only 42%-46% of patients with genotype 1b can achieve sustained virological response (svr) after antivirus therapy, nearly half of them experienced treatment failure. the study aimed to determine hcv-1b sequence evolution in patients experienced treatment failure during and after therapy, and further analyze relations between the mutations and treatment outcome. methods: 19 patients with genotype 1b accepted antiviral treatment of ifn plus ribavirin for 48 weeks, and long-term follow-up after therapy. 2 patients experienced treatment failure were further analyzed (one for relapser, another for nonresponder). sera were reserved at baseline, 12w, 48w and 4-year after therapy. hcv-rna was extracted. hcv full-length orf was amplified by rt-nested-pcr and sequencing. result: 9 of the 19 patients achieved svr (47.4%). from sequence alignments of relapser at baseline and 48w, we find that p7, ns5a and ns4a have higher mutation rate both in nucleotide and amino acid level (7.41% and 6.35%, 4.08% and 5.63%, 4.32% and 5.56%, respectively). but there is no significant difference in the alignments of 48w and 4-year after therapy, the mutation rate is lower. mutation rates of the non-responder among baseline, 12w, 48w and 4-year after therapy are very low. conclusion: antivirus effect is correlated with specific hcv sequences in chronic hepatitis c, mutations in hcv non-structure protein p7, ns5a and ns4a have important impacts on treatment outcome in ifn-based therapy. background: the results of antiviral therapy for hepatitis c (hcv) have improved recently with the use of peg-interferon (peg-ifn)/ribavirin therapy. however, age of patients are concerned because of side effects and safety. as we known, a few studies have targeted therapy in elder with chronic hcv. aim: we reviewed the results of interferon based antiviral therapy in the elderly with chronic hcv at our institution. methods: patients were defined as elderly if they were 65 years and elder who received therapy for hcv. the prescribed treatment duration, end of treatment response were mention. the data recorded included laboratory tests, adverse events (ae), dose modification, and withdrawal rate of therapy. results: 304 of chronic hcv patients treated with peg-ifn/ribavirin between nov 2004 and feb 2008. 50 patients were older than 65 years old. the mean age of the elder patients was 70.3 ± 4.8 years old. 21 were male and 29 were female. histological studies showed 18 with cirrhosis. almost all patients had experienced ae/side effects. the most common abnormalities were anemia and neutropenia. therapy was discontinued in 22% (11/50). the rate of dose modification was 46% (18/39) patients who received 24 weeks therapy. transaminases were normalized in 66% (33/50) after 24 weeks treatment and sustained in 66% (29/44) one year later. conclusion: the elder patients are more at risk of developing ae while on treatment. most patients should be discontinued or decreased dosage of medication. however, the elder patients with chronic hcv can be treated successfully. background: in the general population the incidence of interstitial lung disease is estimated to be 0.03% and has also been reported with the use of interferons. the higher reporting rate of ip in japan has created interest and warrants further investigation. methods: using both data from 12 randomized clinical trials (ex-japan) and the roche world-wide safety database (advent), the frequency of ip was estimated in patients treated with peginterferon alfa-2a ± ribavirin. ip was defined as: interstitial lung disease, alveolitis, pulmonary fibrosis, pneumonitis and pulmonary toxicity. results: one case of ip was reported among the 6180 patients included in the clinical trials (0.02%). in the advent database considering the estimated 926,000 patients with cumulative exposure to peginterferon alfa-2a (42,600 in japan and 883,400 us/row) the 228 reported cases of ip represent a rate of 0.02% with a proportional reporting ratio (prr) of 1.7 (p<0.0001). of these cases, 140 were reported in japan (prr 2.9; p<0.0001), 34 in the usa (prr 0.7; p=0.06) and 54 row (prr 1.2; p=0.11) representing reporting rates of 0.3% in japan and 0.01% in the usa and row. japanese patients with reported ip were older (66 versus 50-55 years) and were more likely to have been treated with peginterferon alfa-2a monotherapy (81% versus 21-39%). furthermore, the yearly incidence rate has remained unchanged. conclusions: the apparently higher rate of ip reported in japan may result from differences in patient demography, diagnostic criteria and treatment patterns. the overall incidence of ip remains low. background: hepatitis c virus (hcv) infection carries a significant risk for development of insulin resistance (ir) and/or diabetes (dm). recently, retinol-binding protein 4 (rbp4) has been reported as a protein contributing to ir. this study aimed to assess the different expression of serum rbp4 between chronic hcv infection (chc) patients and non-chc controls. methods: serum rbp4 was measured in 105 treatment-naïve chc patients and its correlation with the homeostasis model assessment of insulin resistance index (homa-ir), liver histology, virology and metabolic factors was investigated. patients were stratified into different stages of glucose tolerance by oral glucose tolerance test. another 100 sex-and age-matched non-chc adults served as the controls. results: the mean rbp4 level of controls tended to be higher than that of chc patients (32.46 ± 20.92 vs 25.48 ± 13.13 g/ml, p=0.07). the mean rbp4 level of 34 igt control-group subjects was 43.7 ± 24.0 g/ml, which was significantly higher than that of 34 ngt (24.4 ± 13.0 g/ml, p<0.001) and 32 dm controls (29.0 ± 19.5 g/ml, p<0.01). in contrast, the mean rbp4 level (22.2 ± 10.3 g/ml) of 32 dm/chc patients was not significantly different from that of ngt/chc (24.9 ± 10.5 g/ml, n=28) and igt /chc (28.1 ± 15.8 g/ml, n=45) patients. amongst chc patients, there was a significant decreasing linear trend of rbp4 dependent of both histological grading and staging progression, whilst a significant increment of homa-ir was found. conclusion: serum rbp4 is dysregulated in chc patients. introduction: sustained viral response (svr) in hepatitis c treatment with interferon alfa and ribavirin is affected by adherence and compliance due to severe myalgia, fatigue-anxiety and disturbed sleep. pregabalin, an orally effective gabasergic drug is not metabolized via cytochrome p450 and is used in fibromyalgia and fatigue-anxiety syndromes without hepatic toxicity.this study evaluates the addition of pregablin to standard agents in achieving svr by reducing side events. methods: thirty patients with chronic hepatitis c {mean age -46 years, male: female -2:1,genotype(g)1(n=29), g6 (n=1), fibrotic score f2-3 (n=24) and f4 (n=6), mean bmi > 29 kg/m 2 , initial viral load > 800,000 iu/ml} were randomized to pregablin100mg (n=15) or duloxetine 20mg (n=15) both orally daily with interferon alfa 2a 180mcg sq once a week and ribavirin 1200mg daily for 48 weeks. myalgia anxiety scale, modified quality of life score -evaluated at entry and tri-monthly. all were tested for rapid viral response, early viral response and end treatment viral response and svr. results: at the end of 48 weeks, in the pregablin arm, 14(97.3%) completed the therapy without interruption, one stopped due to excessive somnolence. duloxetine arm -10(66.6%) completed with interruptions, 4(22.2%) withdrew from the trial due to side events, one left the country. 9(62.2%) achieved svr in pregablin arm and 5(33.3%) with duloxetine. conclusions: pregablin may be considered with ifn and rbv for better adherence and compliance in achieving svr in treatment of chronic hepatitis c. larger randomized studies are needed to confirm the findings. in this study we extended this treatment approach to on treatment nonresponders (defined as having detectable hcv-rna after at least 24 weeks of soc). methods: so far, 5 pts. hcv-rna pos. after 24 weeks of soc (3 male, 2 female, genotype 1:4; genotype 3a:1, 3 with cirrhosis) participated in this protocol; 4 were treatment naïve pts, 1 relapser to two previous therapies (24 and 48 weeks). 20 mg/kg/d sil was given for 14 days, soc was continued. hcv-rna was quantified by taqman (roche diagnostics, usa) at monthly intervals on standard treatment and weekly after starting sil. results: all patients received at least 24 weeks of soc, at week 24 3 had a log drop < 3, two patients had detectable but unquantifiable hcv-rna (< 15 iu/ml). after 14 days of sil all 5 had undetectable hcv-rna, in one hcv-rna increased to 100 iu/ml and recived after a second course of sil. .all 5 patients are still on soc and are hcv-rna negative. conclusion: sil iv. is an effective "rescue treatment" for on treatment nonresponders to full dose of peginterferon/ribavirin combination therapy. poster exhibition -imaging modalities poster session, hall 5b background: levovist-enhanced ultrasonography using subtractions makes it possible to depict the perfusion of hyperechogenic nodules. our institution performs sonazoid-enhanced ultrasonography using a toshiba aplio80 that is set to a ps low images, as generally recommended. the resulting images, however, are difficult to evaluate the kind of staining image that is obtained from a hyperechogenic nodule. these staining images were then compared to advanced dynamic flow (adf) images of a hyperechogenic nodule recorded using levovist-enhanced ultrasonography. methods: the subjects were five nodules who had undergone sonazoid-enhanced ultrasonography. two patients had experienced a recurrence of hcc after tace, while three patients had a hyperechogenic nodule of hcc that had never been treated. one patient with hcc after tace was imaged at a ps low. the second patient with hcc after tace and the three patients with hcc showing a high echoic nodule, were imaged using adf. results: in the patients with hcc after tace, the remaining tumor was difficult to observe in both the vascular phase and the kupffer phase taken at a ps low. in the other patients, however, images taken using adf clearly showed the residual tumor. also, with regard to the findings from the perfused images obtained from the three patients with hyperechogenic nodules of hcc, the hcc was more easily detectable in the adf images than in those taken at a ps low. conclusion: hyperechogenic perfused nodules are easier to identify in images taken using adf than in images taken using ps low. y. komorizono 1 , t. shibatou 1 , k. sako 1 1 nanpuh hospital background: this study aimed to evaluate the usefulness of sonazoid enhanced radiofrequency ablation under real-time virtual sonography (rvs) guidance in a series of patients with hepatocellular carcinoma (hcc). method: twenty-five patients with a solitary hcc tumor measuring < = 2.5 cm in greatest dimension were enrolled in this study. eight patients received an initial treatment, seven also received an additional treatment for local recurrent tumors, and the remaining ten had distant recurrent tumors. all patients were easy to scan by multiple detector ct (mdct), but not by conventional ultrasound ( conclusions: the combination of the rvs system with sonazoid-enhanced us appears to have a high potential for use on patients that are difficult-to-scan by us examinations for percutaneous radiofrequency ablation. background & aims: contrast enhanced ultrasonography (ceus) with sonazoid can be expected to be useful not only for detection of tumor but also for us guided ablation therapy because kupffer imaging lasts for long time. the aim of this study is to investigate the usefulness of sonazoid in rfa for hcc. material & methods: a total of 716 hcc nodules in 316 patients admitted to receive rfa were studied. the detection ability of hcc was compared between ceus and conventional us using dynamic ct as reference standard. the effectiveness in the treatment was assessed by comparing the mean numbers of treatment session of rfa in patient treated with ceus assistance and that in historical controls matched for tumor and background conditions. results: the detection rate was 83.5% in conventional us and 93.2% in ceus (p=0.04). sixty-nine nodules in 52 patients were not detected by conventional us and detected after injection of sonazoid. the mean increase in detected tumor number with contrast enhanced us were well correlated with serum albumin level (p=0.016). ceus was not superior to conventional us in patients with low albumin level. the mean number of session was 1.33±0.45 as compared to 1.49±0.76 in the historical controls (p=0.0019). conclusions: ceus with sonazoid is useful for detection of tumor in patients with well-conserved hepatic reservoir. the decrease in the mean number of sessions compared to historical controls suggested that sonazoid is an excellent supportive agent in rfa treatment of hcc. direct measurement of peri-operative change in portal blood flow and pressure is difficult in human. in the present study, computational simulation of pre-and post-operative portal blood flow and pressure was performed using computational flow dynamic (cfd) software in patients with primary liver cancers. methods: patients with fibrotic or non-fibrotic livers were analyzed. according to preoperative md-ct, mesh models of portal branches were constructed. cfd software (fluent 6.2, fluent inc.) was employed for flow simulation. on the fluent 6.2, changes in flow dynamics in the remnant portal branches were simulated by virtual cutting of an interested portal branch. the simulation was also performed 14 days after the operation using dicom data obtained at that time. results: relative increase in blood flow in each remnant portal branch was not uniform throughout the liver in each patient. the sudden increase in portal pressure just after the virtual cutting of interested portal branch was almost normalized by day 14 in non-fibrotic liver according to the flow simulation, while the increase in fibrotic liver did not return to the pre-operative values by day 14. these results suggest that responsive dilatation of remnant portal branches and subsequent regional regeneration could normalize the sudden increase in portal pressure after surgery in non-fibrotic livers, while the mechanism is impaired in fibrotic livers. discussion: computational flow dynamic simulation is useful to analyze the differences in the peri-operative portal flow dynamics and liver regeneration between non-fibrotic and fibrotic livers. aim: to determine if roi analysis can characterize washout in hepatocellular carcinoma (hcc) better than visual analysis. methods: surgically proven hccs from a single institution were studied. the patients' gender, age, date of scan, date of surgery were recorded. 94 patients with pre-operative triphasic (n=67) and quadriphasic ct scans (n=27) were included. a representative section containing the lesion was selected for each case. the hu change between the precontrast and arterial (huabsolute hypervascularity) and the hu change between the peak attenuation and late portovenous phases (huabsolute washout) were recorded. cases were deemed positive if the hu change was more than the standard deviation (11 hu). this was compared against visual analysis to determine if our method would increase sensitivity of ct for hcc. results: the mean patient age was 63.7 years (range 19 to 84 years); there were 77 males and 17 females. the mean duration between surgery and the scan was 39.5 days (range 1 to 348 days). peak enhancement was seen in the early portal venous phase in 76.6% cases. the mean huabsolute washout was 23.8 hu (range -5 -54). roi analysis detected 86/94 cases (91.5%). this was 12.8% more than visual assessment, which detected 74/94 cases. this was statistically significant (p=0.014). conclusion: visual assessment of lesion density is subjective. quantitative measurement of lesion attenuation changes between scan phases is a simple and objective method that is more sensitive than visual assessment in determining lesion washout. background: abdominal ultrasonogram(usg) is a common available diagnostic tool to screen and follow up for hepatocellular carcinoma(hcc). but it has been reported that the specificity of ultrasonogram is high but the sensitivity of it is insufficient. we investigated the characteristics of hccs that was missed in the usg but was detected in the ct. methods: total 122 patients who were diagnosed with hcc between december, 2003 and february, 2008 , were enrolled and analysed retrospectively. all patients were performed with a usg prior to a spiral ct. the period between usg and spiral ct was limited within 1 month. we investigated age, gender, cause(hbv, hcv, alcohol), the size of hcc(the length of long diameter), stage(modified uicc), child-pugh grade, cirrhosis, tumor number, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns), and the tumor location at segement 8 as the possible related factors. results: the mean period between usg and spiral ct was 3.04±5.70 days. the diagnostic accuracy rate to hcc was 84.4%(103/122). there was no interobserver variation. in analysis of associated factors, there was no statistical significance in age, gender, cause(hbv, hcv, alcohol), stage(modified uicc), child-pugh grade, cirrhosis, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns) (p > 0.05). there was statistical correlation in the tumor size less than 2 cm, the solitary tumor and location at segement 8. (p > 0.05). conclusion: tumor size less than 2 cm, solitary lesion and location at segment 8 are significant factors to miss hccs in usg diagnosis. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 background: histopathological examination is required in the evaluation of various liver diseases for both diagnosis and prognosis. earlier blinded percutaneous liver biopsy was done commonly but now there are various studies suggesting that sonographic guided percutaneous liver biopsy could be more precise and safer. our aim was to compare the safety and diagnostic utility of sonographic guided versus blind percutaneous liver biopsy. methods: it was a retrospective single center study done between june 2003 and may 2007. trucut liver biopsy needle was used in all patients. demographic, clinical and histological characteristics between the two groups were evaluated. insufficient biopsy was defined as a sample with less than 6 portal spaces. we reviewed the type of complications and if hospitalization was required, or any mortality related to the procedure. results: out of 256 liver biopsies done in this period after excluding 16 patients we included 240 patients, 144 in group a(60%, blind approach) and 96 in group b (40%, sonographic guided approach). mean age was 38±12.4 years and male: female ratio was 1.6:1. biopsy was sufficient in 76% in group a and 94% in group b (p < 0.05). minor complications occurred in 58% in group a and 49% in group b which was not significant. major complications occurred in 2.8 % in group a and 1.2% in group b which was statistically significant. mortality was 1.2% in group a and 0.4% in group b which was statistically significant. conclusion: our study suggest that sonographic guided percutaneous liver biopsy is superior in the diagnosis of liver diseases in all aspects when compared to blind approach as it is more safe, has more diagnostic utility with significantly less complications and mortality. poster exhibition -liver fibrosis poster session, hall 5b background/aims: hmg-coa reductase inhibitors have been shown to reduce hepatic stellate cell proliferation and collagen production and decrease oxidative stress and hepatic vascular tone in cirrhotic patients. therfore, the aim of the present study was to examine whether the lipid lowering agents atorvastatin (ato) or rosuvastatin (ros) would prevent experimentally-induced acute or chronic hepatic damage in rats. methods: liver cirrhosis was induced by thioacetamide (taa, 200 mg/kg, i.p.) twice a week, for 12 weeks. acute damage was induced by two consecutive taa injections (200 mg/kg in a 24 h interval). rats were treated concurrently with taa only or taa and either ato or ros daily by nasogastric gavage. another group was treated with taa+pentoxifyline (ptx), an agent with known antifibrotic effect through a different mechanism and served as positive control. results: presented in the conclusions: the lipid lowering agents used in our study had no effect on the development of acute or chronic hepatic damage in rats or on oxidative stress induced by taa. purpose: the development of hepatic fibrosis in patients with chronic liver disease increases the risk of liver cancer. the present study was conducted to determine whether an easily performed myocardial examination technique can be applied to the assessment of hepatic fibrosis. strain rate imaging is a new method based on tissue doppler imaging (tdi). the usefulness of strain rate imaging in assessing the degree of hepatic fibrosis was evaluated. this time, it mede comparative study with fibroscan in 11 cases. methods: strain rate imaging was performed using a diagnostic ultrasound system (aplio tm , toshiba medical systems corporation, tochigi, japan) in a total of 47 subjects:25 in the chronic hepatitis group, 12 in the cirrhosis group, and 10 in the normal control group. tdi-q, the tissue doppler analysis software installed in the aplio system, was used for analysis. measurement was performed five times from the epigastrium, with the roi size set to 10 mm and the derivative pitch to 3 mm. results: (i): both scores were largely reproducible among the different laboratories. however, compared to the histological findings, the error ratio was 77% for all results calculated by fibrotest and actitest. (ii): calculated scores varied among f2 (9%), f3 (31%), f3-f4 (6%), and f4 (54%) (fibrotest), as well as a1/a2 (48%), a2 (9%), a2-a3 (5%), and a3 (38%) (actitest). results: the mean strain value was 0.156 in the chronic hepatitis group, 0.055 in the cirrhosis group, and 0.26 in the normal control group.the correlation was not thought to be fibroscan. conclusion: the results of the present study suggest that this noninvasive method permits quantitative assessment of the degree of hepatic fibrosis to be performed easily and in a short time. it is expected that the accuracy of the strain rate imaging method in determining the degree of hepatic fibrosis will be improved when it is used in combination with histological examination. conclusion: despite reproducibility of fibro-and actitest results among the six laboratories, large scale investigation (n=64) displayed increasing variability of the results depending on interlaboratory differences that were still in a quality controlled, analytically acceptable range. furthermore, calculated scores coincided with histological findings only in less than 25% of all cases. thus, the diagnostic accuracy of these tests seems low, if histology is accepted as gold standard. background: current knowledge attributes connective tissue growth factor (ctgf/ccn2) a crucial role in enhancing tgf-actions during hepatic fibrogenesis. recently, we demonstrated that caffeine leads to an upregulation of ppar in hepatocytes, thus sensitizing these cells to the well known inhibitory effect of 15-deoxy-12,14 -prostaglandin j2 (15-d-pgj2) on ctgf expression. however, upregulation of the receptor alone is not sufficient per se, its physiological ligand 15-d-pgj2 is required for exerting its inhibitory effect on ctgf synthesis. aim and methods: this study compares serum concentrations of 15-d-pgj2 in caucasian patients with fibrotic liver diseases (n=289), caucasian controls (n=136) and caucasian non-liver disease sick (n=307), as well as of chinese patients with hepatocellular carcinoma (n= 43) and chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with ppar inducing (i.e. ctgf inhibitory) drugs such as caffeine. results: presented data show that caucasian patients with ongoing hepatic fibrogenesis (mean 6.2 ± 5.9 µg/l) display impressingly higher serum concentrations of 15-d-pgj2 than healthy probands (mean 2.3 ± 1.0) and caucasian patients with non-liver disease (mean 2.7 ± 1.4 µg/l). similar results are found in chinese patients with fully developed hcc (mean 1.3 ± 0.7 µg/l) compared to chinese healthy controls (mean 0.4 ± 0.2 µg/l). we identified the predictors of tumor recurrence using cox-regression model. introduction: non-invasive, i.e. serum-based multiparametric panels of biomarkers have been proposed for the diagnostic assessment of liver fibrosis. aims/methods: (i) haptoglobin, alt, ggt, alpha 2-macroglobulin, apolipoprotein a1 and bilirubin in sera of 4 patients with histological proven fibrosis (f1-f4, a1-a3) were determined in 6 different quality-controlled laboratories. interlaboratory variations of the calculated fibrotest score for staging and actitest score for grading (both biopredictive tm ), and their error ratios compared to biopsy results were calculated. (ii) the variability of obtained fibrotest/actitest scores depending on 64 differential combinations of the allowed analyt-specific maximum/minimum permissible values as determined by the external quality control of the german association of laboratory medicine was determined and the frequency distribution of the results calculated. results: a total of 51 patients (mean age, 54.3±9.8 years; male, 78.4%) were included. median follow-up duration was 14.2 months (range, 5.6-37.3) and 20 patients (39.2%) experienced local tumor recurrence during the observational period. multivariable analyses showed that low p2/ms level (relative risk, 0.98; 95% confidence interval [ci], 0.96-0.99; p=0.035) and serum alpha-fetoprotein level >100 ng/ml (relative risk, 5.41; 95% ci, 1.59-18.18; p=0.007) were independent risk factors for tumor recurrence. patients with p2/ms level <45.0 revealed 3.79-fold (95% ci, 1.05-13.76; p=0.042) increase in the risk of recurrence after adjustment for serum alpha-fetoprotein level, as compared to those with p2/ms level >45.0. however, tumor size, child-pugh score, and hepatitis b virus dna level failed to significantly affect the time-to-recurrence. conclusion: our study suggests that lower p2/ms value, which means more severe liver fibrosis, is an independent predictor for hcc recurrence after rfa. background/aims: despite of its high prevalence, osteoporosis is an underestimated complication of liver cirrhosis. the aims of this study is to prove the prevalence of osteoporosis and osteopenia in patients with liver cirrhosis and to identify the principal risk factors associated. methods: the prevalence of osteoporosis and osteopenia was studied in patients with alcoholic or viral liver cirrhosis who were admitted to the institute of gastroenterology and hepatology, cnuh between march 2008 and september 2008. osteoporosis and osteopenia was evaluated by measuring their bone density using dual energy x-ray absorptiometry (dexa) at lumbar spine and femoral head. the variables taken into consideration were: sex, body mass index (bmi), presence of cholestasis, severity and duration of liver disease. results: total 45 patients (male 32 and female 13, respectively) were estimated for association of liver disease and osteoporosis. of these, 39 patients were estimated for bone density of lumbar spine and neck of femur by dual x-ray absorptiometry (dexa). morning blood samples were taken for hormonal and biochemical analysis from all patients. among 39 patients, 25 patients (64%) were found to have osteopenia or osteoporosis. there was no statistically significant correlation between age, bmi, severity and duration of liver disease, pth, vitamin d, alp and igf-1. conclusion: there is high prevalence rate of osteopenia or osteoporosis in liver cirrhosis. although the causes of osteopathy are heterogeneous, the early diagnosis and treatment of osteopathy in patients with liver cirrhosis is important. background: to build and to evaluate mathematical models for predicting liver fibrosis progression by using conventional laboratory indicators in chronic hepatitis b. methods: liver biopsy and routine laboratory tests were performed in 391 patients with chronic hepatitis b. using multiple logistic regression to analyze evidently relevant indicators, then the predicting models were built and analyzed by roc curve. results: after spearman analysis, factors such as age, platelet count(plt), international rate(inr), total bilirubin(tbil), albumin(alb), aspartate aminotransferase (ast), gamma glutamyltranspeptidase (ggt), total bile acid(tba) and cholinesterase(che) were found to be correlated with liver fibrosis p 0.01 . three models (s 2, s 3, s=4, respectively) were built by plt, inr, alb, ggt and che, which were independent predictors after multiple logistic regression analysis.finally, fibrosis score (fs) was calculated to predict different liver fibrosis stages. roc curve analysis revealed that the auc of fs was 0.784 in model1 (s 2), 0.768 in model2 (s 3) and 0.806 in model3(s=4) fig1 .the cut-off fs in model1 was at 7.09 with 67.4% sensitive, 79.3% specificity and the accuracy was 71.1%. the cut-off fs in model2 was at 5.67 with 75.0% sensitive, 67.7% specificity and the accuracy was 72.9%. the cut-off fs in model3 was at 3.65 with 71.4% sensitive, 78.5% specificity and the accuracy was 73.7%. conclusions: the predicting models, built by using conventional laboratory indicators, have fairly well value for diagnosing hepatic fibrosis or hepatocirrhosis in chronic hepatitis b. background: to investigate the effect of liver cirrhosis on the development of atherosclerosis in the rabbits chronically fed with high fat diet. methods: normal male new zealand white rabbits were randomly divided into four groups: a control group, a high fat diet group, a carbon tetrachloride (ccl4) group and a complex group. pathologic changes in ascending aortas and livers were observed. the levels of serum alanine aminotransferase alt , lipid, c-reactive protein (crp) were also determined. results: significant hepatic steatosis, inflammation and fibrosis could be observed in the three treatment groups; while atherosclerosis and typical arteriosclerotic plaques in ascending aortas could only be observed in the two high fat diet groups. compared with the control group, serum alt and lipid levels in ccl4 group were increased significantly (p<0.05), but no difference of arterial intima-media thickness (imt) and i/m ratio between these two groups. the levels of serum alt, lipid, crp and imt in two high fat diet groups were significantly increased compared with the control group (p<0.05). the level of serum alt in the complex group was significant higer than that in the high fat diet group, but the i/m ratio was just opposite (all p<0.05), and there was no difference of imt between the two groups. conclusions rabbits treated with ccl4 can elevate serum lipid levels, but can not induce atherosclerosis. though the activity of liver inflammation was aggravated in the complex model group, it has no effect on atherosclerosis possibly partly because of malnutrition. higher values of liver stiffness in males with mild chronic hepatitis c c. stern 1 , a.c. cardoso 1 , r. moucari 1 , a.d. pumpo 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: liver stiffness (ls) measured by fibroscan (echosens) is a noninvasive method to assess liver fibrosis in patients with chronic liver diseases. we evaluated the impact of factors on ls results in mild chronic hepatitis c (chc). methods: chc patients with metavir fibrosis stage 1 at liver biopsy and a reliable ls exam were eligible. all patients had no prior antiviral treatment. the ls values were compared to clinical and biochemical data. results: 93 patients were included with the following characteristics: mean age 50 11, male gender (46%), mean bmi 23 2.7, median ls 5.8 kpa (3.2-21.8), diabetes (7%), genotype 1 (61%), metavir activity a1 (86%), a2 (10%), steatosis at biopsy 30% (92%), mean glucose 4.9 1, abnormal alt (78%), abnormal ggt (47%), homa (2 1.2). the ls values were associated wtih male gender (median 6.1 in males vs 5.2 in females) (p=0.04), bmi (p=0.03), alt (p=0.006), ggt (p=0.02) and glucose levels (p=0.04). no association was found between ls and activity stage (p=0.34) or steatosis (p=0.14). in the linear regression, the only factor independently associated with higher ls was gender (p=0.038). in men, higher ls was related to levels of alt (p=0.005), but not to necro-inflammation grade (p=0.4). in women, ls was not associated with alt levels, but with bmi (p=0.045) and ggt levels (p=0.035). conclusion: in patients with mild chc, liver stiffness values are higher in males. these results suggest that different cut-off for fibrosis stage 1 should be proposed according to gender. aims: to investigate the effects of shuanghu qinggan granule(sqg) on prevention and treatment of hepatic fibrosis induced by carbon tetrachloride in rats. methods: 60 sd rats were divided into 6 groups, normal control groupamodel groupb, sqg largec1, middlec2small dose groupsc3 and silymarin positive contrast groupd. the rats of bc1c2c3d were injected with carbon tetrachloride for 8 weeks. the rats of c1c2c3 were then administered with sqg for 8 weeks. the rats of d were then administered with silymarin for 8 weeks. results the liver structure of rats of b was severely damagedlarge amount of liver cells became obviously degeneratedand hepatic veins were clearly congested. the hepatic cells fatty degeneration and infiltration of inflammatory cells in rats of c1c2c3d reduced significantly. there was no fiber hyperplasia in liver tissues of rats of c1c2c3d. blood serum ha cp p levels in rats of b were significantly higher than those in ac1c2c3d. conclusion: sqg has remarkable therapeutic effects on rats with hepatic fibrosis induced by carbon tetrachloride, the higher the dosage of sqg was, the more effective the results would be. conclusions: none of sophisticated biomarkers had value in addition to readily available laboratory data for the prediction of significant fibrosis in hbeag positive patients. two markers out of 7 sophisticated biomarkers provide additional diagnostic information in hbeag negative patients. before new biomarkers are accepted, their superiority to routine laboratory data should be meticulously appraised. objective: to evaluate the efficiency and safety of "tinmax" hb-3 herbal compound (cpd) in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. methods: a double-blind randomized method was employed. 60 patients of hepatofibrosis or cirrhosis post hepatitis b were separated into study group ("tinmax" hb-3 group) and control group (natural vitamin group) by randomized method. the course was 52 weeks. patients visited once every 12 weeks and the last visit at 12 weeks after the cessation of treatment. part of patients had liver biopsy before and after treatment. before, during the course and at the end of therapy, clinical symptoms and physical signs were evaluated, hepatic function, and serum markers of hepatofibrosis (such as hyaluronate acid, laminin, serum type iii procollagen and collagen iv) were tested, and ultrasound evaluation was performed. results: 60 patients enrolled in the evaluation. 58 patients completed the evaluation according to the protocol. 20 patients had liver biopsy twice, 10 from the study group and 10 from the other one. at the end of therapy, the total effective rate of hepatofibrosis in histopathology is 74.13% in the study group, much higher than that of 21.95% in the control group (p<0.05). the total effective rate of serum markers of hepatofibrosis at the end of therapy in the study group was 70.10%, much higher than that of 30.24% in the control group (p<0.05). the total effective rate of non-invasion markers of hepatofibrosis at the end of therapy in the study group was 76.08%, much higher than that of 19.41% in the control group (p<0.05). the drugs of adverse event had not happened in both groups. conclusion: "tinmax" hb-3 herbal compound (cpd) is effective and safe in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. w.h. sha 1 , xiaohui zeng 1 , yuyuan li 1 1 gi department, first municipal hospital of guangzhou, guangzhou aim: to investigate the clinical value of serum indices for hepatic fibrosis in chronic liver diseases. methods: competitive radioimmunoassay was used to determine the serum level of collagen type ( c), laminin (ln) and hyaluronic acid(ha) in 193 patients with different severity degree of chronic liver diseases, and in 30 healthy subjects. results: the serum levels of c, ln, and ha in the patients with liver diseases increased to different extent, compared with those in the healthy subjects. of which the highest of c, ln, and ha were found in the patients with primary carcinoma of liver or hepatocirrhosis and the serum level of ha is highlight. the combination detection of serum c, ln, and ha is more valuable than single index. conclusion: joint detection of serum c , ln, and ha is of higher significance in clinical diagnosis and prognosis of hepatocirrhosis, and is also available for successive observation on the development of liver diseases. aims: to investigate the mechanism of fuzheng huayu decoction (fzhy) on hepatic stellate cells (hscs) activation relating to tgf-1 signal transduction pathway. methods: hscs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. at day 4 after isolation, cells were stimulated with 100pm tgf-1 for 24h, then incubated with 10% fzhy pharmacological serum or 10 m t r -i inhibitor (sb-431542) for 24h. protein expression of -sma, smad3 was assayed by immunofluorescent stain; total protein expression of -sma, t r -i, smad2/3 and nuclear expression of smad3 was analyzed by western blotting. results: fzhy pharmacological serum significantly decreased expression of -sma, t r -i, and inhibited smad3 nuclear expression and translocation in tgf-1 stimulated hscs. conclusions: fuzheng huayu decoction can prevent hscs activation through tgf-1 signaling transduction pathway in hscs, which may be the important molecular pharmacological mechanism of fuzheng huayu decoction action against liver fibrosis. background: fatty liver disease has become a health problem related to metabolic syndrome worldwide although its molecular pathogenesis has remained further studied and it is unclear whether advanced fibrosis induced by steatohepatitis will regress when diet is controlled. aim of this study is 1) to study the involvement of endoplasmic reticulum stress (er stress) in the occurrence of seatohepatitis and 2) to obtain the evidence of resolution of fibrosis by changing the diet. methods: non-alcoholic steatohepatitis with advanced fibrosis was produced in rats by giving methionine-choline-deficient diet (mcdd) for 10 weeks. methionine-choline-control diet (mccd) instead of mcdd was given for the last 2 weeks in an experimental group. fibrosis and inflammation was determined by several tissue stainings. gene expression related to fibrosis and inflammation was determined by immunoblotting and real-time pcr. expression of caspase-12, caspase-7, and glucose-regulated protein 78 was evaluated to clarify the presence of er stress aim against liver fibrosis relating to hypoxia and angiogenesis regulation. methods: the rats were divided into normal, model, sa-b and perin control group. rats in sa-b and perindopril group were administrated with sa-b and perindopril respectively. liver fibrosis was induced by ip dimethylnitrosamine (dmn) for 4w. fibrosis degree was observed by sirius red staining. col-i protein expression was analyzed by western blot; col-i , vcam-1, icam-1, hif-1 and vwf expression in liver tissue was checked by immunohistochemistry; gelatinase activities in liver tissue were detected by gelatin zymography and in situ flourescent zymography. result: compared to normal group, col-i, hif-1 , icam-1, results: 1) changing the diet from mcdd to mccd triggered the reduction in fat in hepatocytes, the decrease of inflammatory gene expression and oxidative stress, and the regression of fibrosis accompanied by the disappearance of activated stellate cells and macrophages. 2) immunohistochemistry, immunoblotting, and rt-pcr analysis all indicated the occurrence of er stress in steatohepatitis while it recovered immediately after changing the diet from mccd to mcdd. vwf protein expression and gelatinase activity in liver tissue were increased obviously in model group, while sa-b and perindopril treatment significantly decreased these protein expressions and gelatinase activity. conclusions: this simple experiment clearly shows that the changing diet from steatohepatitis-causing mcdd to mccd triggers the resolution of inflammatory and fibrotic reaction in the liver, suggesting that food intake is a very important factor for controlling the state of fat and pathology of the liver. er stress is involved in the process. background: liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver disease. under injury conditions, hepatic stellate cells (hscs) are activated to transdifferentiate into myofibroblasts, which are capable of secretion of many connective tissue elements, especially collagens i, iii, and iv. gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in asia. gypenosides are the major saponins derived from g. pentaphyllum. in previous study, gypenosides have hepatoprotective and anti-fibrotic activities in rat chronic liver injury induced by ccl4, and anti-proliferative effect in rat isolated hscs. methods: in cultured hscs model, we detected type1 procollagen protein and mrna by western blot and rt-pcr. result: we found that g. pentaphyllum inhibited type1 procollagen protein expression in 66% at 48 hours. furthermore, g. pentaphyllum also inhibited type1 procollagen 1 and 2 mrna expression in 39% and 11% respectively. in addition to transcriptional inhibition, we found that g. pentaphyllum also enhanced the degradation rate of type1 procollagen protein. base on the effect of enhancing protein degradation, we used some protease inhibitors like ca-074 me, z-fa-fmk, aebsf, tpck and tlck to identify the potential target of g. pentaphyllum. on the other hand, in the ubiquitin-proteasome system analysis, we quantified the change of some target proteins of proteasome in the presence or absence of g. pentaphyllum. conclusion: g. pentaphyllum reduced type1 procollagen protein by inhibiting transcription and enhancing protein degradation. aim: excessive oxidative stress in diabetic patients has been implicated in the pathology and complication of liver. the present study was designed to examine whether ginger has a direct hepatoprotective effect in diabetic cases. methods: wistar strain albino rats were selected for this study. the rats were divided into 4 groups: (i) control, (ii) ginger treated (200mg/kg b.w. orally, 30 days) (iii) diabetic (50 mg/kg b.w., i.p.) and (iv) diabetic + ginger treatment. the lipid metabolic profiles such as total cholesterol, triglycerides, phospholipids and lipid peroxidation as stress markers and histopathological studies were carried out to assess the damage in hepatic tissue. results: ginger treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals. diabetic rats have shown increased total cholesterol, triglycerides, phospholipids and lipid peroxidation content in hepatic tissue compared to control, indicate prevailing of oxidative stress and alterations in fatty acid metabolism in these rats. further, degenerative changes of hepatic cells in diabetic group are minimized to nearness in structure by administration of ginger as evinced by histopathological examination. conclusion: we summarize that the hypolipidemic and antioxidant compounds present in ginger may be useful in delaying the complicated effects of diabetes. this results also reveal that ginger possess hepatoprotective properties in diabetic cases. anti-fibrotic action m. naime 1 , s. ali 1 1 hamdard university rhizomes of valeriana jatamansi (family, valerianaceae) have long been used in indian subcontinent by the traditional healers for the treatment of various diseases. this study provides experimental evidence suggesting the therapeutic effect of the crude extract of rhizomes on rat liver fibrosis, and demonstrates its antiproliferative role. crude extract (50% ethanolic) at a dose level of 800 mg/kg body weight was administered to rats to study the effect on biochemical and other markers of liver fibrosis. administration of the extract for 9 weeks could bring down elevated the levels of biochemical markers of liver injury, and modulate several other biochemical responses. morphology and hisopathological examination cooroborated with the biochemical changes, and indicated partial reversal of fibrosis. dpph assay confirmed the antioxidant property of the extract, which is suggested to be due to -ionone, -sitosterol and other chemical constituents. further, treatment could restore depleted glutathione level, inhibit lipid peroxidation, and inhibited elevated xanthine oxidase activity in fibrosis. the study also reports anti-tumour promotion activity of the extract as evident by a significant decrease in [ 3 h]-thymidine incorporation by hepatic dna in extract treated rats. results suggest that v. jatamansi extract has curative effect and can partially reverse biochemical and histological changes associated with liver fibrosis. with chronic hepatitis c c. wongjitrat 1 , s. chainuvati 1 , a. manuyakorn 1 , s. aroonparkmongkol 2 , t. tanwandee 1 1 mahidol university, 2 background: leptin is a peptide hormone that mainly regulates food intake, energy expenditure and reproductive function. leptin also releases from activated hepatic stellate cells and may have a role in regulation of fibrogenesis and inflammation. in human chronically infected by hcv, the role of leptin-associated fibrosis of the liver is still unclear. there is no data in thai patients chronically infected by hcv regarding leptin level and its correlation with hepatic histology and fibrosis.the purpose of this study was to evaluate the relationship between leptin level and severity of liver fibrosis in thai patients chronically infected by hcv. methods: sixty-six patients (31 men, 35 women) with chronic hcv infection and liver biopsy was done within 3 months were enrolled. fasting blood samples were obtained and serum leptin levels were measured by elisa. bmi, blood sugar, liver function test, lipid profile, hcv rna viral load and hcv genotype were also measured and related to histological findings. results: mean serum leptin levels were significantly higher in women than in male. there was a significantly correlation between serum leptin and bmi (r = 0.469, p < 0.001). leptin levels were not associated with hepatic fibrosis (r = 0.166, p = 0.183) and necroinflammation (r = 0.203, p = 0.102). steatosis was significantly associated with severe necroinflammation (r = 0.261, p = 0.034), but not fibrosis (r = 0.22, p = 0.076). conclusions: these findings failed to demonstrate correlation of serum leptin and hepatic fibrosis in thai patients chronically infected with hcv. background and aim: liver cirrhosis is one of the leading causes of mortality in our country as well as in our region. even though deterioration of glucose metabolism and existence of insulin resistance in liver cirrhhosis has been well documented in many studies, it is still unclear how insulin resistance mechanism develops. the aim of the present study is to assess insulin resistance, cytokines and crp levels in patients with liver cirrhosis and control subjects. in additon, we aimed to investigate the relation of insulin resistance in liver cirrhosis with such parameters as age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp. material and method: a total of 79 patients with cirrhosis of different etilogy (49 male, 30 female) were included into the study. as controls, 50 (23 male and 27 female) subjects were taken. the two groups were compared with each other in terms of glucose, insulin, c-peptid, homa-ir, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. in the second part of our study, the liver cirrhosis group was divided into two subgroups: patients with homa-ir value >2.7 as insulin resistance positive, and those with homa-ir value >2.7 as insulin resistance negative. these two groups, i.e. , homa-ir positive and homa-ir negative, were compared in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. results: in liver cirrhosis group, glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp levels were determined to be significantly higher than controls. between patients with homa-ir positive and negative, however, statistically no significant difference was found in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels, but il-8 level was seen to be significantly low in patient homa-ir positive. conclusion: in patients with liver cirrhosis, the levels of glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp increase with respect to normal population. determination of increased homa-ir level in liver cirrhosis supports the view that insulin resistance develops in liver cirrhosis as reported in related studies. in the study, it was also determined that the mechanism of insulin resistance development occurs independent of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels. the determination of statistically lower level of il-8 in patients with homa-ir positive with respect to those with homa-ir negative does not indicate similarity with the studies carried out earlier. ) in patients (48.08%) than in controls(14%)in group i hla-b5 significantly increased in patients (60%)as compared to controls (14%) . in group ii hla -b5 significantly higher in patients (45.46%)than controls (14%) also hla-aw19 significantly higher (40.91%) in patients than controls (12.67%).in group iii hla-aw19 significantly increased in patients (46.67%) compared to controls.no significant association between hla antigens and cases with hbv or hcv infection. conclusion: the significantly high association of hla-aw19 and hla-b5 in patients with hepatic schistosomiasis as compared to normal controlstogether with the lack of any association with active intestinal schisto . antigens predispose to liver affection.individuals possessing hla-aw19 appear to be more prone to severeform of liver disease background: atp8b1 mutation is one of the factors that result in cholestasis and progress to chronic liver disease, but has never been reported in the mainland china before. the aim of this study was to elucidate the role of atp8b1 mutation in mainland chinese patients with progressive intrahepatic cholostasis and low ggt. methods: 24 children who presented with progressive intrahepatic cholostasis and low ggt were admitted in a tertiary pediatric hospital in eastern china. abcb11 gene was analyzed firstly to exclude bsep deficiency. afterwards, all the encoding exons and their flanking areas of atp8b1 gene were sequenced in the remaining 19 patients in whom only one or no mutations of abcb11 were found. results: 10 mutations of atp8b1 gene were found in 9 patients. i694n had been reported in taiwanese patients with pfic1, and the others were novel. p209t and ivs6+5t g were linkage and found in 4 of 9 patients, including 2 homozygote and 2 heterozygote. liver biopsy had been performed in 6 patients with atp8b1 mutations and 5 with abcb11 pe408 mutations. variety portal fibrosis was showed in 2 patients with atp8b1 mutations and 4 patients with abcb11 mutations. giant cell transformation was detected in one patient with atp8b1 mutations and 4 patients with abcb11 mutations. 1 laboratory of exercise biochemistry, taipei physical education college, jhongcheng rd., no. 101, sec.2, taipei city-11153, taiwan, roc background/aim: generation of reactive oxygen metabolites are depends on the consumption of oxygen and their cumulative effects may be different from lean to obese population. this study was designed to investigate the deleterious effects of oxidants on hepatic antioxidant defence system in lean and obese rats under hypoxic condition. methods: zucker rats lean (200±10gms) and obese (450±15gms) were divided into control and acute hypoxia groups. the acute hypoxia treatment was performed in a hypoxic chamber at 14% oxygen consumption. objectives: to compare the diagnostic value of morning urine copper to zinc (copper/zinc) ratio and 24 hour urinary copper excretion in wilson's disease (wd) children. results: in the results, acute hypoxia caused a significant (p<0.05) decrease in major antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px), glutathione reductase (gr) and glutathione (gsh) content in lean groups when compare to their controls. the decrease in the activities of all antioxidant enzymes was also noticed in obese group with hypoxia treatment. however, this decrease was not significant in case of cat, gsh-px activities and gsh content. the mda levels (lipid peroxidation marker) were higher in obese rats compare to lean rats. methods: morning urine and 24 hour urine were collected from 96 patients over three years age who were hospitalized in a tertiary pediatric liver service. each patient was re-evaluated according to wd scoring system, and was assigned to one of the three groups: wd, suspecting wd, and non-wd. 24, 6, and 64 cases were assigned to wd, suspecting wd, and non-wd respectively. urine copper and zinc concentration was determined simultaneously by using inductively coupled plasma mass spectrometry. conclusions: the higher hepatic mda values observed in obese rats indicate that accumulation of free radicals may be more in obese rats thus leads to promote the lipids oxidation. from this study it is concluded that decrease of antioxidant enzymes (except gr) with acute hypoxia treatment were more in lean group compared to with that of obese group. results: the morning urine copper/zinc ratio and 24hr urinary copper excretion correlated well (r=0.758, p < 0.001). the median of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, 24h copper excretion, and 24h zinc urinary excretion were 0.370, 0.394, 87.1 and 398.7 in wd group, and 0.051, 0.061, 24.2 and 358.9 in the non-wd group respectively. the differences of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, and 24h copper excretion were significant (z-value -6.502, -6.020 and -6.208 respectively, all p values < 0.000 chd1l is a recently discovered oncogene localized at 1q21, one of the most frequently amplified chromosomal regions in hcc. herein, by yeast-two hybrid assay, we demonstrate that the anti-apoptotic ability of chd1l is associated with its interaction with nur77, a critical member of a p53-independent apoptotic pathway. as the first cellular protein identified to bind nur77, chd1l inhibits the nucleus-to-mitochondria translocation of nur77, and subsequently hinders the release of cytochrome c and the initiation of apoptosis ( figure 1 ). further study found that c-terminal macro domain of chd1l is responsible for the interaction with nur77, and a chd1l mutant lacking residues 600-897 failed to interact with nur77 and prevent nur77-mediated apoptosis. we also find that chd1l confers cellular chemoresistance to drugs that induce apoptosis via the nur77-mediated pathway, which may lead to the identification of new therapeutic targets for hcc treatment. background/aim: accumulation of oxidative damage to proteins, lipids and mitochondria could increase with advancing of age. the current study was aimed to test the hypothesis that swimming exercise training could revert the age dependent oxidative damages in liver. methods: sprague-dawley rats of young (3 months) and old (12 months) were divided into four groups; young control (n=5), young exercise (n=5), old control (n=5) and old exercise (n=5). 90 minutes of swimming exercise was given to the exercise group for a period of two weeks. results: the estimated antioxidant enzyme activities including, superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px) and glutathione reductase (gr) were decreased with age and significantly (p<0.05) increased with exercise training. however, elevated protein carbonyls and mda levels were noticed in old animals, which indicate that old liver had greater accumulated oxidative damages. the significant drop in protein carbonyl content and increase in mitochondrial succinate dehydrogenase (sdh) activity was observed with response to swim training in old rats. conclusions: this data implied that swim exercise training could revert the oxidative damages in liver. this was also proven by enhanced antioxidant enzyme status with response to exercise training in old rats. to sum-up these results it is cleared that age induced detrimental effects to the liver might be reversed by regular swimming exercise training in old rats. results: peg10 was expressed in l02/peg10 (fig.1) . peg10 accelerated the growth of l02. after treatment with 400mm h2o2 for 24 h, the inhibitory rate of l02/peg10 cells was 32.5%; the chromosomal condensation and ladder-like dna fragmentation were not observed (fig.2) . methods: hepg2 or hepg2.2.15 were co-cultured with jurkat cells, with blocking test by adding anti-pd-1 antibody. the pd-1 expression was detected by flow cytometry (fcm); cytokines in culture supernatant in blocking groups and controls were measured by enzyme-labeled immunosorbent assay (elisa); cytotoxic test of t cells were measured by methyl thiazolyl tetrazolium (mtt). conclusions: over-expression of peg10 can significantly promot l02 proliferation and ameliorate apoptosis-inducing effects of h2o2 on l02. results: the pd-1 expression on jurkat cells was induced by hepatoma cells, the expression rate were 16.17±6.5% (by hepg2) and 17.43±6.8% (by hepg2 2.2.15), respectively. the cytokines il-2 level (202.9±53.0 pg/ml), inf-level (88.6±4.6 pg/ml) and il-10 level (63.7±13.4 pg/ml) in culture supernatant of blocking groups were significant higher than that of controls (il-2, 102.9±53 pg/ml, inf-, 39.3±4.2 pg/ml and il-10, 34.6±13.7 pg/ml, respectively. p < 0.05). the cytotoxic test (od value) was markedly higher in blocking group (0.29±0.06) than that of control group (19±0.09 p<0.05) . conclusion: the pd-1 expression on lymphocytes can be induced by hepatoma cells, and cytokines expression and cytotoxic test were recovered by blocking pd-1/pd-l1 interaction. background: hedgehog (hh) pathway is well known as a positive regulator for tissue construction( during development) and reconstruction (in adults). our aim to observe the expression change of hh pathway on rat hepatic regeneration . materials and methods: adult male sprague-dawley rats underwent approximately 70% partial hepatectomy (ph) or sham operation (so). liver specimens were collected at 2, 6, 12, 24, 36, 48, 72 , and 168h after ph or so. hedgehog expression was determined in mrna level by rt-qpcr as well as in protein levels via immunohistochemical staining and western-blotting. results: so treatment did not induce remarkable changes in hedgehog expression; however, the level of transcript for hedgehog was significantly upregulated after ph. we found sonic hedgehog(shh )and glioblastoma (gli1-3) mrna expression in the regenerating liver arrive at its peak at as early as 24 h and returned to its physiologyical level 168 h later. it is similar to the change of proteins (shh and gli1) .as seen from immunohistochemistry experiments; shh protein was expressed uniquely in regenerating hepatocytes. similarly, ph induced over expression for shh protein occurred from 12 h with a peak level at 36 h after surgery. but gli protein mainly located in nucleus and no significantly changes in the phrase of liver regeneration. conclusion: hedgehog pathway may play a role in the activation of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as ph. background: to investigate whether peg10, an imprinted gene with an active paternal but silent maternal allele, was involved in hydrogen peroxide (h2o2) induced cellular apoptosis. methods: peg10 gene was stable transfected into l02. cellular gene expression was determined by rt-pcr, western blot and immunocytochemistry. cell proliferation was analyzed by mtt. after treatment with different concentrations (50-400 mm) of h2o2, cell proliferation inhibition rate was measured by mtt. morphological changes of apoptotic cells were determined by hoechst33342 staining, dna fragmentation was observed by agarose gel electrophoresis. hua tang 1 , xiao-yan tang 1 , min liu 1 , xin li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china we determined how afp modulates the proliferation of hepatoma cells. a recombinant adenovirus expressing sirna against afp (adv-afpsirna) was created and found that it reduced expression of afp specifically in hepatoma cells, and markedly inhibited the proliferation of hepatoma cells in vitro. local treatment using adv-afpsirna caused significant repression of the growth of hepatoma derived hepg2 cells in xenograft in nude mice. knockdown of afp resulted in an obvious delay in the g1/s transition of cell cycle, but did not affect apoptosis in hepg2 cells, as analyzed by flow cytometry and tunel assay. also, differential expressions of some genes related to the cell cycle, including skp2, cyclin d1, csk and ebag9 were identified by microarray and rt-pcr in hepg2 cells and hepg2 cells with knocked down afp. these results suggest that endogenous afp is a critical determinant of the growth of hepatoma cells. hematopoietic stem cell (cd 34+) therapy can improve liver function in patients with cirrhosis. these cells can be mobilized into peripheral blood using granulocyte colony stimulating factor (gcsf). this study was undertaken to assess feasibility and safety and of gcsf induced cd 34+ cell mobilization and its impact on liver function in patients with cirrhosis. patients with liver cirrhosis (cryptogenic or alcoholic with 6m abstinence) with cpt > 7 and < 12, and splenic diameter < 17 cm were included. gcsf injection was given for 5 days (5mcg/kg/dose). baseline & day-6 cd 34+ counts in peripheral blood were done by flow cytometry. follow up was weekly for 4 weeks and then monthly. cpt was compared at baseline and 6 months. 9 patients (median age 51 y, range 33-64 y, 8 males; etiology: 6 alcohol, 3 cryptogenic; median cpt 10, range 8-12) were included. cd 34 + cell counts at baseline and day 6 were 2(0-3) and 15 (13-41) respectively (median, range). side effects were fever in 8, allergic reaction in 1 and increase in splenic size in 1 (excluded). in follow up, 3 patients died (1, 3 & 4m after therapy, 1 after olt), 1 lost to follow-up. 4 patients showed improvement in 10 -7) at 6-month follow-up. gcsf treatment is safe and yields adequate cd 34+ cells in peripheral blood. in short term it results in improvement in liver function in patients with cirrhosis pe415 molecular cloning and transcriptional analysis of kctd9 gene promoter b. pi 1 , j.s. wang 1 , m.f. han 1 , y.y. zhou 1 , x.j. liu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science and technology aim: our previous work has shown that high expression of kctd9, a potassium channel associated gene, correlated with the disease severity of patients with severe chronic hepatitis b(schb). to further understand the gene transcription and regulation, kctd9 promoter was cloned and gene transcription was studied. methods: a full length of isolated promoter and series of 5' truncated promoter of kctd9 gene was subcloned into the luciferase report vector pgl2-basic to form the promoter-report constructs. the kctd9 promoter-report construct upstream of the luciferase report gene was cotransfected with constructs expressing hbv x,c and s protein respectively or stimulated with cytokines (il-2, ifn and tnf ) in 293 t cells to investigate kctd9 gene regulation upon both viral factors and host cytokines. result: a 759bp kctd9 segment upstream of atg translation start site was evidenced to contain potential regulative domains. an important regulation site located between -268bp and -81bp upstream of atg translation start site. based upon the luciferase activity assay, il-2 was able to upregulate the transcription of kctd9 whereas there was no effect from neither hbv viral proteins nor ifn and tnf . conclusion: here we first successfully cloned the full length promoter of kctd9. il-2 significantly enhanced the transcription of kctd9, a gene which has been shown to be involved in t cell activation and disease severity of schb from our group. this work was supported by nsfc(30571643, 30672380, 30700702) 1 1 istanbul university, cerrahpasa medical school, 2 marmara university, 3 okmeydani teaching hospital, 4 background: the treatment in chronic hepatitis c virus (hcv) is not highly effective, and cost, duration, and side effects are challenging. predicting favorable factors of response to treatment would make it possible to give it only responsive patients. recent studies report more conclusive results about the role of apoptosis in inflammation and fibrosis seen in chronic viral hepatitis. hepatocyte damage in hcv is mediated by cytotoxic t-cells. apoptosis primarily developed by the interaction between fas antigen on hepatocyte and fas ligand on t-cell corresponds to a main mechanism for hepatocyte damage. methods: in this study, we aimed to detect any relationship between apoptotic markers (fas, fas ligand, fas-associated death domain, caspases 3,8, and 9, insitu apoptosis) in liver biopsy taken before the treatment and response to the treatment of interferon+ribavirin. additionally, any relationship between these parameters and the other ones predicting the response to therapy including alt level, viral load, genotype, and gender were studied. results: the study includes the patients in 4 centers managing chronic hcv infection. all parameters were studied in 180 patients. study results revealed that histological activity index is correlated with cd95 staining density, caspase 8 intensiveness, and portal and parenchymal fas ligand scores. fibrosis is also seen to be correlated with the same parameters. apoptotic parameters of the responsive cases were not significantly different from unresponsive ones. conclusion: apoptotic parameters studied in the liver tissue is associated with inflammation and fibrosis, however these parameters may not predict the response to the treatment. s. gao 1 , d. xi 1 , j.w. guo 1 , c.l. zhu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science objective: this study was designed to explore the opportunity of microrna interference technique in the inhibitory application of human fgl2, human fas and tnfr1 expression. methods: the eukaryotic expression plasmids of human fgl2, fas and tnfri genes were constructed and have successfully expressed hfgl2, hfas and htnfri protein. mirna expression plasmids of hfgl2, hfas and htnfri complimentary to the sequence responsible for hfgl2, hfas and htnfri respectively were also constructed, meanwhile an irrelevant mirna plasmid was used as control. by respective cotransfection of p-hfgl2mirna and pcdna3.1-hfgl2, p-hfasmirna and pcdna3.0-hfas, p-htnfri mirna and pcdna3.0-htnfri expression construct into 293t cells, the inhibition of hfgl2, hfas and htnfri expression were analyzed by quatitative real time pcr and western blot. results: the experiments showed the significantly inhibitory effect of p-hfgl2mirna on hfgl2, p-hfasmirna on hfas and p-htnfri mirna on htnfri expression at 48h post-transfection both at rna level and at protein level, as well in 293t cell lines the inhibitory efficiency reached as high as 89.3% for hfgl2, 87.5% for hfas and 80% for htnfri, respectively. conclusions: the study demonstrated the constructs of p-hfgl2mirna, p-hfasmirna and p-htnfri mirna successfully interfered their target genes expression in vitro, which provides the foundation for further investigation of these constructs' application in vivo and further more as a therapeutic strategy for a targeting intervention in the diseases which the gene fgl2, fas and tnfri contribute to. this work was supported by nsfc30571643, 30672380, 30700702; 2005cb522901, 2007cb512900 pe418 influence of the id2 on the anti-tumor activity of histone deacetylase inhibitor in hepatocellular carcinoma cells r. tsunedomi 1,2 , s. harada 1 , n. iizuka 1 , m. oka 1 1 dept. of digestive surgery and surgical oncol., yamaguchi univ. , 2 research fellow of the japan society for the promotion of science for young scientists background: our recent study revealed that levels of the inhibitor of dna binding/differentiation 2 (id2) were associated with the progression of hcv-related hepatocellular carcinoma (hcc) and can affect susceptibility of hcc cells to histone deacetylase (hdac) inhibitors. we here aimed to investigate how and whether id2 expression affected on the anti-tumor activity of sodium butyrate (nab), one of hdac inhibitors. methods: two hcc cell lines, hle and huh-7, were used for gene targeting experiments. the id2 over-expressing and knockdown cells were subjected to mts assay to evaluate the susceptibility to nab. time-course of the expressional change of bcl-2 and bcl-xl genes after nab administration was measured by real-time rt-pcr. result: upregulation and downregulation of id2 levels in hcc cells resulted in decreased and increased susceptibility to nab, respectively. we observed that after nab administration, the id2 expression was induced gently, the bcl-2 expression was greatly increased immediately, and the bcl-xl expression was decreased to less than half once and then recovered. these increase and recovery of the expression of anti-apoptotic genes were inhibited in the id2 knockdown cells. in the id2 overexpressing cells, the bcl-2 expression was more upregulated than mock-transfected cells. conclusion: in hcc cells, id2 influences the susceptibility to the hdac inhibitor by regulating the expression of anti-apoptotic genes caused by the hdac inhibitor stimulus. we suggest that id2 could serve as a fascinating marker predictive of response to hdac inhibitors. the role of zinc finger protein 146 in liver cancer m.m.y. waye 1,2 , t.l. yeung 1,2 , j.l. zhu 1,2 1 the chinese university of hong kong, 2 croucher laboratory for human genomics background/aims: the aim of this research project is the characterization of a krüppel zinc finger protein, zinc finger protein 146 (znf146) using hepatocellular carcinoma as a disease model. zinc finger protein 146 (znf146), also named as only zinc fingers protein (ozf), is a 33kda nuclear zinc finger protein consisting solely of ten c2h2 zinc finger motifs of the krüppel type. like most of krüppel proteins, it is assumed to be the transcription factor and involved in the regulation of gene expression. the znf146 gene is amplified in 15-25% of pancreatic carcinomas and overexpressed in more than half of the tumors including liver cancer. it is thus proposed that overexpression of the znf146 may contribute to the development or progression of hepatocellular carcinoma. methods: we used flow cytometry, microarray, green fluorescent recombinant protein, rt-pcr site-directed mutagenesis, and transfection to study the effect of expression of znf146. results: our results shown that znf146 was over-expressed in two human hcc cell lines hepg2 and hep3b. expression profiles of znf146 over-expressing shown that genes related to the p53 tumor suppressor activity or dna damage, repair response and control were deregulated upon overexpression of znf146. conclusions: znf146 is possibly involved in liver carcinogenesis by affecting dna repair and cell cycle control upon induced dna damage. background: in the present study, we reported the establishment of a real-time monitor system for directly observing the catalytic, kinetic characteristics of dnazyme 10-23 in vitro cleavage on the target rna molecules as well as for rapid, accurate, high-throughout evaluation of varied dnazymes on their counterpart rna molecules. methods: dnazyme named dz-hcv-9 specific to hepatitis c virus (hcv) orf aug were designed and synthesized. dz-hcv-mis-9 with mismatched substrate-recognition domains, dz-hcv-mut-9 with mutant catalytic domains, antisense oligonucleotide ason and nonsense oligonucleotide nson were synthesized respectively as controls. a chimeric oligonucleotide of 29nt containing both rna and dna bases was designed and synthesized as the substrate: 5' fam-gt agaccgugcaccaugagcacgaaucct-bhq 3', corresponding to the330-354 nt (underline) of hcv genome(gi: 329873) the reporter fam/bhq was incorporated at the 5' and 3' end, respectively. under simulated physiological conditions (37 ), kinetic characterization of rna-cleaving dnazyme was analyzed in a real-time way. factors that influencing dnazyme cleavage were analyzed. results: dz-hcv-9 specific to hcv orf aug could cleave target rna at a•u site, a continuous change of fluorescence intensity was monitored. while the control oligonucleotides couldn't cleave rna, there were no change of fluorescence intensity. factors that influencing dnazyme cleavage concluded different substrate-recognition domain, mg 2+ concentration and ph. conclusion: a real-time monitoring system for kinetic characterization of rna-cleaving dnazyme was successfully established in the first time. the study on the apoptosis of hepatoma cells synergeticly induced by plasmid-mediated anti-angiogenesis and immunopotentiation therapy p.y. li 1 , q. zhang 1 , y. chang 1 , j.s. lin 1 , d.a. tian 1 1 background: angiogenesis is improtant to hepatoma and decreasing of host immunity promotes the development of tumor. we want to study the effect and mechanism of apoptosis of mice implanted hepatoma cells induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. methods: mouse endostatin eukaryotic plasmid (pseces) and mouse il-12 (interleukin 12) eukaryotic plasmid (pmil-12) were extracted and purified from e. coli. h22 hepatoma cells were inoculated into the leg muscle of mice, which was divided into four groups and injected with pseces, pmil-12, pseces+pmil-12 or pcdna3.1 naked plasmid dna respectively into implantation sites repeatedly. tumor formation and its weight was evaluated. tumor microvessel density, tumor infiltrating lymphocytes and apoptosis of tumor cells were assayed by cd31 staining, he staining and tunel assay respectively. results: inoculated mice received pseces, pmil-12 injection formed tumor slowly with less microvessel density, more tumor infiltrating lymphocytes in the latter and more tumor apoptosis cells in both groups compared with pcdna3.1 injection. there were much more tumor apoptosis cells in pseces+pmil-12 group (19.9±5.5 per 400× microscope field p<0.05) than any other single plasmid injection group (400× microscope field: pseces 11.3±4.1, pmil-12 14.6±3.2, pcdna3.1 1.4±1.3). conclusion: tumor cells of implanted hepatoma in mice could be synergeticly induced to apoptosis by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocytes to infiltrate, by which mice implanted hepatoma was inhibited. (20, 40, 80, 160, 200 ng/ml) in a serum-free medium for 24 h. cell proliferation was measured by brdu incorporation analysis, untreated wb-f344 cells were taken as controls. after treatment with wnt3a (160ng/ml) for 24 h, subcellular localization and protein expression of -catenin in wb-f344 cells treated and untreated with wnt3a were examined by immunofluorescence staining and western-blot analysis. cyclind1 mrna expression was determined by semi-quantitative reverse-transcript polymerase chain reaction (rt-pcr). mrna levels of some phenotypic markers (afp, ck-19, alb) and two hepatic nuclear factors were measured by rt-pcr. expressions of ck-19 and afp protein were detected by western-blot analysis. results: wnt3a promoted proliferation of wb-f344 cells. stimulation of wb-f344 cells with recombinant wnt3a resulted in accumulation of the transcriptional activator -catenin, together with its translocation into the nuclei, and up-regulated typical wnt target gene cyclind1. after 3 d of wnt3a treatment in the absence of serum, wb-f344 cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as afp, ck-19 following activation of the canonical wnt signaling pathway. conclusion: the canonical wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells. the expression level of bid and other pro-and anti-apoptotic proteins were detected by immunoblotting. results: hbx/51 showed the most sensitive towards dox treatment, and truncated bid (tbid) was also only detected in this cell line. the level of bax was also increased in hbx/51 cells. conclusions: the carboxy-terminal of hbx may enhance the processing of bid into tbid, which may contribute to increased sensitivity of the cell towards the dox treatment. cell homeostasis were performed with concentrations of oxysterol (3x10 -5 -10 -4 m) faraway from the physiological and/or pathological one (0.2 and 2x10 -7 m). in our study, we asked the effects of oxysterols (7k and 5'6s) on hepatoma cell lines homeostasis. to this purpose we used concentrations similar to those described in physiological or pathological conditions. sub-physiological (10 -9 m) to pathological (10 -7 m) oxysterol (7k and 5'6s) concentrations were used to stimulate hepg2 cells. a surprising pro-proliferative effect of 5'6s at sub-physiological (10 -9 m) concentration was observed. this behaviour was confirmed by the synergic increase of erk1/2 levels. facs analysis revealed an early progression of cells in s phase at the lowest concentration of 5'6s, while all the remaining concentrations of the two studied oxysterols induced a weakly accumulation of cells in g2/m phase. apoptosis was absent at all concentration used, except for the highest one (10 -5 m). at this point we asked if cells didn't undergo apoptosis but acquired a senescent profile. effectively, both 7k and 5'6s, at all concentration used (except for 10 -9 m), induced cell senescence (revealed by sa-ß-gal staining and sirt1 and p21 over-expression). in conclusion the two oxysterols analyzed have different and in same case opposite effects on hepatocellular line. the main effect is surely the senescence induction, but it is important to highlight the proproliferative effects of 5'6 secosterol at low concentration. mortalin, a member of hsp70 family protein, has been shown to play an important role in hepatocellular carcinoma (hcc). it has been reported that mortalin is binding to the c-terminal of p53, which acts as a safety guard and is a commonly mutated gene in hcc. in this study mortalin was silenced by specific shrna in plc/prf/5, a hcc cell line constitutively expressing p53ser249, and normal liver cells miha, and we found that suppression of mortalin can selectively trigger the mitochondria mediated apoptosis pathway by p53 dependent way in plc cells. tunel staining positive cells were only found in the plc cells mortalin knockdown group, and apoptosis associated protein, such as p53, bax, bcl-xl, cleaved-caspase 3, have been screened by western blot after transfection. quantitative-pcr data also showed that p53 mrna level are upregulated about 2 folds in mortalin knockdown group compared with the control groups in liver tumor cells. two p53 inhibitors, pft-and pft-, which can reverse this apoptosis was applied to demonstrate p53 dependent way. in summary, knockdown mortalin can selectively kill liver cancer cells through reactive apoptosis by sensitizing mutant p53 in plc cells, but had no effect on normal cells. the clinical application of this study suggested that motalin specific shrna might be a potential anti-cancer drug for hcc. background: nafld can proceed to nash and are at risk of cirrhosis and hcc. aim was to study profile of bangladeshi nafld patients. methods: 52 patients with nafld were included. of them 59.6% were males and 40.4% females. patients were between 12-60 years of age. they presented with dull right upper abdominal ache and/or incidental detection of raised alt/ast and/or fatty liver on ultrasonography. all tested negative for hepatitis b and c. none had history of alcohol. all underwent per-cutaneous liver biopsy for histopathology. they were also tested for dm, dyslipidaemia, insulin resistance, hypothyroidism and hepatitis c. their bmi and bp were recorded. results: 88.5% had nash. 63.0% of them were males and rest 37.0% females. 11.5% had nafl. of them 50% each were males and females. majority had nash. 47.8% were obese and 41.3% had dyslipidaemia. 28.3% had hypertension, 28.3% insulin resistance and 13% were diabetics. 6.5% had hypothyroidism. none had hepatitis c. alt was raised in 72% and ast in 40%. although all patients with nash did not have elevated alt, it was raised in majority, contrary to ast, which was normal in most. conclusion: majority nash patients in bangladesh are obese. other leading causes of nash include dyslipidaemia, hypertension and insulin resistance. some nash also had diabetes and hypothyroidism. this study also reveals that elevated alt in patients with nafld is suggestive of fibrosis, although normal serum alt does not exclude nash. the study further suggests that alt is superior to ast in predicting nash. background: non-alcoholic fatty liver disease is prevalent in obese patients. liver biopsy remains the best diagnostic tool for confirmation. we tried to find out the correlations of laparoscopic parameters with histology and laboratory data. besides, we also evaluated the effectiveness of laparoscopy in liver disease diagnosis. methods: in the period of one year and five months,126 morbidly obese patients submitted to laparoscopic bariatric surgeries at our institutions were prospectively studied. results: laparoscopic parameters of significant correlations with histologic steatosis, inflammation and fibrosis were summarized in table 1 . besides, important parameters with relationships to laboratory data were summarized in table 2 department of internal medicine, seoul national university hospital gangnam healthcare center, seoul, south korea, 2 department of internal medicine and liver research institute, seoul national university college of medicine, seoul, south korea background/aims: hepatic fibrosis is associated with poor prognosis in non-alcoholic fatty liver disease (nafld). recently, many non-invasive fibrosis markers have been studied to overcome the limitations of liver biopsy. among them, bard score and guha's simple panel are easy to use in clinical practice. in this study, we evaluated the efficacy of bard score and guha's simple panel as a noninvasive fibrosis marker in korean nafld patients. methods: data from 79 patients with biopsy-proven nafld in seoul national university hospital from 2000 to 2007 were used. bard score and guha's simple panel were calculated by using clinical and biochemical data and were compared with the histological fibrosis stages. results: stage 0 fibrosis were found in 67 patients, stage 1 in 4, stage 2 in 2, stage 3 in 1 and stage 4 in 5. the relationship between fibrosis stage and bard score ( = 0.43, p < 0.001) was statistically significant. all patients with advanced fibrosis (stage 3-4) had bard score greater than 2. mean values from original guha's simple panel for no fibrosis were not different between the patients with and without fibrosis. however, after adjusting coefficients by logistic regression analysis, the differences in mean values became statistical significant (p < 0.001). conclusions: our data suggest that bard score may be effective for detecting high risk patients for advanced fibrosis, and modification of coefficients within the guha's simple panel may be needed to use as a fibrosis marker in asian nafld patients. s.k. mohan 1 , s. subramaniam 2 , s. subramaniam 3 1 assistant professor, department of biochemistry, saveetha medical college & hospital, saveetha university, t.n, india., 2 consultant, department of biochemistry, apollo hospitals, chennai, t.n, india. , 3 department of biochemistry, apollo first med hospitals, chennai, t.n, india. background: non-alcoholic fatty liver disease (nafld) covers a spectrum of liver diseases from simple fatty infiltration to progressive fibrosis. non-alcoholic steato hepatitis (nash) is a severe form of nafld and progresses to the end stage of liver disease. it is becoming the leading cause for referral to liver clinics in most areas. the prevalence of nafld in indian population is estimated around 7 -13%. the nafld has the potential to progress to hepatocellular carcinoma or liver failure, both events that ultimately lead to early death. aim: to evaluate the combination of inter cellular adhesion molecule -1 (icam -1), adiponectin and type-iv collagen, a new biomarker profile for nash in patients with nafld. methods: 76 patients with nafld and age & sex matched 68 normal healthy individuals as controls were selected for this study. levels of serum icam -1, adiponectin, type-iv collagen, lipid profile and liver function test parameters were estimated in patients and compared with controls. results: serum icam -1 & type -iv collagen levels were significantly increased in patients with nash among the nafld patients compared to controls. the serum adiponectin levels were significantly reduced in patients with nash among the nafld patients compared to controls. compared to liver function test parameters and lipid profile levels, nash profile has got positive negative predictive value among the nafld patients. conclusion: in patients with nafld, nash profile test -a simple, noninvasive and reliable to predict the presence or absence of nash. background/aim: oxidative stress and cytokines plays an important role in the pathogenesis of nonalcoholic fatty liver disease (nafld). aim of study was to assess lipid peroxidation, serum levels of transforming growth factor-( tgf-) and tumor necrosis factor-( tnf-) in patients with nafld and compare it with patients of chronic viral hepatitis (cvh) and healthy controls (hc). methods: lipid peroxidation was studied by estimating plasma malondialdehyde (mda) levels as per the methodology described by buege and aust and tgf-& tnf-levels were measured by elisa kits (ray biotech, usa, & diaclone, uk) in the stored sera in 10 biopsy proven patients with nafld (m: 4, f: 6, mean age: 41.7 ± 11.0 yrs), 15 patients with cvh ( m:14, f:1, mean age: 33.7 ± 11.4 yrs) and 5 hc (m:5, mean age: 25.2 ± 2.7 yrs). results: there was no difference in mean plasma mda levels amongst patients with nafld (17.28 ± 3.6 mol/l), cvh (15.29 ± 2.3 mol/l) and hc (16.79 ± 1.2 mol/l). serum tgf-levels between nafld (0.56 ± 0.41 ng/ml) and cvh (0.52 ± 0.25 ng/ml) patients and hc (0.58 ± 0.57 ng/ml) were also comparable. though patients with cvh (17.2 ± 27.0 pg/ml) and nafld (7.5 ± 6.3 pg/ml) had higher levels of tnf-than hc (5.5 ± 1.1 pg/ml), the difference was not significant statistically. conclusion: lipid peroxidation, tgf-and tnf-need to be studied in a larger number of patients with nafld. background/aim: burnt out nonalcoholic fatty liver disease (nafld) may be responsible for cirrhosis and hepatocellular carcinoma (hcc) in the absence of other causes. aim of this study was to evaluate the surrogate markers of nafld in patients with cryptogenic cirrhosis (cc) and cryptogenic hcc (chcc). methods: sixty five patients with cc and 31 patients with chcc were analyzed for the presence of abnormal body mass index (bmi) and type 2 diabetes mellitus (dm aim: to investigate the relation of phosphatidylethanolamine n-methyltransferase pemt gene g175a single nucleotide polymorphism (snp) with the susceptibility to nonalcoholic fatty liver disease nafld . methods the genotypes and allele frequencies of pemt exon 8 snp g175a were analyzed by using pcr-rflp in 51 nafld patients and 50 controls. results: the g to a variation of the pemt gene g175a snp was significantly higher in nafld group compared with controls. the frequencies of gg ga and aa genotypes were 58.8 39.2 and 2.0 in nafld and 78.0 22.0 and 0.0% in controls (p=0.038 . the a allele of the pemt gene was significantly more frequent in nafld group (21.6%) than that (1.0%) in controls p=0.042 .there were significant differences in serum levels of cholesterol, triglyceride, hdl-c and ldl-c between gg and ga/aa genotypes p <0.05 . n. assy 1,2 , g. lipez 3 , s. korem 3 , m. grozovski 3 1 sieff hospital, safed, israel, 2 2technion institute, faculty of medicine, haifa, israel, 3 ort braude college, karmiel, israel background: previous studies reported increase in serum protein c and decrease in serum paraoxonase levels in patients with non alcoholic fatty liver diseases (nafld). conclusion people with pemt gene g175a snp were more susceptible to develop nafld aim: 1) determine whether there is a relationship between nafld, protein c and paraoxonase levels in quiescent and in regenerating rats fatty liver 2) determine the effect of isa on hepatic "protein c" and paraoxonase mrna. pe439 methods: forty-eight sd rats were treated with fructose enriched diet (fed), or fed with metformin (2 mg/kg/d), fed with rosiglitazone (3 mg/kg/d), or the combination of both drugs for 5 wks. 30% phx was performed at wk 5. protein c, paraoxonase mrna expressions, lipids, mda were measured before and 24 hours after phx. results: hepatic "protein c" mrna was higher in rats with fatty liver than control rats (+105%, p<0.01) whereas hepatic paraoxonase mrna was lower in rats with fatty liver than control rats (-28%, p<0.005). hepatic protein c and paraoxonase mrna increased in rats with fatty liver in regeneration (+116%, p< 0.01, and +15%, p<0.01 respectively). the combination of metformin and rosiglitazone decreased hepatic protein c expression at 24 hours after phx by -170% (p<0.001) and increase paraoxonase mrna by +50% (p<0.01). serum paraoxonase correlates with serum protein c (r=-0.2), mda (r=0.4), background: non-alcoholic steatohepatitis (nash) is a type of non-alcoholic fatty liver disease (nafld), and may progress to hepatic fibrosis and cirrhosis. the pathogenesis of nash remains unclear. the aim of this study was to explore the arginase change in the progress of steatohepatitis in rats. methods: male sd rats weighing 70-80g were obtained. twenty animals were randomly divided into two groups. in the model group, five animals were fed with high lipid forage that includes 3% cholesterol and 20% lard for 6 weeks, five were fed for 12 weeks, while the control group ate normal foods. the animals were sacrificed after 6 weeks. the animals were sacrificed after 6 weeks and 12 weeks. liver and blood serum were collected while the serum levels of alt, ast, tg and tc were measured. the pathology of liver was observed by he staining. western blot was used to investigate the expression of arginase in control and model group. tg (r=-0.23). conclusion: hepatic "protein c" mrna levels are high at baseline, up regulated during liver regeneration and decrease after treatment with (isa) whereas hepatic paraoxonase mrna levels are low at baseline, up regulated during liver regeneration and increase after treatment with isa. results: vacuolization were observed extensively in hepatic cells in the model group after 6 weeks and 12 weeks of high-fat diet. it is demonstrated that rats fed with high-cholesterol food are indeed fatty liver models. western blot showed that the level of arginase ii increase in the liver of model group rats as compared to the control group. furthermore, the level of arginase was higher in liver samples obtained from model rats that were 12 weeks on a fat diet as compared to rats that were only 6 weeks on the same diets. conclusion: the level of arginase ii was altered in the progress of non-alcoholic steatohepatitis in rats suggesting that arginase ii is putative biomarkers and may represent new targets in the development of therapeutic strategies against fatty liver disease hepatic fibrosis and cirrhosis. methods: c57bl6/j mice were fed with mcd diet to induce hepatic fibrosis and rosiglitazone was given in treated group. effect of rosiglitazone was assessed by comparison of the severity of hepatic fibrosis in liver sections, expression of mmp-2/9, timp-1/2 mrna and protein detected by rt-pcr and western blot respectively. the ethanolic extract of fructus schisandrae chinensis decreased hepatic triglyceride level in mice fed with a high fat/cholesterol diet results at week 8, fibrosing nash models showed severe hepatic steatosis, infiltration of inflammation and fibrosis, which is associated with down-regulated mmp-2/9 mrna and protein, up-regulated timp-1/2 mrna and protein. rosiglitazone significantly reduced mcd-induced fibrosis by induced mmp-2/9 expression and reduced timp-1/2 expression by activating ppar . s.y. pan 1 , z.l. yu 2 1 beijing university of chinese medicine, 2 hong kong baptist university effects of the ethanolic extract of fructus schisandrae chinensis (etfsc) on serum and liver lipid contents were investigated in mice fed with normal diet or high fat/cholesterol diet for 8 or 15 days. single dose of etfsc (1 or 5 g/kg/day, i.g.) increased the serum triglyceride (tg) level (40 and 142%, respectively), but decreased hepatic total cholesterol (tc) level (15 and 16%, respectively) in normal mice. the hypertriglyceridemia produced by etfsc was suppressed by the co-administration of fenofibrate. the induction of hypercholesterolemia by high fat/cholesterol diet caused significant increases in serum and hepatic tc levels (up to 165%) and hepatic tg levels (up to 528%) in mice. etfsc treatment (1 or 5 g/kg/day for 7 days, i.g.) significantly decreased the mouse hepatic tg level (by 35%) and slightly increased the hepatic index (by 8%). whereas fenofibrate treatment (0.1 g/kg/day for 7 days, i.g.) significantly lowered the hepatic tg level (by 61%), it significantly elevated the hepatic index (by 77%) in hypercholesterolemic mice. the results indicate that etfsc treatment can invariably decrease hepatic tg in hypercholesterolemic mice, suggesting its potential use for fatty liver treatment. aim: to investigate the influence of multiple gene polymorphisms in the susceptibility of nafld. methods: the data of single nucleotide polymorphisms (snps) in 201 nafld patients who had at least one of the genetic variations at the sites of tnf--238, adiponectin -45 and leptin-2548 were analyzed. the genotypes were determined by using pcr-rflp. our previous studies showed that the variations of these sites increased the susceptibility of nafld. results: the prevalence of nafld in adiponectin variation alone group (n=45) was 35.6%; in tnf-alone group (n=33) 42.4%; in leptin alone group (n=54) 35.2% (p>0.05). in comparison with the above groups with single snp, the prevalence of the groups with two gene variations of tnf-plus adiponectin (57.9%, n=19) increased significantly (p<0.05). however the prevalence of other two groups i.e. adiponectin plus leptin (34.6%, n=26) and tnf-plus leptin (40.0%, n=15) did not differed significantly from those of groups with single snp (p>0.05). the prevalence in the group with three gene variations (55.6%) differed significantly from all (p<0.05) except that of tnf-plus adiponectin group (p>0.05). the metabolic features of the nafld patients in the 7 groups mentioned above were not different significantly (p>0.05). conclusion: nafld is a polygenic disease. multiple gene polymorphisms may, but not always, increase the susceptibility of nafld. chronic hepatitis b patients with nonalcoholic fatty liver disease r.d. zheng 1 , c.r. xu 1 , j. chen 1 , b.f. chen 1 1 southeast hospital background: to investigate clinical pathological characteristic in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). methods: we measured fasting blood glucose, insulin, triglyceride, cholesterol, alanine aminotransferase (alt), aspartate aminotransferase (ast) in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). and we detected hepatitis b virus marker, hbv-dna, counted body mass index, insulin resistance index and observed pathological characteristic. all these patients with diagnosis were confirmed by clinical and pathological evidence. result : the body mass index, homeostatic model assessment (homa) of insulin resistance, fasting blood glucose, insulin, triglycerides, cholesterol, were significantly higher in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld) than hbeag negative chronic hepatitis b patients. but the alanine aminotransferase (alt), aspartate aminotransferase (ast), hbv dna levels were significantly lower in hbeag negative chb patients with nafld than in hbeag negative chronic hepatitis b patients. histologic features in hbeag negative chronic hepatitis b(chb) patients with nonalcoholic fatty liver disease (nafld) are in zone 3 predominate macrovesicular steatosis and mild inflammatory infiltrate in portal region. conclusion: the hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease, whose hepatic steatosis changes are mainly caused by the metabolic factors. to carry out liver biopsy selectively for the patients with hbeag negative chronic hepatitis b having metabolic factors, which is helpful for early diagnosis in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). aims: to investigate the preventive effect of cordyceps sinensis and its possible mechanism on apoptosis of nafld. methods: rats were randomly divided into basic diet group (b group), pathologic group (nash group) and cordyceps sinensis group(cs group).the latter two groups were administered with high-fat diet to establish nafld animal models. cs group were treated with cs at the 9th week after high fat diet. rats were sacrificed at the end of the 18th week. biochemical examination were used to detect superoxide dismutase (sod) of liver tissue. hepatocyte apoptosis was assessed in each group using the tunel assay and immunohistochemistry for activated bax bcl-2 caspase-3 and nf-kb p65. results: (1) compated with the b group, severe hepatosteatosis, inflammative necrosis and local fibrigenisis were showed in liver of nfsh group. sod lever was significantly decreased (p<0.01) and tunel-positive cells were significantly increased (p<0.01). immuunohistochemistry test demonstrated active bax caspase-3was increased (p<0.01) while no apparent change was observed in bcl-2. (2) in cs group, only diffusive steatosis but not inflammation or fibrosis was found. sod lever was increased than that of nash group (p<0.05). tunel-positive cells and active bax caspase-3 were significantly decreased (p<0.05 p<0.01) that those of nash group. bcl-2 and nf-kb p65 were increased (p<0.01) than those of nash group. conclusions: hepatocyte apoptosis is a prominent feature of nafld. cordyceps sinensis may be useful as an antiapoptosis theraphy in this syndrome through increasing activity of sod, decreasing express of bax and increasing express of bcl-2 and nf-kb p65. background: non-alcoholic steatohepatitis (nash) is a leading cause of chronic liver disease. insulin-sensitizing , anti-inflammatory and anti-fibrotic effect of thiazolidinediones support their use in the treatment of nash. we aimed to evaluate the efficacy of thiazolidinediones in the treatment of nash. methods: we have identified randomised clinical trials, evaluating the efficacy of thiazolidinediones versus placebo in nash, through medline, embase, ami, cochrane central register of controlled trials. data were abstracted from each study and disagreements were resolved by consensus. dichotomous outcomes were reported as relative risk with 95% confidence interval based on fixed-effects model. results: we included three trials, two evaluating pioglitazone and another rosiglitazone. a total of 171 patients were involved in the analysis. thiazolidinediones was noted to improve liver function tests. it was effective in the reduction of steatosis among patients with nash (rr 0.67, 95% ci 0.52-0.87). it was found to be beneficial in improving ballooning necrosis (rr 0.79, 95% ci 0.66-0.95). it was also found to improve lobular inflammation (rr 0.72, background: it is well known that the weight reduction is effective for alt normalization in patients with non-alcoholic fatty liver disease (nafld). the necessary condition for alt normalization is still unclear. to clarify the necessary and sufficient condition for alt normalization, we investigated the effects of body fat decrease in nafld patients by body composition analyzer. methods: forty-six nafld patients (23 male, 23 female, mean age 49.8±12.9 years old) with abnormal alt levels were evaluated. the volume of skeletal muscle, body fat and bmr were examined by using the body composition analyzer (in body 720; biospace co. ltd., tokyo japan). all patients were received an individualized diet consultation by dietician every 4 weeks for 6 months. daily energy was bmr (basal metabolic rate) x1.2 kcal and protein was 1.0-1.2g per ideal body weight. result: twenty-eight of 46 patients (60.9%) were achieved normal alt level. in alt normalized group, the body weight and fat loss were 3.6±2.3 kg, 3.0±1.5kg (2.8±1.7 %body fat) respectively. on the other hand, in cases with alt remained abnormal level, the body weight and fat loss were 0.5±1.5kg, 0.4±1.6kg (0.5±1.6 %body fat). conclusion: our results demonstrate that the fat loss of 3 kilograms or more was necessary to normalize alt level in nafld patients. a. somani 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background : nafld is often clustered within families and the causes include both genetic and environmental factors. family studies done thus far have been limited by small sample size. to examine the familial patterns , we performed a prospective study to see (a) whether nafld is more common in first degree relatives (b) genetically determined risk factors associated for clustering. methods: first degree relatives of histologically confirmed nafld patients and spouses (controls) were included after excluding other causes of fatty liver. those having raised transaminases >3 months or sonographic examination consistent with fatty liver, had undergone liver biopsy for histological confirmation. they were divided into three groups. group i patients group ii first degree relatives group iii spouses results: nafld was more prevalent among first degree relatives then spouses (37% and 17%, p<0.01). anthropometric measurements, systolic and diastolic blood pressure, lipid profile and liver function tests were comparable in three groups. homa-r was similar in group i and ii (p=0.073), but was significantly different in group i and iii (p= 0.0001) and group ii and iii (p=0.0001) respectively. metabolic syndrome was present in >70% of patients and were comparable in three groups except for fasting glucose > 110, which was present in 76%, 77% and 57% of patients in group i, ii and iii respectively. majority (>50%) of our patients among groups i, ii and iii were having only steatosis while nash was present in 20%, 13% and 14% of patients. a. somani 1 , v. dixit 2 , a. jain 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha background: normal levels of alanine aminotransaminase (alt) have been demonstrated in nafld patients. alt levels are also modulated by age, gender, bmi, fasting glucose, and serum triglyceride levels. we performed a prospective study of patients with histologically confirmed nafld and having alt < 1.5 times and compared them with those having raised alt to determine (a) clinico-pathologic features of nafld patients with normal alt (b) to observe any differences between them. methods: patients with fatty liver on sonography had under gone biopsy for histological confirmation after excluding other causes of fatty liver. participants were divided into two groups (a) those having alt > 1.5 times normal (n=97) (b) those having normal alt (n=47) results: mean age was comparable with slight male predominance. there were significant differences in anthropometric measurements like bmi (p=0.0001) and whr (0.90±3.57 and 0.88±3.23, p=0.0071). mean bp, lipid profile, fasting glucose, insulin, and homa r were comparable. there were significant differences in both mean ast (50.2±5 and 37.4±3.21, p=0.0001) and alt (104±7.29 and 69.9±11.5, p=0.0001) levels. metabolic syndrome was present in >75% of patients and individual components were comparable except for increased waist circumference which was significantly more in those with raised alt (78.35% and 44.68%, p<0.001). majority of our patients were having only steatosis, while nash was present in (27.82% and 8.5%, p<0.05) of patients. conclusion: nafld can exist in patients with normal alt values. although more work is needed to determine who should be screened for nafld and how such individuals should be evaluated, this study is a step toward the identification and characterization of nafld patients with normal alt. we can suggest that patients having metabolic syndrome or insulin resistance, despite having normal alt, should be screened for nafld. also alt values should be adjusted for variables like bmi to appropriately screen nafld patients. background: scientific evidence has demonstrated that traditional chinese medical (tcm) approaches and products can be beneficial for managing non-alcoholic fatty disease (nafld), but few rigorous criteria of patterns of tcm therapy are available to guide practitioners in deciding the cam interventions. objectives: to evaluate criteria of patterns of tcm therapy for the management of nafld identified by biomedicine. methods: literature research, clinical epidemiological investigation and mathematical statistics were employed to make information collecting tables and to establish database. descriptive analysis, factor analysis, and cluster analysis were involved. results: (1) background/aim: serum uric acid level has been suggested to be associated with factors that contribute to the metabolic syndrome. the aim of this study was to investigate the association of serum uric acid level with nonalcoholic fatty liver disease (nafld). methods: a cross-sectional study was performed among the employees of zhenhai refining & chemical company ltd., ningbo, china. results: the study included 8925 subjects (6008 men) with a mean age of 43 years. the prevalence rate of nafld and hyperuricemia was 11.78% and 14.71%, respectively. nafld patients had significantly higher level of serum uric acid than controls (370.3 ± 86.6 vs. 321.1 ± 82.6 mol/l; p < 0.001). the prevalence rate of nafld was significantly higher in the subjects with hyperuricemia than those without hyperuricemia (24.75% vs. 9.54%; p < 0.001), and the prevalence rate increased along with serum uric acid levels (p value for trend < 0.001). multiple regression analysis showed that hyperuricemia was associated with increased risk for nafld (odds ratio [or]: 1.291, 95% confidence interval [ci]: 1.067 -1.564; p < 0.001). conclusion: serum uric acid level is significantly associated with nafld, and increased serum uric acid level is an independent risk factor for nafld. background: development of fatty liver is believed to be an early and reversible consequence of excessive alcohol consumption. however, the cellular and molecular events in the early development of alcoholic liver diseases (ald) and the contributory effects of a high fat diet are not fully understood. methods: this study was designed to quantify specific enzymatic and cytokinetic activity as well as the development of hepatic steatosis in a rat model of alchohol-induced liver injury without high fat diet. results: ethanol-fed rats exhibited high blood ethanol levels (0.85+0.31%) and significant increases in serum alt (67.7+ 13.4 unit/l), ast (136.3+ 60.2 unit/l), and alp (400+108.9 unit/l) when compared with control rats (p<0.01, respectively). histopathological examination found unevenly raised knodell scores (5.33+1.15 in the ethanol-fed livers vs. 0.33+0.58 in control), which were characterized by scattered hepatocyte ballooning, portal inflammation and collagen fiber deposition. however, typical steatosis lesions were absent. qpcr demonstrated up-regulation of genes in the ethanol-fed livers, including hepatocyte metabolism enzymes/receptor (adh1, p<0.05; cytochrome p450 2e1, cyp2e1, p<0.05; gsta2, p<0.01; ppar , p<0.05), and genes coding for pro-inflammatory cytokines (il-1 , p<0.01 vs. control livers; tnf-p<0.05; tgf -, p<0.05; rantes p<0.05), ecm components and proteinases (collagen-1, p<0.01; sma, p<0.01; mmp -9, p<0.05 and timp-1, p<0.05). conclusion: chronic administration of ethanol to rats without high fat diet productively induces alcohol hepatitis in the absence of fatty liver, suggesting that alcohol hepatitis may precede steatosis in the development of ald. the aim of the present study was to evaluate the changes of several cytokines associated with inflammatory liver disease and liver regeneration by molecular adsorbent recirculating system (mars) in 15 aclf patients versus 15 patients treated with medical standard therapy (smt) that presented alcoholic liver disease etiology and similar model end-stage liver disease (meld). methods: mars group: fifteen (10 male and 5 female) patients were treated with mars® (gambro). five patients were excluded by study.the number of mars applications was about 9, the length of applications was about 10h. smt group: fifteen patients (10 male and 5 female) were treated medical standard therapy such as prophylaxis against bacterial infections, albumin and fresh plamsa and judicious use of diuretics. three patients were excluded by the study. the patients were valued during 30 days from inclusion with a survival follow up a three months. results: mars group: we observed a significant changes in levels of il-6(p<0.01), il-1(p<0.002), il-8(p<0.04) and tnf-alfa (p<0.03) in association with improvement of hepatic growth factor (p<0.001). the patient's survival at three months was 50%. smt group: we observed only a significant changes in il-1(p<0.01) and tnf-alfa (p<0.2). the patient's survival at three months was 33%. conclusion: the mars liver support device has corrective effects on disturbed pathophysiology of aclf and may be used to enhance spontaneous recovery or as bridge to transplant. a study of protective effect of centella asiatica in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury n. haleagrahara 1 , s. chakravarthi 1 , p. kumar 1 1 international medical university, malaysia background: centella asiatica has been used for centuries as a medicinal herb for wound healing, memory enhancement, cancer, vitality, respiratory ailments, psoriasis and eczema, revitalizing connective tissue, burn and scar treatment, skin infections, arthritis, rheumatism, periodontal disease, varicose veins, hypertension, sedative, anti-stress, anti-anxiety, aphrodisiac, and as immune booster. results: ppc significantly reduced hepatocyte damage, hepatitis, and hepatic fibrosis, but did not affect steatosis. phosphorylation of apoptosis signal-regulating kinase 1, p38 mitogen-activated protein kinase, and protein kinase c, as well as activation of nuclear factor-kappa b, were markedly suppressed by ppc. these effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ros)-generating enzymes, including cytochrome p450 2e1, acyl-coa oxidase, and nadph oxidases, in addition to restoration of ethanol-induced increases in toll-like receptor 4 and cd14. ppc also decreased the pro-apoptotic proteins bax and truncated bid, thus inactivating mitochondrial permeability transition. furthermore, ppc suppressed overexpression of transforming growth factor-1 and hepatic stellate cell activation, which retarded hepatic fibrogenesis. conclusion: ppc exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ald as a result of inhibition of alcohol-induced ros production. background: dysctamnus dasycarpus has used for the promotion of health in south korea. but, there were rare a report concerning the hepatotoxicity. we report cilinical features of liver injury by dysctamnus dasycarpus. method: eighteen patients diagnosed as acute toxic hepatitis by dysctamnus dasycarpus in chungnam national university hospital between january 2005 and arpil 2008 was enrolled. toxic hepatitis was diagnosed by rucam score ( 4). the medical records were reviewed, retrospectively. result: eleven patients (61%) were female and the mean age was 53.5. most common symptom was jaundice. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4 g/dl, platelet 212×10 3 /l,alt 1375iu/l, total bilirubin 11.3 mg/dl, alkaline phosphatase 159 u/l, ggt 244u/l, prothrombin time(inr) 1.1. the mean hospitalization was 21.5days. peak laboratory findings were as follows: alt 1382iu/l, total bilirubin 15mg/dl. recovery time of each biochemical finding was as follows: alt 37days, total bilirubin 39.4 days. recovery rates of alt and total bilirubin were 27.8% and 38.9%, 89.9% and 89.9% at 4 weeks, 8 weeks, respectively. the main biochemical pattern of hepatotoxicity was hepatocellar (72.2%) type. prednisolone was prescribed in six patients. progressive anemia and thrombocytopenia were detected in one patient diagnosed as pure red cell aplasia. other one patient had prolonged jaundice (117 days). but, all patients had recovered without sequelae. conclusion: in south korea, liver injury by dysctamnus dasycarpus was more frequent in women. the main pattern of hepatotoxicity was hepatocelluar type. most patients had prolonged icteric phase and hospitalization. patients were recovered by supportive management after drug cessation or prednisolone therapy. in korea, traditional medicine that is based on the use of herbal medicine developed from a long time ago. however, clinical study of the herbal medicine is not conducted in a structured manner. we report three cases of toxic hepatitis caused by the intake of dictamnus albus. the first patient, a 44 year old woman was admitted due to nausea after ingestion of liquor containing dictamnus albus for 2 months. total bilirubin was 2.55 mg/dl ast/alt 774/1,424 iu/l on admission. liver biopsy observed hepatocyte necrosis and cholestasis. the elevated bilirubin and transaminase returned to normal 2 weeks later after cessation of dictamnus albus. the second patient, a 64 year old man was admitted due to jaundice after ingestion of boiling dictamnus albus for 3 months. total bilirubin was 11.37 mg/dl ast/alt 2,010/2,522 iu/l on admission. liver biopsy observed pericellular fibrosis and necrosis. the bilirubin decreased slowly compared to the transaminase and normalized 3 months later after cessation of dictamnus albus. the third patient, a 48 year old man was admitted due to jaundice after ingestion of liquor containing dictamnus ablus for 1 month. total bilirubin was 12.8 mg/dl ast/alt 1,097/1,869 iu/l the hepatocyte necrosis was observed by liver biopsy. the elevated bilirubin and transaminase levels normalized 1 month later after cessation of dictamnus albus. all patients had negative viral markers and non-specific ultrasonographic findings. the above mentioned three cases demonstrate that liver may have been damaged by dictamnus albus, which indicated clinical characteristics. background/aims: cmili poses a diagnostic challenge as no tests are available to confirm the causality. the aims of this study were 1) to evaluate clinical features and patterns of cmili and 2) to assess the likelihood of causality among patients with liver impairment and exposure to chinese medicine (cm) by a multidisciplinary approach. method: between 5/2005 and 8/2007, patients who had liver derangement and cm or proprietary cm exposure within six months managed in the united christian hospital were studied. clinical features and the cm were reviewed by a multidisciplinary team involving a hepatologist, a toxicologist and cm experts. literature search of relevant herbs in chinese and western journals were performed. cm samples or residue were sent to toxicology laboratory for analysis to look for any toxic constituents, adulterant or contaminant. the likelihood of causality was ranked by various experts independently and disagreements were settled by a consensus meeting. results: there were forty-six cases of suspected cmili, nineteen cases with alternative causes of liver diseases were excluded. twenty-seven cases of cmili proceeded to detailed analysis. median age of patients was 51 (21-76) with female predominance. the median duration of cm exposure to presentation was 20 (1-150) days. majority of them (82%) had hepatocellular liver injury pattern. one case of adulteration with nsaid and erroneous substitution of herb was identified respectively causality were classified as unlikely, possible, probable and highly probable in 3, 12, 8 and 3 patients respectively. conclusion: a multidisciplinary approach allows systemic evaluation of suspected cmili. mouse model i. nassar 1 , t. pasupati 1 , i. segarra 1 , j.p. judson 1 1 international medical university, kuala lumpur, malaysia background: imatinib, a selective tyrosine kinase inhibitor, exhibits drug interactions with other drugs that are metabolised via the cytochrome p450 pathway. acetaminophen, a widely used analgesic and anti-pyretic drug is also metabolised via p450 pathway. this study aimed to evaluate the nature of hepatotoxicity after co-administration of imatinib and acetaminophen in a preclinical mouse model. methods: four groups of male icr mice (30-35g) were used. the mice were administered either saline solution orally, imatinib 100 mg/kg orally (control), acetaminophen 700 mg/kg intraperitoneally (positive control) or co-administered imatinib 100 mg/kg and ip acetaminophen 700 mg/kg (study group). the mice (n=4 per group) were fasted overnight, dosed respectively and sacrificed at pre-determined time intervals of 15, 30 minutes, 1, 2, 4, 9 and 12 hours and liver samples obtained by dissection. h&e stained liver sections (3µm thick) were histopathologically analysed. results: the liver samples showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, with both imatinib and acetaminophen, administered separately. the damage increased gradually with time, peaked at 2 hours and then resolved completely by 6 hours. liver samples showed irreversible damage (cytolysis, karyolysis and karyorrhexis) when both drugs were administered concurrently, the damage increased with time and had not resolved after 12 hours duration. conclusion: co-administration of acetaminophen and imatinib increased the hepatoxicity caused by acetaminophen and imatinib to become irreversible. this may be due to the fact that both drugs are metabolised by the cytochrome p450 pathway in the liver. background: a higher risk of antituberculosis drug (att) induced hepatotoxicity has been reported in indian subcontinent compared to the western counterparts. slow acetylator genotype of n-acetyltransferase2 (nat2) and ci genotype of cytochrome p4502e1 (cyp2e1) gene are two known risk factors associated with this disease. cyp2e1 gene encodes a rifampicin inducible enzyme which increases hepatotoxicity. therefore slow acetylation of isoniazid and simultaneous use of rifampicin may augment the toxicity of isoniazid. objectives: to analyze the allelic distribution of nat2 and cyp2e1 gene in patients of pulmonary tuberculosis who developed att induced hepatitis materials and methods: the study included cases of pulmonary tuberculosis (190) and att induced hepatitis (95). polymorphism of nat2 and cyp2e1 gene was studied by pcr-rflp method in both these groups. results: occurrence of att hepatotoxicity was 17.89%. there was a higher prevalence of slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 in patients with hepatotoxicity compared to patients without hepatotoxicity (72.09% vs 44.2%, p value < 0.05). no association of cyp2e1 rsai polymorphism could be considered with att hepatotoxicity. however, drai c/d genotype of cyp2e1 appears as a risk factor for predicting the occurrence of antituberculosis drug induced hepatitis (or 5.06, p value < 0.05). conclusion: the study demonstrates that patients with slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 and heterozygous mutant c/d genotype of cyp2e1 gene are predisposed to develop antituberculosis drug induced hepatotoxicity. regular monitoring of clinical and biochemical profile may be considered in these patients when they receive antituberculosis treatment. background: drug-induced liver injury is the most common adverse drug reaction. we often use two kinds of diagnostic scales to evaluate suspected patients. however, we still can't diagnose accurately without the direct drugs history and the pathological evidence. methods: twenty-seven drug-induced liver injury cases with liver biopsies from 2001 to 2008 were reviewed retrospectively by maria and japanese scale. result: there were 11.1% of cases with increasing eosinophils. herbs (29.63%) were the most common suspected drug and unknown drugs intake history (14.81%) were described in these cases. the high possibility and possibility were 25.93%, 29.63% by maria scale and 85.19%, 3.7% by japanese scale, respectively (p=0.015). conclusions: japanese scale seems more sensitive than maria scale in these cases. however, there are still some definite cases ignored as low possibility due to absence of obvious drug using history. early treatment and suspected drugs prohibition interferes the outcomes of the two diagnosis systems and lead to a false result. it is still a clinical challenge without strong drug using history or pathological evidence of liver biopsies to diagnose the drug-induced liver injury quickly and accurately. background: previous study suggested that oxidative stress may be an important mediator of methamphetamine-mediated tissue injury. the study was to examine the mechanism of antioxidant activity and methamphetamine-mediated liver injury. materials and methods: the 25 days old male sprague-dawley rats were subcutaneous injected daily with methamphetamine (10 mg/kg body weight) for 15, 30, 60 and 120 days. control group received equal volumes of vehicle. the liver tissues were extracted to measure the activities of sod, catalase, glutathion reductase (gr), and glutathione peroxidase (gpx), and the level of glutathione. western blot were used to measure the expression of rho and phosphor-ezrin-radixin-moesin (p-erm). results: compared with vehicle group, treated with methamphetamine for 15 and 30 days, the activities of liver sod, gpx, and catalase were significantly decreased. in 60 and 120 days group, the activities of antioxidant enzymes of methamphetamine-treated liver was not different from that of vehicle group. the levels of glutathione production also had the same trend. the activities of gpx and catalase on vehicle group gradually reduced following the days of treatment. however, administration of methamphetamine resulted to a lower activity of catalase through the treated days. there was no difference on the activity of gr between vehicle and methamphetamine group. the expression of rho and p-erm were also increased by methamphetamine treated for 30 days. conclusion: these results suggested the methamphetamine lead to liver remodeling via decreased antioxidant activity. finally, the situation of mechanism needs taking in advantage discussion. background: to observe intervening effects of preventive and theraptical treatment of radix sophorae tonkinensis's polysaccharides(rstp) on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: kunming mice intoxicated with anit 50mg/kg orally and treated with rstp 50mg/kg for 7 days before anit exposure and for 2 days after anit exposure respectively, the general condition,mortality rate and serum alt activity are obeverated. result: it was found that by preventive treatment the general condition and mortality rate were improved, serum alt activity reduced.by therapeutic treatment,the general condition deteriorated,mortality rate and serum alt activity increased. conclusion: the preventive treatment of rstp reduce the liver damage due to increasing the anti-stress ability such as the antioxidant capacity,its therapeutic treatment increase the injuried liver damage due to increasing the non-specific immune response and aggregating the preexisting liver inflammation. background: the product's instruction pointed out that in some patients polyphenolic acids' salt from salvia miltiorrhiza(ppas-sm) may lead to a temporary increase in serum alt activity.so we observe effects of ppas-sm on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: 24 hours after intoxicated with anit 50mg/kg orally, kunming mice were treated with ppas-sm 50, 25,10mg/kg/days for 2 days orally, then serum alt activity was measured. result: all doses of ppas-sm led to rise of serum alt activity in mice, most obvious in group of high dose.but the general situation and mortality rate did not increase significantly. conclusion: ppas-sm lead to rise of serum alt activity in mice with damaged liver.the auther suggests as a double-edged sword,the antioxidant ppas-sm may have a prooxidative effect in some condition too. *this project was supported by grants from shanghai municipal education commission under high school high-tech characteristic development programme (no smec finance (2005) 81) pe467 a. somani 1 , a. jain 2 , v. dixit 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha introduction: hepatic encephalopathy, a complex neuropsychiatric syndrome secondary to acute liver failure, chronic parenchymal liver disease or portal-systemic shunting, may possibly develop through mediators of endotoxin and tumor necrosis factor-alpha (tnf-). several studies have shown that serum levels of (tnf-) are significantly elevated in patients with acute and chronic liver diseases, where these elevations are independent of the etiology of the underlying disease. it has been shown that plasma levels of tnf-correlate with the severity of hepatic encephalopathy (he) in fulminant hepatic failure. however, still there is very few published data regarding the relationship between serum levels of tnf-and the presence or severity of he in patients with chronic liver failure. methods: the aim of this study is to determine the relationship between serum levels of tnf-and clinical grades of he in patients with chronic liver failure. this prospective study included 149 consecutive male patients with alcoholic cirrhosis in various clinical grades of he (according to west haven criterion). detailed clinical, biochemical and sonographic examination was done in all patients. circulating levels of tnf-was measured using solid-phase elisa. results: the mean±sem values of serum tnf-at presentation in patients with mhe (n=37), grade 1 (n=17), grade 2 (n=41), grade 3 (n=44), and grade 4 (n=10) were 6.2±0.4, 9.5±0.6, 15±0.7, 26.3±1.7, and 46±5 .9 pg/ml, respectively. significant positive correlation was found between serum levels of tnf-and severity of he (correlation coefficient = 0.7). conclusion: from the present study we can suggest that there is significant relationship between tnf-and he in patients with alcoholic cirrhosis and it could be involved in its pathogenesis. background: acute hepatitis a (aha) is one of the most common infectious diseases and usually a self-limiting disease. although extrahepatic manifestations are not common, a few cases associated with acute renal failure (arf) have been reported. methods: we reviewed clinical features of aha patients complicated with arf (group a) and compared with non-complicated aha patients (group b). medical records of 191 patients with aha were reviewed between january 2003 and december 2007. we experienced 11 patients (5.8%) with arf associated aha. result: there were no differences between group a and group b in sex ratio and age. the peak value of alt (median: 6133 iu/l vs 1685 iu/l, p<0.001), alkaline phosphatase (median: 235 iu/l vs 201 iu/l, p=0.03), prothrombin time (inr, median 1.72 vs 1.09, p<0.001) was significantly higher in group a than b. nine patients (81.8%) recovered completely with hemodialysis (6 patients, 66.7%) and only conservative management (3 patients, 33.3%), while 1 patient underwent liver transplantation and 1 patient died due to fulminant hepatic failure. there were 4 patients who underwent kidney biopsy. two patients were diagnosed as acute tubular necrosis and 2 patient as acute interstitial nephritis and iga nephropathy. conclusion: aha patients with arf had higher alt and more prolonged prothrombin time. the prognoses were poorer than those without arf. however, arf patients with nonfulminant aha had a good prognosis with a proper treatment and should not be confused with hepatorenal syndrome. background/aims: to investigate the hev infection among different animals and people with special profession, and to analyse the genotype of hev isolated in this study. methods: serum and fecal samples were collected from various animals and people with special profession in the south suburbs of beijing. hev antigen and anti-hev antibody were detected by das-elisa. hev rna was extracted from fecal samples and amplified by rt-npcr. the nucleotide sequence homology and phylogenetics of hev strains isolated from swine were analysed. results: the anti-hev antibody positive rate of adult swine, cow, sheep and younger swine were 98.23% (222/226), 29.35% (54/184), 9.80% (20/204) and 60.73% (99/164), respectively. the hev antigen positive rates of adult swine, cow, sheep and younger swine were 2.65% (6/226), 4.35% (8/184), 1.45% (3/207) and 9.75% (16/164), respectively. the hev antigen and anti-hev antibody positive rate of professional group was 0.40% (1/247) and 42.51% (105/247) respectively. the hev rna positive rate of fecal samples from younger swine was 22.89 %(19/83). 16 of 19 samples were hev rna positive by pcr with primers of hev orf1 and orf2. the sequence analysis of the 16 samples showed that there were 2 groups designated as bj-1 (11/16) and bj-2 (5/16). the nucleotide homology of bj-1 and bj-2 was 99%. phylogenetic analysis of hev orf2 indicated that both of them belonged to genotype 4d. conclusion: phylogenetic analysis of hev orf2 indicated that hev isolated in the south suburbs of beijing belonged to genotype 4d. 6 bracops hospital brussels , 7 st. jan hospital, bruges , 8 chu brugmann, 9 chu sart tilman, liège , 10 gent university hospital , 11 zna middelheim, antwerp hepatitis delta virus is a subviral satellite requiring hepatitis b virus to propagate, usually leading to severe, chronic liver disease. as data on epidemiology and management practice of hdv infection in belgium are lacking, a retrospective and prospective, multi-centric questionnaire-based registry is performed in 2008. results of 32 patients are reported. background/ aims: hepatitis a is an acute infectious disease that is transmitted by fecal-oral root. because the incidence of hepatitis a has been increased in gwangju and chonnam province of korea recently, hepatitis a patients in chonnam national university hospital employees had been increased. so we investigated the seroprevalence of igg anti-hav in hospital empolyees less than 40 years old. methods: we analysed seroprevalence of anti-hav igg from 1,002 hospital employees (men: 190, women: 812) . serum alt and bilirubin at admission were 2,979 1,711 iu/l and 3.9 2.4 mg/dl, respectively. these levels were elevated up to 3,397 1,789 iu/l and 6.8 3.7 mg/dl, respectively. ana was positive in 81 patients (64.3%). age, duration from peak-alt day, duration from peak-bilirubin day, alt level, and peak-bilirubin level were not different between ana(-) patients and ana(+) patients. in the while, sex, duration from symptom-onset day, and bilirubin level, and peak-alt level were significantly different. in 51 (62%) of 81 patients with positive ana, ana was followed after 1 month and ana became negative in 38 patients (74.5%). among 13 patients with positive ana after 1 month, titer decreased from the baseline in 7 patients, showed no interval change in 4, and increased in 2. conclusions: positive ana result is not rare in patients with acute hepatitis a. it is considered that ana transiently appear during the course of acute hepatitis a and then, disappear with the improvement of acute hepatitis. (89), 2007(107). the clinical data such as sex, admission period, ast, alt, total bilirubin, prothrombin time, crp, alt normalization time did not show difference. just wbc and gtp were higher on 2008 group. the older age patients were more on 2008 group. the patients admitted mainly on april, may, june, july (84%) on 2008 while admitted even on past years. conclusion: acute hepatitis a ptients is increasing. it is occurring in older age people and mainly on specific period. the more concern to prevention should be needed. background: we analyzed the 5' non-translated region (5'ntr), non-structural proteins 2b and 2c of hepatitis a virus (hav) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all of our data and examine whether genomic differences in hav are responsible for the range of clinical severities. methods: our accumulated hav strains of 5'ntr (nt 200 and 500), entire 2b and 2c from 25 japanese patients with sporadic hepatitis a, consisting of 7 patients with fulminant hepatitis (fh), 5 with severe acute hepatitis (ahs), and 13 with self-limited acute hepatitis (ah), in whom the sequences of all 3 regions were available, were subjected to phylogenetic analysis. results: fh patients had fewer nucleotide substitutions in 5'ntr, had a tendency to have more amino acid (aa) substitutions in 2b, and had fewer aa substitutions in 2c, than ah patients. four fh and 2 ahs with higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis a and genomic variations in 5'ntr, 2b and 2c of hav. conclusions: our study suggests that genetic variations in some parts of hav might cooperatively influence replication of the virus, and thereby affect virulence. viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis a. aims: the incidence of acute viral hepatitis a in adults is increasing very much in south korea, 2008. the aim of this study was to the clinical features and course in daejoen and its surrounding area. methods: forty seven patients admitted as acute viral hepatitis a in chungnam national university hospital between january 2008 and june 2008 were enrolled. the medical records were reviewed, retrospectively. results: the mean age was 30.5. common occupations were company employee and studuents. most common symptom was jaundice. presumptive infection sources were raw fish or shellfish and raw meat. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4g/dl, platelet 212×10 3 /l, ast 2268iu/l, alt 2755iu/l, total bilirubin 5.2mg/dl, alkaline phosphatase 207u/l, ggt 379u/l, prothrombin time(inr) 1.2. hospitalization was 21.5days. peak laboratory findings were as follows: alt 3181iu/l, total bilirubin 8.8mg/dl. leukopenia (<4000/ul) and thrombocjtopenia (<10×10 5 /l) were ocurred in sixteen and six patients, respectively. recovery time of each biochemical finding was as follows: alt 20.2 days, total bilirubin 26 days. recovery rates of altand total bilirubin were 74.4% and 74%, 88.5% and 92% at 4 weeks, 8 weeks after diagnosis, respectively. prolonged jaundice (115days) was detected in one patient. all patients were recovered by supportive management. conclusions: in south korea, acute viral hepatitis a was more prevalent in young adults, recenlty. presumptive infectious sources were raw fish or 1 guangxi center for disease prevention and control shellfsh and raw meat. if it can not change the food style that many korean enjoy raw seafood, vaccination for adults must be considered to prevent it. objective: to assess the safety and immunogenicity of a new inactivated hepatitis a vaccine (vero cell). pe479 methods: 1507 subjects were selected in gongcheng city of guangxi zhuang autonomous region, and the clinical trail was carried out according to the random, double-blind and parallel principle from january to august, 2005. after vaccination by 0, 6 schedule, adverse events of the subjects were observed, the seroeonversion rate and geometric mean titer (gmt) were tested by the competitive inhibition elisa. results: after immunization, the systemic and local reaction rates of adults were 8.80% and2.67%, which was no significantly statistical difference compared with control group, 12.41%and 4.41%; while the rates of children were 10.60and 2.28%%, and no significant statistical difference compared with control group, 10.71%and2.86%. one month after first dose of vaccination, the seroconversion rates of children and adults were 88.2% and 93.8%, and one month after second dose of vaccination, the rates were all 100%, the gmts of children and adults were 16447miu/m1 and 8555miu/ml, which was significant statistical difference in children compared with control group, 1946 miu/ml and 5881 miu/ml, respectively. methods: igg anti-hav was measured in a total of 3905 subjects under the age of 50, who visited hanyang university seoul and guri hospitals between january 2001 and may 2008. results: fig. 1 shows the relatively low positive rates of the antibody in ages of 11 to 30 and the lowest rates of 9.9% and 9.2% in the age group of 16 to 20, following the ages of 21 to 25 with rates of 12 background: some viruses encode proteins that affect their cap-independent internal ribosomal entry site (ires)-mediated translation and their replication. it was recently reported that hepatitis a virus (hav) proteases interact with intracellular dsrna-induced retinoic acid-inducible gene (rig-i)-mediated signaling, but it remained unknown whether hav proteins have any effects on hav ires-independent translation. in this study, we investigated the effects of 7 hav non-structural proteins on their ires-mediated translation using a reporter assay. pe480 methods: the bicistronic reporter constructs, termed psv40-hm175-ires, psv40-a1-ires, psv40-a2-ires, psv40-f1-ires, and psv40-f2-ires, contain the sv40 promoter that controls the expression of a bicistronic message coding for renilla and firefly luciferases separated by hav ires, and are derived from strain hm175, acute convalescent hepatitis clones a1, a2, fulminant hepatitis clones f1, f2, respectively. human hepatoma cell lines were co-transfected with psv40-hav-ires and each hav protein-expression vector. luciferase activity was determined 48 h after transfection. were from other countries within asia, africa, middle east, and eastern europe. patients of a wide age range were affected by hepatitis delta (mean age 46.0, median 47.0, range 19-79). 18 (40%) of 45 were co-infected with hcv. hepatitis b virus (hbv) dna was detectable in 43 (78%) patients and negative in 8 (15%) patients. all hepatitis delta patients were extracted from a prior study conducted by this collaboration. there were 1,191 chronic hbv carriers. 18 (1.51%) were hbv/ hcv/hdv infected. 32 (58%) of 55 patients carried a diagnosis of cirrhosis compared to 262 (22%) of 1191 chronic hbv patients. 13 (72%) hcv co-infected patients had evidence of cirrhosis while 4 (22%) patients did not. conclusion: individuals with hbv/hdv co-infection have higher rates of cirrhosis. individuals with hbv/hcv/hdv infection have rates of cirrhosis significantly higher than individuals with either chronic hbv infection or hbv/hdv co-infection. testing for hdv should be performed in all patients, especially those with advanced liver disease or high risk behavior. clinical characteristics were compared between the patients with significant endoscopic findings (group a) and without such findings (group b). peak ast and alt level were higher in group a (p<0.01). there were no statistical differences in age, gender, comorbidity, and etiology of acute hepatitis between group a and group b conclusion: significant endoscopic findings were found in considerable proportion of patients with acute hepatitis. severity of acute liver injury was associated with significant upper gastrointestinal endoscopic findings. in patients with severe acute hepatitis who complain of upper gastrointestinal symptom, esophago-gastro-duoenoscopy should be performed. background: in japan, hepatitis e virus (hev) testing is not allowed as routine one. to study the role of hev testing, we checked 22 sera of the patients diagnosed as etiology-obscure acute liver injury. methods: we have seen 54 cases of acute liver injury from january 2004 through december 2007 in our hospital and 25 cases of them were etiology-obscure. in 25 cases, 22 were retrospectively tested for hev-igm, hev-iga and hev-rna (rt-pcr) by direct sequence method on stored sera taken at the time of presentation. result: two of 22 cases (9.1 ) were positive for both hev-igm and hev-iga and one case was positive for hev-rna. in 54 cases of acute liver injury, the cause of virus was 31 cases (57.4%) and unknown was 8 cases (14.8%). hev was occupied in 3.7% in all cases and 6.5% in the cases caused by virus. one of the two cases had been misdiagnosed as "drug induced hepatitis". hev of genotype 3 was detected in one case and its nucleotide sequences of hev showed quite a high degree of similarity to the reported one at closed city in the same year. conclusion: hev is not rare in japan and the hev testing can reverse the diagnosis of acute liver injury. hev testing sould be used as routine one for acute liver injury. association of progesterone receptor gene with hepatitis e disease severity in pregnancy p.d. bose 1 , b. das 2 , a. kumar 1 , p. kar 1 1 maulana azad medical college, 2 background/aims: incidence of fulminant hepatic failure (fhf) in hepatitis e is high in pregnancy particularly during 3 rd trimester when there is an altered status of hormone and immunity. progesterone receptor (pr) up regulation provides fetal protection via immunosuppression but lower immune status in pregnancy may add to the disease severity. till now, no data is available whether pr can play any role in hepatitis e disease severity during pregnancy. progins, a haplotype of pr consisting of 320-bp insertion in intron g together with point mutations in exons 4 and 5 is associated with increased stability and higher transcriptional activity. the aim of the study is to analyze pr mutation (progins) and m rna expression in hepatitis e virus infected pregnant women with avh and fhf. methods: a total of 68 avh and 32 fhf cases were studied. blood and placental tissue were collected from the medicine and gynecology wards of lnjp hospital, new delhi. cases were screened for acute viral markers by commercially available elisa kit. extraction of dna from blood and rna from placental tissue was done by qiagen kit. mutation in pr was detected by pcr-rflp. semiquantitative rt-pcr for pr expression was performed in placental tissue using beta-actin as internal control. results: pr mutation (progins) was significantly more in fhf compared to avh (28.1% vs 10.3%, p value<0.05). protein expression was found higher in progins carriers. conclusion: progesterone receptor mutation (progins) may have a role in the hepatitis e disease severity in pregnant women. results: the hepatitis e was predominantly sporadic, some patients superinfected with other viral hepatitis, especially hepatitis b. in the old patients, jaundice lasted longer and the length of stay was longer, the incidence of complication was higher than the young men. the incidence of complication in the superinfected group was higher than the simple infection. the transaminase in the simple infection group was obviously raise than superinfected with liver cirrohsis. methods: liver sample were paraform-glutaral fixed, paraffin-embedded, sectioned and immunohistochemical stained, and positive samples were selected for histological analysis and rt-pcr detection. result: positive rate of hev immunohistochemistry ranged from 90% to 100% (fig. 1) . hepatocyte degeneration, scattered singled karyopyknosis, lymphocytic infiltrate, hyperplasia of bile canaliculus at the portal area and fibrous connective tissue hyperplasia been observed during histological analysis (fig. 2) , and two genotype 4 hev which closely related to many strain isolated from patients with sporadic acute hepatitis been detected. conclusion: the patients infected with hepatitis e of young men were frequently. jaundice lasted long in the old patients, the incidence of complication was higher in the superinfected men and the old men. conclusion: additional public-health concerns might be placed on pork safety and the risk of hev infection via the consumption of undercooked pork products. poster session, hall 5b aim: esophageal varices (ev) recurs frequently after endoscopic variceal ligation (evl) or endoscopic injection sclerotherapy (eis). we retrospectively investigated risk factors for early recurrence of ev after endoscopic treatment. methods: we treated 110 patients with ev, who had no past history of ev, at ehime prefectural central hospital from october 2005 to june 2008. of those, 78 (71%) were observed for at least 2 months after treatment and enrolled. we divided them into rupture cases at initial endoscopic treatment [(bleeding group; n=25 (32%)], and cases with preventive evl or eis performed [preventive group; n=53 (68%)]. all received periodic upper endoscopy examinations to confirm recurrence or no recurrence of ev. results: recurrence of ev occurred in 18 of all subjects and the average period after treatment was 6.5±3.0 months. the recurrence rate was significantly higher in the bleeding group (11/25) as compared to the preventive group (7/53) (p=0.0026). there was a significant relationship between recurrence of ev and hepatic reserve function (child-pugh a+b, c; 11/64, 7/14 respectively; p=0.0083). in logistic multi-variant analysis, ev rupture at initial treatment and child-pugh c were risk factors for recurrence. in contrast, age, sex, hepatocellular carcinoma, portal tumor thrombosis, continuous alcohol consumption, therapeutic modality (evl or eis), number of treatment sessions, and operator experience did not have a significant relationship with recurrence. conclusion: in cases with ev rupture at initial treatment or child-pugh c, the risk for early recurrence must be considered and patients carefully observed in follow-up examinations. endoscopic cyanoacrylate injection: less oil for less ectopic embolism c.z. li 1 , l.f. cheng 1 , z.q. wang 1 , f.c. cai 1 , q.y. huang 1 , e.q. linghu 1 1 general hospital of chinese pla background and aim: endoscopic injection sclerotherapy with n-butyl-2cyanoacrylate (nbca, histoacryl) has been reported to be effective for hemostasis of bleeding gastric varices, but occasionally the gel flows to other organs and causes ectopic embolism. the present study aimed to determine whether less amount of iodized oil preload in nbca injection helps in decreasing ectopic embolism. methods: from january 1997 to april 2006, 2 different methods of endoscopic nbca injection, "sandwich method" and "modified sandwich method" (in which iodized oil preload was minimized), were applied on 635 gv cases, to evaluate if decrease of iodized oil preload resulted in less ectopic embolism. results: altogether 5 cases of ectopic embolism occurred in the whole group (0.8%), including 3 cases of splenic infarction, 1 case of transient paralysis and 1 case of minor infarction of the lung. the modified sandwich method showed some superiority over original method in decreasing ectopic embolism (0/276 vs. 5/359, p=0.049). less cough during procedure was also found with the modified method (3/276 vs. 18/359, p=0.006). conclusions: less amount of iodized oil preload in endoscopic nbca injection is beneficial to decrease ectopic embolism. background: portal hypertension is closely associated with serious complications of liver cirrhosis which contribute to bad prognosis. hepatocellular carcinoma (hcc) and low serum sodium (sna) are manifestations of end-stage liver disease (esld) and are associated with poor survival in decompensated cirrhosis patients. therefore, we aimed to determine the relationship between hepatic venous pressure gradient (hvpg) and the development of hcc or low sna in decompensated alcoholic cirrhosis patients. methods: child-pugh scores, meld scores, and hvpg at baseline, and the development of low sna (sna <130 meq/l) or hcc during follow-up were analyzed prospectively in 170 patients with decompensated alcoholic liver cirrhosis. the predictive values of different risk factors for the progression to the esld were investigated by multivariate analysis and the kaplan-meier method results: twenty-four patients developed hcc during the follow-up period. in the multivariate analysis, only baseline hvpg>15mmhg was an independent predictive factor for the development of hcc (relative risk (rr)=1.128, p<0.05). those with hvpg >15 mmhg showed a significantly shorter time for the development of hcc on kaplan-meier analysis. twenty patients developed low sna during follow-up. initial hvpg was also an independent predictive value for the development of low sna in the multivariate analysis (rr=1.169, p<0.05). those with hvpg>15 mmhg also showed significantly shorter times for the development of low sna on kaplan-meier analysis. conclusions: in decompensated alcoholic cirrhosis, hvpg may be a useful predictive factor for the development of hcc and low sna, both of which are characteristic of esld and poor prognosis. the effectiveness of the treatment of octreotide on chylous ascites after liver cirrhosis d.x. zhou 1,2 , h.p. hu 1,2 background: octreotide is a crucial drug used for treating patients with chylous ascites; however, there have been few reports related to octreotide that are being used in cirrhotic patients. thus, this thesis is designed to determine the effects of octreotide on patients with chylous ascites after liver cirrhosis. methods: eight patients were diagnosed with chylous ascites, on the basis of laboratory findings on ascites samples, between january 2003 and may 2008. octreotide was given to the six patients, while the remaining two were treated as a control. all patients had persistent peritonea drainage with the quantity and quality of the drainage fluid observed once every other day. all the necessary care was individually given to the patients during the therapy results: all patients properly received combined therapy including low fat and sodium diet, and diuretic and peritoneal drainage. the volume of the peritoneal drainage was reduced to zero in one of the six patients who received octreotide therapy, while the other five had the drainage volumes decreased from 2000 ml to 50 ml with a clear appearance and negative qualitative analysis of chyle for those two patients who did not receive octreotide therapy, the conditions of peritoneal drainage seldom changed both from the qualitative and quantitative aspects. conclusion: octreotide, along with combined therapy, can rapidly relieve portal hypertension and reduce fat absorption from intestinal mucosa. it appears to be an effective therapy available for the treatment of chylous ascites caused by liver cirrhosis. albumin <38g/l were the best predictors of large varices. a model using these predictors in a validation cohort study is planned. background-aim: cirrhosis is associated with raised acute phase proteins (app), irrespective of infection. it is, however, unclear whether their values differ significantly or whether a particular app might be more indicative of infection, and these questions were addressed in our study. methods: we measured serum crp, fibrinogen, ferritin, haptoglobin, 2-microglobulin, c3, c4, and c1 inhibitor in 88 consecutive, cirrhotic patients, on admission. all patients were investigated according to a standard protocol for infection. child-pugh scores (cps) were calculated. results of app were expressed as means sem and compared with the mann-whitney test. results: 19 (21,6%) patients, median age 60 years, (cps: a=0; b=7; c=12), were diagnosed with infection (spontaneous bacterial peritonitis=7; pneumonia=5; septic shock=4; extensive cellulitis=1; listeria monocytogenes meningitis=1; viral infection=1), while 69 (78%) patients, median age 59 years, (cps: a=25; b=28; c=16), showed no infection. although most app values were raised, there was no statistically significant difference between patients with or without infection, or among different cps groups, except for crp, which was significantly more raised in patients with infection (p<0.01). this difference remained even after cps a cases in the non-infection group were excluded from analysis. interpretation: a significantly raised crp in cirrhosis would seem to be independent of cps staging and should prompt a thorough work up to exclude infection. by contrast, the discriminating power of all other app in the face of possible infection is negligible. the predictive value of crp towards infection is under investigation prospectively. although bleeding from ectopic varices such as duodenal, jejunal, ileal, colonic, and rectal varices is less common, it can also cause life-threatening problem, which is often difficult to diagnose and treat successfully. here we present a novel endoscopic approach for hemorrhagic rectal varices using endoscopic injection sclerotherapy with ligation (eisl). patients and methods: in 2000-2008, we performed endoscopic treatment in 215 patients with portal hypertensive varices. among those, four cases of hemorrhagic rectal varices were treated with the combined evl and sclerosing technique. the etiology of portal hypertension included oen idiopathic portal hypertension and three hcv cirrhosis. all patients had a history of prior abdominal surgery or endoscopic treatment for gastro-esophageal varices. results: hemostasis was obtained easily by the evl initially. furthermore, to avoid recurrent bleeding, the patients underwent endoscopic varicerography injection sclerotherapy (evis) using 5% ethanolamine oleate with iopamidol and the feeding vein was sclerosed successfully with no major complication occurred during the entire course of the treatment. conclusions: it is important to recognize the possibility of ectopic varices as a cause of gastro-intestinal haemorrhage especially in patients with a history of variceal therapy or abdominal surgery. the eisl technique is useful to control the initial and recurrent bleeding from rectal varices. t. hirano 1 , t. okada 1 , j. yamanaka 1 , y. iimuro 1 , n. kuroda 1 , k. oh 1 , y. yoshida 1 , j. fujimoto 1 1 aim: interferon (ifn) therapy is a powerful treatment for hcv-related hepatitis and is known to decrease the incidence of progression of hepatocellular carcinoma (hcc). however, thrombocytopenia is a common side effect of ifn treatment, often leads to discontinuance without insufficient therapeutic effect. in this study, we investigated the efficacy and safety of laparoscopic splenectomy ( ) in reversing thrombocytopenia in patients with hepatitis c cirrhosis and portal hypertension. patients and methods: out of 41 patients who underwent ls in our department during aug 2003 and december 2007, 13 patients associated with portal hypertension. among these patients, three patients had hcc, and they were simultaneously underwent partial hepatectomy after splenectomy. platelet count, operative time, blood loss, complications and length of stay were calculated. results: thirteen patients underwent laparoscopic splenectomy; their mean age was 59 years (range 20 to 68 years). six patients were child's class a and seven patients were class b. mean operative time was 178 minutes (range 97 to 255 minutes). blood loss was little, and none required transfusion with packed red cells. a hand-assisted laparoscopic technique was used in four cases (30.8%). average length of stay was 12.7 days. there have been no major complications during follow-up. platelet counts improved from a preoperative mean of 62000/ul (44000 to 108000) to 273000/ul (126000 to 469000) postoperatively. six patients are ongoing ifn treatment without remarkable thrombocytopenia. conclusion: laparoscopic splenectomy is safe and in patients with portal hypertension and thrombocytopenia. it may allows these patients by reversing thrombocytopenia. background: hepatic encephalopathy (he) is a significant cause of mortality in advanced cirrhosis patients. l-acyl-carnitine has been suggested as an alternative treatment for patients with he patients. to assess the clinical efficacy of acetyl-l-carnitine in the treatment of hepatic encephalopathy in cirrhotic patient, especially in diminishing the recurrence and reduction serum ammonia level. methods: we performed a randomized placebo-controlled, cross-over study. we administered acetyl-l-carnitine to group 1 during 3 months first then placebo during later 3 months, and administering acetyl-l-carnitine to group 2 alternatively. results: between january 2008 and february 2008, thirty two selected cirrhotic patients were enrolled in this study. following randomization, the patients were divided into two groups (group 1=14, group 2=18). during administering acetyl-l-carnitine period, serum ammonia level was decreased significantly in both groups significantly (p=0.005, vs. p=0.001 respectively). however, during administering placebo period, serum ammonia level changes were not significant. in group 1, the first recurrence cases of hepatic encephalopathy were more than group 2(group 1=5, group 2=2), and the first recurrences were occurred during first 3 months in all groups. conclusion: our study demonstrates that acetyl-l-carnitine administration reduced serum ammonia level, but not definitely diminishing the recurrence of hepatic encephalopathy. sodium (na + ) and water retention are the most common abnormalities in cirrhotic patients and the magnitude varies from patients to patients. aim: to assess the relationship between the meld score and urinary excretion of na+ in non-azotemic cirrhotic patients. methods: fifty four cirrhotic patients with ascites and normal serum creatinine (<1.5 mg/ml) were admitted and placed on a low sodium diet (4g/day), while all diuretics were withdrawn for 3 days. the electrolytes (na + , k + , na + / k + ) were measured in a random urine and both the volume and na + concentration of urine collected for 8 h after administration of furosemide 80 mg i.v. were determined. results: table. conclusions: the meld score was significantly correlated with the degree of impairment of urinary na+ excretion. the ratio of na + /k + in a random urine specimen and furosemide-induced na+ excretion reflect the degree of impaired natriuresis in non-azotemic cirrhotic patients with ascites. background: portal hypertensive gastropathy (phg) is common finding in patients with liver cirrhosis and portal hypertension. despite portal hypertension remains the crucial trigger for the development of phg, the relationship between portal hypertension and phg has not been widely investigated. methods: fifty-three cirrhotic patients (48 males, mean age 51 years) who were performed hepatic vein catheterization between november 2006 and august 2008 were prospectively included in this study. the degree of phg was assessed according to the third baveno international consensus workshop, and classified three degrees as no, mild and severe. the hepatic venous pressure gradient (hvpg=whvp-fhvp) measurements were performed by triplicate in each case, and results were given as arithmetic means of the three determinations. result: hvpg values did not differ between the patients without phg (13.28±4.72 mmhg) and those with phg (14.91±3.84, p=0.187), nor between those with mild (15.31±3.69 mmhg) or severe phg (14.27±4.12mmhg, p=0.323). the degree of phg and hvpg did not differ regarding the etiology of the cirrhosis(p=1.0, p=0.085) nor regarding the child pugh classification(p=0.085, p=0.738). no correlations were found between the degree of phg and child pugh score, age, with or without ascites, albumin, bilirubin, creatinine, meld score and the degree of gastroesophageal varices. conclusions: our data show that the presence and the severity of phg does not correlate with the degree of hvpg, and that correlate with esophageal varices in patients with liver cirrhosis. introduction: phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the colonic wall due to fibrous degeneration of the submucosal layer and fibrotic sclerosis of the venous wall. there are a few reports those this entity might be related to portal hypertension with disturbed venous return from the colon and mesentery. case description: a 61-year old man with alcoholic liver cirrhosis presented with right lower abdominal pain/tenderness and bloody diarrhea. a colonoscopy revealed multiple circumferential ulcerations in the transverse colon and the scope could not get through the ascending colon due to luminal stenosis, showing histologic finding of ulcerative inflammation with inflammed granulation tissue. abdominal computed tomography demonstrated liver cirrhosis with splenomegaly, multiple portosystemic venous collaterals, diffuse vascular engorgement and the wall thickening of right proximal to mid ascending colon with increased density in the surrounding fatty tissue. a follow-up colonoscopy performed one month later showed still remained multiple ulcerations in the transverse colon and could not further advance to ascending colon. superior mesenteric angiography revealed no main branch occlusion but pooling at the venous phase on ascending colon. a right hemicolectomy was performed because of the colonic obstruction. gross findings on operation showed thickening of the cecum and ascending colon. microscopic examination showed fibrous thickening in the submucosa, abundant neurovascular bundles in the mesentery and several intravascular hyaline thrombi of the mesenteric vessels. here we report the first case of early stage of phlebosclerotic colitis in a cirrhotic patient in korea. spontaneous bacterial peritonitis (sbp) is one of the severe complications in advanced cirrhotic patients with a high mortality rate. although a more rapid diagnosis should lead to the better survival, it takes several days to detect the causal bacteria from ascitic fluid cultures. furthermore, despite the use of sensitive methods, ascitic fluid cultures were negative in more than 50% of patients with suggestive clinical manifestations of sbp. therefore, diagnosis of sbp is based on the polymorphonuclear leucocytes (pmn) cell count in the ascitic fluid. the hybrizep kit (fuso pharmaceutical industries, osaka, japan) detects the dna of bacteria that have been phagocytized in neutrophils and macrophages, using in-situ hybridization method within one day. here we present a case of the patient for whom the hybrizep kit was used to detect the causal pathogen of sbp. a 76-year-old man had been admitted for the treatment of ascites and esophageal varices. one week after the admission, he complained abdominal pain and fever. because the pmn cell count in ascites fulfilled the criteria of sbp (1141/mm 3 ), we started an empirical antibiotic therapy without waiting for a result of the culture, and his symptoms improved within a few days. on the following day of the onset, in situ hybridization showed the positive signals by the ek probe, which detected the genomeic dna of e.coli species. however, the ascitic fluid culture was negative. this case suggested that the hybrizep kit was useful for the rapid diagnosis of sbp with high sensitivity. background: it has not been known that the hemodynamic effect of a portal hypertension for splenomegaly or esophageal and gastric variceal formation. this study was performed to access the parameters of doppler ultrasonography associated with splenomegaly or varices in patients with cirrhosis. patients and methods: from may 2007 to may 2008, 144 cirrhotic patients were performed the doppler ultrasonography. 91 of these patients were accessed the severity of varices endoscopically. the three dimensional volume of spleen was measured from a length, width and thickness on sonography. results: the splenic volume (415.2ml vs 505.6ml, p=0.048) and blood flow of main portal vein (12.6cm/s vs 15.1cm/s, p=0.012) were statistically significant different in alcoholic (38/144) and non-alcoholic (105/144) cirrhosis groups. the splenic volume (600.1ml vs 384.4ml, p=0.001), damping index (0.52 vs 0.38, p=0.024), and blood flow of main portal vein(12.5cm/s vs 15.7cm/s, p=0.006) were statistically significant different in esophageal variceal groups (55/91) and non-esophageal variceal groups(36/91). the only splenic volume (669.4 ml vs 461.7 ml, p=0.004) were statistically significant different in gastric variceal groups (24/90) and non-gastric variceal groups (66/90). the hemodynamic parameters venous ammonia and cff at baseline and after one month of treatment with lactulose. mhe diagnosed by abnormal psychometry and/or p300erp.response defined by normalization of abnormal test parameters. results: mhe diagnosed in 35(58%) patients. of 35 patients 26 (74%) had both abnormal psychometry and p300erp whereas30 (86%) alone had abnormal psychometry, 31 (89%) had abnormal p300erp.cff was <39hz in 28(80%) patients. mhe recovered in 55% with treatment and cff >39hz was seen in 22(69%) of 32 patients. cff sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy before and after treatment is shown in table. conclusions: critical flicker frequency is a simple and accurate test without any age or literacy dependence for the diagnosis and recovery of patients with mhe. background/aims: endoscopic injection of n-butyl-2-cyanoacrylate (histoacryl) is an effective treatment of varix bleeding. but nontarget embolizations and septicemia are unwanted complications. we evaluate the risk factors for complications. methods: thirty-three patients with esophageal or gastric varix bleeding received endoscopic histoacryl therapies (54 procedures). baseline varix size, ctp score were checked. serum leukocyte, blood culture and body temperatures were repeated checked within one week after procedure. average volume of histoacryl per each session was 1.6 ml, and dilution volume ratio of histoacryl/lipiodol was 1/1 or 1/2. results: average of ctp score was 8.0 ± 1.7. three cases of septicemia were correlated with ctp score rather than session frequency or injection volume. two cases of systemic embolizations (pulmonary and splenic arterial embolism) were correlated with high lipiodol dilution ratio (1/2) and lipiodol volume rather than histoacryl volume or ctp score. conclusion: ctp score, lipiodol volume and dilution ratio of histoacryl/lipiodol were significant risk factors for complications. detection of circulating toll-like receptor 2 and 4 and cd4+cd25+ regulatory t cells in patients with hbv-related liver cirrhosis x.q. wang 1 , y. zhang 1 , x.f. bai 1 , j.q. lian 1 1 background : to detect circulating cd4 + cd25 + regulatory t cells and toll-like receptor(tlr)2 and tlr4 expression on the peripheral blood mononuclear cells (pbmcs) of patients with hbv-related liver cirrhosis (lc), and to explore the correlation between them. methods: pbmcs isolated from 14 lc patients , 21 chronic hepatitis b (chb) patients and 16 normal controls(nc) were stained with fluorescent labeling anti-tlr2-pe, anti--tlr4-apc, anti--cd14-fitc monoclonal antibodies and anti-cd4-percp anti-cd25-fitc anti-cd127-pe. samples were collected and detected of three-color immunofluo rescence by flow cytometry. results: the expression of tlr2 and tlr4 were significantly up-regulated in patients with lc than those in the controls.the expression of tlr2 was significantly increased in patients with lc than those in patients with chb, but there were no differences of tlr4 expression between lc and chb.treg/cd4 + t cells were significantly increased in patients with chb than those in patients with nc and lc, but there were no differences between lc and nc. there were no correlation between the expression of tlr2,tlr4 and treg in patients with lc . the expression of tlr2 and tlr4 on pbmcs in patients with lc were positive correlation.the expression tlr4 and hbv dna level were negative correlation in patients with lc. conclusion: the expression of tlr2 and tlr4 were up-regulated on pbmcs in patients with lc. it seems to be expression of tlr2 and tlr4 invovlved in the pathogenesis of lc. evaluation of 13c-phenylalanine breath test for the measurement of hepatocyte function in patients with chronic liver disease z.j. bao 1 , d.k. qiu 2,3 , x. ma 2,3 , g.s. zhang 1 , y.q. huang 1 , z.p. fan 2,3 , s.m. yin 1 1 huadong hospital, fudan university, 2 renji hospital, shanghai jiao tong university school of medicine, 3 background: the objective is to investigate whether the 13c-phenylalanine breath test(pbt) would be useful for the evaluation of hepatic function in patients with chronic hepatitis b, liver cirrhosis and minimal hepatic encephalopathy (mhe). methods: l[1-13c] phenylalanine was administered orally in a dose of 100 mg to 80 patients with liver cirrhosis, 20 with chronic hepatitis b and 20 healthy subjects. the pbt was measured at 8 different time points (0, 10, 20, 30, 45, 60, 90, 120 min) to obtain the values of delta over baseline, percentage 13co2 exhalation rate and cumulative excretion (cum). the relationships of the cumulative excretion with the 13c-%dose/h and blood biochemical parameters were investigated. results: the 13c dose h -1 at 20 and 30 min combined with the cumulative excretion at 60 and 120 min showed correlations with the chronic liver diseases, especially child-pugh score and mhe or not. and the data showed correlations with serum albumin hemoglobin platelet and child-pugh score. prothrombin time, total and direct bilirubin were significantly increased, while serum albumin, hemoglobin and platelet, the cumulative excretion at 60 and 120 min values decreased by degrees in healthy controls, child-pugh a, b, and c patients (p<0.01). similar results of pbt were in the patients with and without mhe, while only prothrombin time prolonged and total bilirubin increased (p<0.05). conclusions: the pbt can be used as a non-invasive assay to evaluate hepatic function in patients with liver cirrhosis and mhe. the %13c dose h-1 at 20 min, %13c dose h-1 at 30 min and cumulative excretion at 60 min may be the key value for determination at a single time-point. branched chain amino acids in improving survival and decreasing risk of liver failure among cirrhotic patients: a meta-analysis h. flores 1 , e.l. ang 1 , n. iv estanislao 1 1 philippine general hospital background: the state of a patient's nutritional status greatly affects disease outcome. among cirrhotic patients, approximately 60-90% are in a state of protein-energy malnutrition. hence, adequate nutritional support is essential to improve their general medical condition and long term prognosis. several studies have shown than branched chain amino acids (bcaa) may be of benefit for this purpose. it is the aim of this study to evaluate the effectiveness of diet plus bcaa compared to diet alone in improving survival and in decreasing liver failure among cirrhotic patients. methods: pubmed, cochrane, and embase search was done for articles which compared the clinical effects of bcaa supplementation versus diet alone among patients with liver cirrhosis. the following free-text terms and mesh words were used -"branched chain amino acids", "amino acids, branched chain", "bcaa", "liver cirrhosis", "cirrhosis", "randomized controlled trials'" and "meta-analysis". after critical appraisal of the included studies, a random effects model using odds ratio was used to synthesize the results (revman 4.2). results: 3 rcts were included for analysis with a total study population of 836. combination of the studies showed a significant decrease in the risk of liver failure (or 0.45, 95% ci 0.25-0.82, p=0.009) and a trend towards benefit in improving survival (or 0.57, 95% ci 0.27-1.17, p=0.12). conclusions: the overall trend appears to show benefit in the use of branched chain amino acids for patients with cirrhosis with respect to liver failure and survival. background: the proxisome prolifrator-activated receptor gamma (ppar ) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation, carcinogenesis and gastric ulcer. on the other hand, the frequency of gastrointestinal ulceration is higher in cirrhotic patients compared with the normal population. the present study was designed to investigate the effect of specific ppar ligand, pioglitazone, on the mucosal lesions induced by ethanol in cirrhotic rats and the possible involvement of nitric oxide in the pioglitazone effect. methods: cirrhosis was induced by surgical ligation of bile duct and sham-operated rats served as controls. both cirrhotic and sham rats were kept for 28 days after the operation. different groups of sham and cirrhotic animals received saline, or 5, 10 or 15 mg/kg pioglitazone, daily during last 5 days of the fourth week after the surgery. another 2 groups of bdl or 2 groups of sham rats received l-name, a non selective inhibitor of nitric oxide synthase, alone or along with 5 mg/kg pioglitazone for 5 days. on day 28, rats were killed 1 hour after ethanol administration and the area of gastric lesions was measured. results: the ethanol-induced gastric mucosal damage was significantly more sever in cirrhotic rats than sham-operated ones (p < 0.001). pretreatment with pioglitazone dose dependently attenuated gastric lesions induced by ethanol in both sham and cirrhotic rats, but this effect was more significant in cirrhotic ones. concurrent treatment of l-name and pioglitazone decreased the ulcer index in bdl rats more than the groups that received l-name or pioglitazone alone. conclusion: we conclude that chronic treatment with pioglitazone exerts a potent gastroprotective effect on the stomach ulcers of cirrhotic rats probably due to inhibition of nitric oxide synthase. inhibition of phosphodiesterase 5 -a novel therapeutic strategy for portal hypertension l. halverscheid 1 , p. deibert 2 , b. pannen 1 , r. schmidt 2 , m. roessle 2 , w. kreisel 2 1 university hospital duesseldorf, germany, 2 university hospital freiburg, germany introduction: the no-cyclic gmp system is a key factor in the regulation of splanchnic and hepatic blood flow and may be a target for medical treatment of portal hypertension. clinical data have shown that inhibitors of phosphodiesterase 5 (pde5) lower portal pressure in cirrhotics. methods: we monitored in rats the effects of the pde5 inhibitors vardenafil and sildenafil on systemic and hepatic hemodynamic parameters up to 60 minutes after the drug. the drugs were administered intravenously into the tail vein at 1 (group a), 10 (group b), and 100 g/kg body weight (group c). 0.9% nacl was the control. n = 7 for each group. results: the most prominent changes were observed in the vardenafil b group: mean arterial and portal venous pressure decreased (-9%, -8%), as well as portal venous, hepatic arterial, and systemic vascular resistance (-31%, -30%, -12%). portal venous and sinusoidal flow increased (+31%, +13%). in the vardenafil c and sildenafil b and c groups there was an increase of portal venous flow by 20-30%, an increase of sinusoidal flow by 12-30%, and a decrease of portal venous resistance by about 25%. there was a trend for reduction of portal venous pressure. conclusions: vardenafil and sildenafil influence portal hemodynamics in the rat. portal venous flow increases by 20-30%, portal venous resistance decreases by >25%. dependent on the dose, portal venous pressure decreases significantly. these data yield further evidence that pde5 inhibitors may be a novel therapeutic option for portal hypertension. groups of liver cirrhosis e. havrilyuk 1 1 lviv national medical university introduction: rupture of esophageal varicose resulting in posthemorrhagic anemia is a common life-threatening complication of liver cirrhosis. but it is not clear, why the other patients, having the same degree of sclerosis and histologic activity index, die from hepatocellular failure or other reasons. aims & methods: 3713 autopsy cases performed in lviv regional hospital in 2004-2007 were analyzed. screening of slides with liver tissue allow to select 580 cases (15,6%) with cirrhosis (complete and incomplete), which are examined in order to evaluate the frequency of lethal portal hypertension complications in the different etiologic groups of liver cirrhosis. results: according to the etiologic factor the following groups of liver cirrhosis were examined: alcoholic disease (49,8%), viral hepatitis (7,8%), nonalcoholic steatohepatitis (14,8%), secondary biliary cirrhosis (2,9%), cardiac sclerosis (0,9%), combined lesions (11,9%) and cryptogenic cirrhosis (11,9%). analysis shows that in 287 cases (49,5%) patients die from cirrhotic complications (hepatocellular failure, jaundice, portal hypertension) and only in 105 cases (18,3%) -from posthemorrhagic anemia caused by rupture of esophageal varicose. in the latter cases correlation between the etiologic types of cirrhosis is almost the same, as in the main group and only alcoholic lesions (60%) and biliary cirrhosis (6,7%) are more frequent. conclusion: analysis shows that development of lethal complications of portal hypertension can not be explained only by etiologic factor. probably additional stimuli are more important for morphogenetical variants of cirrhotic transformation. patients with viral cirrhosis k. mumtaz 1 , s. ahmed 1 , h. ali shah 1 , s. hamid 1 , w. jafri 1 background and aims: increased nitric oxide (no) production is incriminated in the pathogenesis of arterial vasodilation and hyperdynamic circulatory state in non cirrhotic models of portal hypertension (pht). we investigated the relative roles of constitutive nos (enos) and inducible nos (inos) isoforms in the development of rabbit models of endotoxemia induced portal hypertension (eipht) methods: eipht was induced by chronic injection of lipopolysaccharide via an indwelling cannula placed in the gastrosplenic vein of rabbit and maintained for 6 months. the concentration of no, expression of nos (enos and inos) mrna and protein was measured in eipht and sham operated control animals. results: rabbits with eipht compared with controls had raised portal pressure (in mmhg-14.34±1.78 vs 6.30±0.60;p<0.05;1mo; 14.91±0.56 vs 7.04±0.42; p<0.05, 3mo; 19.8±3.10, vs 10.2±4.80;p<0.05), arterial hypotension (in mmhg-65.40±3.2 vs 80.04±1.40, p<0.05, 1mo; 62.90±5.40 vs 76.05±2.60,p<0.05, 3mo; 65.85±2.50 vs 79.1±5.10, p<0.05,6mo), splenomegaly (in g-0.92±0.14 vs 0.60±0.13,1mo; 0.90±0.16 vs 0.62±0.04, 3mo; 0.97±0.12 vs 0.67±0.07, 6mo), normal liver functions and preserved hepatic architecture at 1,3 and 6 mo. serum levels of no2 as well as the no3 were significantly elevated in eipht rabbits as compared to the controls. the expression of enos, at the level of mrna, was significantly increased in eipht rabbits consistent with increased levels of expression of inos as compared to the controls. the enos but not inos protein expression was elevated in eipht than control rabbits. conclusion: vascular dysfunction in the splanchnic circulation during the development of endotoxemia induced portal hypertension is predominantly characterized by enos and partly by inos gene up-regulation. liver cirrhosis contributed to the immunocompromised status by shedding the membranous tnfrii t.n. lin 1 , c.h. chao 1 , i.s. sheen 1 , y.p. ho 1 , w.t. chen 1 , c.j. lin 1 , c.t. yeh 1 , c.y. lin 1 1 liver research unit, linkou medical center, chang gung memorial hospital, chang gung university, taoyuan, taiwan background & aim: patients with decompensated liver cirrhosis (dlc) were regarded as immunocompromised, reflected by high incidence of bacterial infection. paradoxically, the proinflammatory cytokine like tnfincreased significantly in patients with dlc even in the face of this immunocompromised status. on the other hand, regulatory t cell (treg cell) is believed to play an important role in inhibiting immune responses, including innate immune responses like blockade of tnf-effect through soluble tnfrii. here, we studied the role of treg cells and tnfrii in patients with decompensated liver cirrhosis. patients and methods: 33 healthy volunteers and 78 cirrhotic patients were enrolled. the percentage of treg cells were enumerated by flow and serum levels of il-10, tgf-and tnf-by elisa. results: the percentage of treg cells increased significantly in patients with dlc associated with increased serum levels of il-10 and tgf-. in addition, these treg cells were mainly memory type reflected as high cd45ro. furthermore, the tnfrii expression increased significantly on these treg cells of dlc. interestingly, these membranous tnfrii on treg cells could be shed-off. lastly, we found the serum soluble tnfrii concentration increased significantly in patients with dlc when compared with normal volunteers. conclusion: our results demonstrated memory treg cells with high tnfrii expression increased significantly in patients with decompensated liver cirrhosis that could possibly blocked the biological effect of tnf-by shedding membranous tnfrii and contributed to the immunocompromized status of dlc. background: portal pressure measured as hepatic venous pressure gradient (hvpg) correlates with severity of portal hypertension and the development of complications. hvpg measurement is invasive. recently, liver stiffness measurement has been shown to correlate with liver biopsy and helps predict outcome in chronic liver disease patients. this study was conducted with the aim to study the correlation between portal pressure as measured by hvpg and liver stiffness as measured by fibroscan among patients with portal hypertension due to various causes. methods: between august and september 2008, consecutive patients with portal hypertension were included and were subjected to hvpg measurement and fibroscan (echosens, france). results: of the 18 patients with portal hypertension, both hvpg and liver stiffness were measurable in 12[9 (75%) males; mean age 37.5(10.6) years]. the etiological distribution was hbv related cirrhosis in 3 patients, hcv cirrhosis in 3, cryptogenic cirrhosis in 2, alcoholic cirrhosis in 2, hbv and alcoholic cirrhosis in 1and primary extra-hepatic portal vein obstruction in1. the mean hvpg and liver stiffness of this group were 13.9 (5.1) mm hg and 26.6 (16.5) kpa respectively. there was a strong positive correlation between hvpg and liver stiffness [r = 0.708; p = 0.01]. conclusions: non-invasive measurement of liver stiffness correlates well with invasive measurement of portal pressure. liver stiffness measurement could be used as a prognostic indicator to predict the severity of portal hypertension. endpoints were rate of rebleeding and mortality till day 30 after inclusion and to see for any adverse events. results: the bleeding was stopped in all 15 patients (100%). rebleeding till day 30 was observed in 4 (26%) patients (2 each in group a and b). total 2 patients (13%) died (1 each in both groups) due to rebleeding. transfusion needs were higher in group a (4.2±2.8 versus 2.3±2.1, p<.05). serious adverse effects leading to treatment discontinuation were not seen in any patients in both groups. conclusion: prolonging terlipressin treatment did not confer any significant decrease of mortality or bleeding recurrence. however transfusion requirements were significantly decreased in patients receiving prolonged treatment. serious adverse effects leading to treatment discontinuation are rare. poster exhibition -miscellaneous poster session, hall 5b background: it is important to know the prevalence of atp7b gene mutations of different geographical areas to justify the local screening strategies for wilson disease (wd). materials: eleven unrelated lithuanian families, including 13 wd patients were tested. genomic dna was extracted from whole venous blood using a salt precipitation method. firstly, semi-nested pcr technique was used to detect the c.3207c>a (p.h1069q) mutation. patients not homozygous for c.3207c>a (p.h1069q) mutation were further analyzed. the 21 exons of the wd gene were amplified in a thermal cycler. direct sequencing of the amplified pcr products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer. results: total of 13 wd patients (mean age 26.4 years; range 17-40; male/female, 3/10) presented with hepatic disorders and 16 their first degree relatives were studied. some of wd patients in addition to hepatic symptoms have had extrahepatic disorders (haemolytic anaemia 3; fanconi syndrome 1; neurophsychiatric and behavioural disorder 2). twelve of 13 (92.3%) wd patients have had c.3207c>a (p.h1069q) mutation, 6 of them in both chromosomes, 5 were presented as compound heterozygotes with additional c.3472 -82delggtttaaccat, c.3402delc or c.3122g>a (p.r1041q) mutation. for one patient with liver cirrhosis and psychiatric disorder no mutations were found. out of 16 first degree wd relatives 11 (68.7%) were heterozygous for c.3207c>a (p.h1069q) mutation. conclusion: c.3207c>a (p.h1069q) missense mutation is characteristic for lithuanian wd patients. even 92.3% of wd patients with hepatic presentation of the disease are homozygous or compound heterozygote for this mutation. background: diabetic dyslipidemia is a crucial problem of diabetic patients with inadequate control. we investigated the relationship between glutamic-pyruvic transaminase (gpt) and high-density lipoprotein cholesterol (hdl-c) in diabetic patients. methods: with informed consents, we recruited outpatients with diabetes at a hospital in rural area in taiwan in [2004] [2005] [2006] [2007] . anthropometric measures, blood tests and urine screening were examined in diabetic patients. results: overall, there were 1241 diabetic patients aged 19-91 years enrolled in this study and 660 (53.2%) of them had low hdl-c. diabetic patients with the highest quintile of gpt had higher average of body mass index (p<0.0001), diastolic blood pressure (p = 0.003), but lower average of hdl-c (p<0.0001) compared with diabetic patients with the lowest quintile of gpt. the prevalence of obesity (45.7% vs. 24.8%, p<0.0001) and low hdl-c (64.6% vs. 48.3%, p<0.0001) were higher in diabetic patients with highest quintile of gpt than in diabetic patients with lowest quintile of gpt. in the multivariate logistic regression, diabetic patients with highest quintile of gpt had higher odds ratio (or) of low hdl-c compared with diabetic patients with lowest quintile of gpt (or = 1.88, 95% confidence interval [ci] = 1.21-2.92). the corresponding or of low hdl-c in patients aged 70 years and older was 3.30 (95% ci = 1.15-9.47). conclusion: high gpt is one of factors associated with low hdl-c in diabetic patients. the effect of desferrioxamine as supplement to cefotaxime in the treatment of spontaneous bacterial peritonitis na. seda 1 , m. el-hamamsy 2 , r. el-wakil 3 , m. al azizi 4 1 ain shams specialized hospital, 2 faculty of pharmacy,ain shams university, 3 faculty of medicine,ain shams university, 4 faculty of pharmacy ,ain shams university background: oxidative damage lead to cell damage, organ dysfunction and death in sepsis. desferrioxamine (dfx), an antioxidant iron chelators. the aim was to assess the efficacy of desferrioxamine supplemented to cefotaxime in the treatment of spontaneous bacterial peritonitis (sbp) in cirrhotic patients. methods: thirty patients divided into two groups: group i (n=15) with sbp and receiving cefotaxime (1g iv every12 hours) alone and group ii (n=15) with sbp receiving cefotaxime (1g iv every 12 hours) with desferrioxamine (500mg im twice daily).all patient were monitored for seven days, their vital organs were screened and their ascitic fluid was assessed completely including microbiological investigations. results: the concomitant administration of desferrioxamine with cefotaxime significantly at (p<0.001)and(p<0.01) improved the therapeutic outcome and the cure rate after 5 days of treatment as compared to patients using cefotaxime only. conclusions: desferrioxamine can improve the therapeutic outcome by preventing iron-induced organ damage and inhibiting bacterial growth. oligella ureolytica is a gram-negative, nonfermenting rod that is infrequently recovered from clinical specimens and is most commonly isolated from the urine of patients with chronic indwelling urinary catheters or other urinary drainage systems. bacteremia due to this organism is an extraordinary finding. we describe here a case of oligella ureolytica being detected in the blood of a patient with decompensated cirrhosis. a 51-year-old male man was admitted to hospital with 9-month duration of debility, poor appetite and abdominal distension. decompensated cirrhosis was diagnosed based on clinical findings such as hepatic face, ascites, edema of lower limbs and icteric sclera. laboratory results showed positive serum anti-hcv and high serum hcv rna level. the patient received a therapeutic regimen of pegylated interferon alpha 2a plus ribavirin after being admitted to hospital. during hospital stay, a fever of 2-day duration with shivering occurred to the patient. three blood cultures were drawn, which all grew oligella ureolytica in pure culture. the organism was identified by the viteck 2 compact (biomerieux, france). additional tests for identification resulted positive for nitrate reduction and urea hydrolysis, strongly positive for phenylalaninedeaminase activity and showed no growth at 42.7c. tests for nitrite reduction and motility resulted negative. the organism was resistant to amikacin, cefoperazone, levofloxacin, piperacillin/tazobactam, trimethoprimsulfamethoxazole, aztreonam, cefotaxime, piperacillin and was susceptible to gentamicin, imipenem, meropenem, and netilmicin. a 14-day of combined therapeutic regimen with cefminox and isepamicin was administered to the patient. within 2 days, the patient became afebrile. background: endoscopic ultrasound (eus) is often performed in patients with unexplained liver tests to assess the gallbladder, bile ducts and pancreas. an unremarkable eus exam and negative hepatology workup often leads to a liver biopsy. eus may provide histopathologic evaluation of the liver in these cases under direct, real-time visualization. aim: to assess the feasibility and efficacy of eus guided core biopsy of the liver in a porcine model. methods: female pigs were used and live procedures were performed under general anesthesia. a linear echoendoscope was used and the liver identified endosonographically. transgastric core biopsies of the liver were obtained with a 19 gauge quick-core ultrasound biopsy needle (wilson-cook) and sent for histopathologic evaluation. live animals were euthanized at the end of the procedure and necropsy performed. results: core biopsies of the liver biopsy were obtained in 4 animals (1 cadaver and 3 live anesthetized). a total of thirteen needle passes were made (mean 3.25; range 2 -4 needle passes per animal) and a visible core of tissue obtained. the maximum length of liver tissue obtained was 10 mm and considered adequate for assessment as more than one such specimen could be obtained. microscopic evaluation confirmed liver tissue. no complications were noted. necropsy did not show any evidence of bleeding, perforation or damage to surrounding structures. conclusion: eus-guided liver biopsy is feasible and can be performed at the time of routine echoendoscopic exam in select patients undergoing eus examination for abnormal liver tests. background: as the common indexes, alt, ast and plt play an important role in disease diagnosis, treatment and prognosis. many researchers suggested that there was inflammatory changes and fibrosis in chronic hepatitis b and c patients whose alt level was persistently normal. a large sample investigation showed that the serum level of alt in healthy persons is lower than the normal reference value. this study re-evaluated the normal serum level of alt, ast and plt. methods: 3815 people were enrolled in the study between sep. and oct. 2007. the platelet count and serum alt and ast levels were measured. frequencies, one-sample kolmogorov-smirnov test and nonparametric tests were used to analyze the difference between age groups, male and female, glucose groups, cholesterol groups and triglyceride groups. result: in the five groups, there is significant difference in alt and ast levels between male and female. in group 1, the alt and ast levels showed a significant difference between different age groups, between different glucose groups and triglycide groups. in the three groups the plt level is significantly different between male and female, and the serum level in male is higher than female. there is significant difference between different age groups conclusion: the serum levels of alt, ast and plt are all significantly different between male and femal. there is significant difference between different genders and age groups for plt. the serum level of plt is higher than the reference value. background/aims: myeloproliferative disorders (mpd) (like polycythemia vera, essential thrombocythemia and primary myelofibrosis) are responsible for 50% cases of hepatic venous thrombosis (hvt) and 35% of portal venous thrombosis (pvt) in western series. latent form of mpd lacks the characteristic blood picture and may be classified as idiopathic thrombotic disorder. a point mutation at val617phe of janus kinase 2 tyrosine kinase gene (jak2 v617f mutation) occurs in high proportion of the patients with mpd. this non-invasive test with high positive predictive value is now considered to be essential for diagnosis of various mpd. this test may be useful in diagnosing latent form of mpd in splanchnic venous thrombosis methods: 232 patients with confirmed pyogenic liver abscesses admitted from 1995 to 2007 in our institution were included. there were 145 men and 87 women ranging in age from 19 to 91 years. the medical records were reviewed for clinical, laboratory and radiographic characteristics. results: among 232 patients, 81 (34.9%) experienced at least one complication. there were 67 pulmonary (pleural effusion, pneumonia, empyema) complications, 16 septic shock, 12 acute renal failure, 2 abscess rupture, 2 pseudomembranous colitis, and 2 pericardial effusion. the predictive factors for its complications were: systemic inflammatory response syndrome (sirs, 2 factors), thrombocytopenia ( 80,000/ml), hypoalbuminemia ( 3.0g/dl), elevated ast or alt (>200 iu/l), hyperbilirubinemia ( > 2.0 mg/dl), k. pneumonia, air within abscess cavity (p<0.05). conclusions: the incidence of complications in the pyogenic liver abscess was 34.9%. the various predictive factors of complication should be monitored carefully. further large scaled study should be warranted. background/aims: hepatic iron deposition is a common feature in chronic hepatitis c (ch-c), however, whether it could enhance the progression of fibrosis or not is controversial. the aim of this study was to evaluate the status and significance of hepatic iron deposition in the korean patients with chronic hepatitis c. methods: untreated, 78 ch-c patients who underwent liver biopsy were included. the hepatic iron was assessed by scheuer's scoring system, and activity, fibrosis, and steatosis were scored by a pathologist in a blind manner to the clinical features. clinical and laboratory data including serum iron indices, virological, biochemical results were analyzed to search for significant factors associated with hepatic iron deposition. results: hepatic iron staining was positive in 26(33%). among 26 patients with hepatic iron deposition, serum levels of ferritin (p=0.005) and -fetoprotein (p=0.002), and body mass index(bmi) (p=0.039) were significantly elevated. there was no significant association between the degree of hepatic iron deposition and fibrosis stage (p=0.321), although elevated levels of serum hyaluronic acid (p=0.049), -glutamyl transpeptidase (p=0.028), and prothrombin time (p=0.012) were associated with advanced fibrosis. conclusions: hepatic iron deposition in asian-pacific ch-c patients seemed to be neither frequent nor related to hepatic fibrosis, but related to obesity. therefore, phlebotomy might not commonly applicable to this area. further studies on the pathogenic role of iron in ch-c in asian-pacific countries are warranted. a late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture, necrosis of hepatocytes and the formation of regenerative nodules contributes to cirrhosis. limitations like organ donors shortage, high cost, absence of proliferation in cultured hepatocytes, inherent risks of infection, rejection in xenogenic cells and other socio-economical complications emerges advanced regenerative human hepatic stem cells(hhpscs) transplantation. hhpscs are located in the ductal plates in fetal and canals of hering in adult livers [schmelzer et al.(2006) ]. hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [schmelzere etal(2006) ].hhpscs express cd326(epcam)marker. scjelzer etal demonstrated that during embryogenesis 90% of the epcam positive cells had hepatoblast phenotype. in animal study, on transplantation of freshly isolated hhpscs in scid mice results in mature liver tissue expressing human-specific proteins. recently, we(aleem etal 2008)have shown clinical improvement in study in patients with crigler-najjar syndrome, biliary atresia using hhpscs infusion. in the present study we transplanted hepatic progenitors to five subjects of end stage liver cirrhosis with meld score >30. hhpscs were sorted using macs with cd326 antibody microbeads and infused through hepatic artery via femoral artery catheterization, a safe procedure provided portal pressure to monitor cell infusion route in order to prevent vascular thrombosis. all the patients showed improvement clinical and functional biochemical parameters after first month of cell infusion. ascites was decreased and changed encephalopathy grade into normal level was observed. meld score system falling to normal level from >30 to <22 after infusion. 1 the aga khan university patients. the apri of 1.5 in combination with a cut-off ha of 300 ng/ml can best detect patients with moderate to severe fibrosis (stages 2-4). it has a ppv of 93.7%. also, for patients without moderate to severe fibrosis, the test is hardly ever positive (specificity of 98.9%). but the apri of 1.5 in combination with different ha as cut-off points is not possible to detect patients with no or mild fibrosis. objectives: terlipressin is used in esophageal variceal bleed (evb) along with endoscopic band ligation (ebl) for 3days (uc). due to its high cost, it was stopped <3days (sc) who could not afford & were stable after achieving hemostasis with ebl. we retrospectively assessed the efficacy of sc vs uc of terlipressin for control of evb and length of stay. conclusion: the apri of 1.5 in combination with ha 300 ng/ml as cut-off points to predict patients with moderate to severe fibrosis (stages 2-4) is an easy and accurate method. methods: patients with evb who had achieved hemostatsis with ebl from jan 2004-dec 2005 were included. all were managed on standard protocol on hospital variceal bleeding pathway. the course of terlipressin as sc or uc was based on patient's inability to afford the cost of hospitalization and terlipressin. the efficacy of terlipressin in the control of evb was defined based on baveno iii criteria. results: total of 117 patients were admitted during the study period. out of them, 66 received uc & 51 sc of terlipressin. the base line characteristics were comparable except younger age in sc. there were 2 re-bleed (3%) in uc and 1 (2%) in sc terlipressin group. the length of stay was shorter in sc group. (2.47±0.57 vs 6.15±2.92 days). conclusions: sc seems as effective as uc terlipressin in the control of evb after initial control of hemostatsis with ebl and may reduces the length of hospital stay. rcts are needed to assess this as all stable patients may not need to continue terlipressin for 72 hours. background/aims: to analyze the relationship between conventional laboratory results and death risk in patients with esophageal varices bleeding due to cirrhosis (cevb), and establish a simple model for timely predicting death rsik of the patients. outcome of patients with gastro-oesophageal bleeding in a tertiary center j. wat, w.h. li, m.t. cheung methods: the medical documents of cevb patients were reviewed retrospectively and the data were collected. univariate and multivariate logistic regressions were performed, in which the discharged results (survival or death) as dependent variable and the results of liver function, kidney function, serum electrolytes and blood cell analysis as independent variables. the multivariate regression equation was as the model for the prediction of patient outcome and its predictive performance was evaluated. objectives: to determine the rebleeding rate, mortality and long term survival in cirrhotic patients presented with acute gastro-oesophageal variceal bleeding. method: this is a retrospective review of adult patients who were admitted to our hospital with the diagnosis of acute gastro-oesophageal variceal bleeding for the first time regardless of their underlying causes for cirrhosis. the study period was from january 2000-october 2008. data were collected from our hospital computer system and records. results: in univariate regression, the significant positive variables for death outcome were dbil, akp, k, wbc and plt, and the significant negative variables were tp, ap, a/g, na, cland ca 2+ . the variables entered the multivariate regression are alt, tbil, dbil, gp, a/g, cr, na + , cl -, ca 2+ , wbc, hb, plt. the sensitivity, specificity and accuracy of the regression model for predicting death of cevb patients were 97.1%, 95.1% and 95.8%. results: a total of 172 patients were included in this study, with 122 male and 55 female. their mean age was 61.9. the initial failure rate in endoscopic haemostasis was 15.1%. the 5-day and 6-week mortality rates were 11.8% and 21.9 %, respectively. poor child's grading, multiple columns of oesophageal varices, high grade of varices, failed initial endoscopic haemostasis, presence of inoperable hcc, low platelet count on admission, and short duration from index bleed to rebleed were factors associating with increased risk of 6-week mortality (p < 0.05). mean duration from index bleed to first rebleed was 15.1 months. poor child's grading and presence of inoperable hcc were associated with both early or multiple rebleed (p <0.05). overall, 37.2% of our patients developed rebleed before their variceal eradication. 5-year survival in patients with child's a, b and c were 65%, 22%, and 10%, respectively (log rank test p 0.000). conclusions: the liver function, kidney function, serum electrolytes and blood cell analysis are generally independent factors for cevb patient death risk, especially dbil, a/g and ca 2+ . the established model shows a excellent predictive performance. an imbalance in plasma amino acids of advaced cirrhotic patients impairs the maturation of dendritic cells via mtor/s6k signaling pathway e. kakazu 1 , y. ueno 1 , y. kondo 1 , k. fukushima 1 , m. shina 1 , j. inoue 1 , k. tamai 1 , m. ninomiya 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university hospital conclusion: although endoscopic haemostasis is an effective treatment modality; rebleeding is still commonly seen among patients with poor child's grading and inoperable hcc. this will result in significant bleeding-related death and poor overall survival. further advancement in treatment strategies for this group of patients are required to improve their outcome and prognosis. background: we have demonstrated that extracellular branched-chain amino acids (bcaas), especially valine, regulate the maturation and function of monocyte-derived dendritic cells (j immunol. 179: 2007) . however, it is not clear whether an imbalance in plasma amino acids of advaced cirrhotic patients influence the function of dendritic cells (dcs). methods: we used human pbmcs and cd1c+dcs in this study. we made two mediums: a serum free culture medium consistent with the average concentration of the plasma amino acids from a healthy volunteer (n=100) was defined as the healthy control medium (hcm); whereas that from advanced cirrhotic patients (n=50) was defined as the advanced cirrhotic active hepatitis b replication is defined as hbeag + or hbv dna > fibrosis was scored according to the metavir system. alt levels were characterized as being normal, < 2 x normal, and > 2 x normal. conclusions: pe494 frequency of portal hypertensive gastropathy and its non-invasive predictors in patients with viral methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent for screening egd for varices in last 2 years was reviewed. phg was defined endoscopically by using mccormack classification. noninvasive markers such as spleen/platelets ratio, meld score and child score of all the patients who underwent for egd were recorded. results: out of 360 patients 226(62.77%) were males. out of 300(83.3%) patients who had phg, 136(45.3%), 93 (31%) and 71(23.7%) had mild, intermediate and severe phg respectively. higher proportion of esophageal varices (89.7%) was present among those who have phg (p<0.001).196(65.3%) with phg has child score of 7. meld score >15 and 15 were seen in 32.3% and 67.7% of patients with phg, respectively. platelet/spleen ratio was 916.08± 400 in patients with phg as compared to 1476 methods: retrospective analysis of cirrhotics undergoing surveillance endoscopy was undertaken assessing for oesophageal varices. clinical, biochemical and radiological indices were analysed. results: 81 cirrhotics underwent surveillance endoscopy during the study. childs pugh scoring (cps) to assess prognosis of liver disease showed cps a(58%), cps b(27%) and cps c(14%). 68% were male albumin (42g/l vs 35g/l significant factors on multivariate analysis were albumin (p=0.003) and platelet count (p=0.026) conclusion: in our cohort there were significant biechemical and radiological differences in differentiating patients with large varices (grade 2-4) on surveillance endoscopy aims and objectives: to study the frequency of ev in patients with cirrhosis due to viral etiology and its correlation with different non-invasive markers. methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent screening egd for varices in last 2 years was reviewed. ev were divided in two grades (small and large) as proposed in consensus development workshop. noninvasive markers such as spleen/platelets ratio, meld and child turcotte pugh (ctp) scores of all patients were recorded. results: out of 360 patients, 226 (62.77%) were males on multivariate analysis ctp score of 7 (or 2.06, p<0.01), meld score >15 (or1.63, p<0.05) and platelet/spleen ratio 900(or 2.35, p=0.005) were found as significant predictors of large ev. conclusion: the frequency of ev is high in viral cirrhosis patients on screening egd. meld score>15, ctp score 7 and spleen/platelets ratio 900 can be used as non-invasive predictors of large ev. pe519 treatment outcome and prognostic factors of spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with hepatitis b virus-related thirty-seven (28.5%) patients had sbp while 91 (71.5%) had cnna. except the higher proportion of renal failure at admission in patients with sbp than cnna (32.4% vs. 7.5%, p=0.001), no significant difference in the clinical and laboratory data related to liver and renal function was observed. overall mortality during hospitalization was higher in patient with sbp than that of cnna evl was repeated every 2-weeks till varicial eradication.bb dose was titrated to achieve a resting heart-rate of 55bpm or a maximum dose 320mg/d or when side-effects began to appear.primary end-points were rebleed and death.secondary end-points were complications as a result of evlor beta-blocker,variceal recurrence afterevl,and decrease in variceal grade inbb limb. results: 71 patients (median age 14[range2-68] yrs, males65%) were included (evl arm[n=37]and bb arm[n=34]. median grade of varices was iii(range ii to iv) nitric oxide synthase isoforms play distinct roles in the evolution of hyperdynamic state in endotoxemia induced portal hypertension m.r. rizvi 1 , m. shahid institute of genomics and integrative biology, mall road, delhi 110 007, 4 department of physiology a seconderc was performed after two or more years to assess the progression of the disease. results: 79 ehpvo patients(median age 20[range 5-62]yr, males 67%) were studied.history of present or previous jaundice was present in 24%,ascites 18% and pain 9%. on erc,92% had portal biliopathy.the type of bile duct involvement was categorized as: type 3 b(51%) and type 1(42%).the pattern of involvement included indentations(49%) and dilatation and strictures(36%).20%of the patients had bile duct stone and 9% had history of cholangitis.the mdian bilirubin was1.1(range0.3-15.9) mg/dl and median serum alkaline phosphatase 171(range 42-617) iu/l.all patients were treated endoscopically by endoscopic stone extraction, dilations with/without stenting. 24(30%) patients underwent second erc after a median interval of 21(range 1-111)months.the type of involvement progressed,75% patients developed type-3 involvement compared to54% at the baseline (p=ns).indentations progressed to develop strictures, from46% to71%.the frequency of new bile duct stones per year was 4%(p= ns). conclusions: portal biliopathy is very common in ehpvo, often remaining asymptomatic.however,it is slowly progressive leading to development of biliary strictures objective: to investigate the mechanisms of angiotonin ii (angii)-induced ca (2+)-independent pathways mediated by rho kinase in hepatic stellate cells (hscs) various vasoactive drugs that reduce portal pressure are used in treatment of esophageal variceal bleeding alongwith endoscopic treatment. terlipressin use decreases both, recurrent bleeding and mortality. it is given usually for 3-5 days, nevertheless there is very little data comparing different time periods. our aim was to compare the efficacy and safety of 5-days versus 10 days of terlipressin treatment in bleeding esophageal varices. methods: out of 15 patients who presented with variceal bleeding, 8 were randomized to receive terlipressin 2 mg 8 hrly, i.v. daily for first 5 days and placebo for next 5 days (group a) and 7 to receive terlipressin 2 mg 8 hrly, i.v. daily for 10 days (group b). both groups were both age and sex matched. (svt) {consisting of hvt and pvt}. there is no such data from india a comparative study of male vs background: mucosal lesions are frequently observed.gender differences are expected due to food habits, nature of job, mobility related to work, consumption of alcohol, tobacco etc. material and methods: procedure done in 1395 m & 19(3.4%) f& duo 48(5.73%) m & 73%)f.varices (48) eso varices 31 (4.3%) m &10(1.8%) f & gastric varices 1 (.11%) m & 1(.11%) f .growth (17)eso growth 5 (.59%) m &1(.17%) f & stomach growth 9(1.07%)m & 1(.17%) f & laryngeal 1(.11%)m. eso monoliasis (7) 3(.35%) m & 4(.71%) f.polyp (8) eso polyp 4(.47%) m & 1(.17%) f & gastric polyp 1(.11%)m & 1(.17%) f & moniliasis and ulcers are more common in female lee 1 1 department of internal medicine, gyeongsang national university school of medicine background/aims: although the pyogenic liver abscess is a common intraabdominal inflammatory disease, this complications are not rare. however, reports dealing with this complications are not good enough and results are often variable. the aim of this study was to identify the predictive factors of complication in the pyogenic liver abscess. pe538 effects of saikosaponinsd (ssd) on expression of c-myc and pcna in experimental hepatocarcinoma of all rats were killed in the 18th week, then general conditions of rats were recorded, the serum alt akp ggt afu was detected and pathological examination was made. the expression of pcna and c-myc were tested by immunohistochemistry. results: he staining showed that rats were induced to hepatocellular carcinoma,the results of liver function in the 18th week displayed that alt akp ggt and afu of all groups were increased than that of normal control group conclusion: ssd can inhibit development of hepatoma induced by den, possibly by down-regulating the expression of pcna and c-myc protein lethal endotoxic shock was induced by single endotoxin (e.coli) 6mg/kg injection into abdominal cavity. ppc in 5% gs was given via tail vein at 1ml/100g (232.5mg/kg) 24h and 6h before endotoxin. rats' behavior and 24h survival were recorded, venous blood taken for ast and alt, liver preserved for he staining and liver/body weight ratio l/b and liver wet/dry weight ratio (w/d) calculated. intercellular adhesion molecule-1 (icam-1) expression in liver tissue was observed with 2-step immunohistochemistry assay. results: rats of ns and ppc group demonstrated similar normal activity, histology and other characters (p>0.05), while lps group showed sag and less water-intak and severe inflammation in liver including inflammatory cell accumulation, parenchymal cells edema and tissue exudation. p+l group turned tired but could drink water the role of insulin resistance, adipokine and cytokine pro-inflammatory in non-alcoholic fatty liver disease n. ratnasari 1,2 , s. anam 2 , p. bayupurnama 1,2 , s. maduseno 1,2 , s. nurdjanah 1, 2 1 dr sardjito general hospital yogyakarta indonesia, 2 background: non-alcoholic fatty liver disease (nafld) is a benign disease during 5-10 years period, with 67%-59% survival. nafld can progress to fibrosis, cirrhotic and cancer of the liver. the etiopathogenesis of nafld is still unknown, however genetic and environment factors are predicted. objective: to know the role of insulin resistance, adipokine and cytokine pro-inflammatory on nafld. methods: the cross sectional study was performed on general check-up population at dr. sardjito general hospital yogyakarta, indonesia. the study was begun from january 2007 until november 2007 at internal medicine outpatient department. inclusion criteria: adult, alcohol consumtion 20g/day, metabolic syndrome patients, and healthy subjects. exclusion criteria: the diseases with increasing liver enzymes (hbv, hcv, ischemic hepatitis, congestive liver), a "bright liver" on ultrasound examination (malnutrition, rapid weight decreased, post gut surgery on obesity patients, and drug induced). based on liver ultrasound subjects were devided into steatosis group and non-steatosis group. data were analyzed by t-test and non-parametrical test. results: 101 subjects that were enrolled the study 61 steatosis (60.4%) and 40 non-steatosis (39.6%) and the subjects who completed cytokine and adipokine examination were 48 steatosis and 30 non-steatosis. there were significantly different on homa -ir and adiponectin level in steatosis group (homa-ir 3.367±4.901 vs. 1.430±1.889, p=0.019; adiponectin 4.049±1.929 vs. 7.034±3.777, p=0.000). there were not significantly different on tnf-, il-6, leptin and visfatin level (p>0.05). conlusions: there were significantly different on homa-ir and adiponectin level in nafld patients compared non-nafld patients. background: to optimize management of nonalcoholic fatty liver disease (nafld), a simple screening tool is necessary. in this study, we aimed to devise a simple index that reflects the presence of nafld in the korean population. methods: a cross-sectional study was conducted on 10,724 health check-up subjects at a healthcare center (5,362 cases with nafld versus age-and sex-matched controls). study subjects were randomly assigned to a derivation cohort or a validation cohort. an index reflecting the presence of nafld was derived in the derivation cohort and validated in the validation cohort. results: multivariable analysis indicated that body-mass index (bmi), serum alanine aminotransferase (alt) to serum aspartate aminotransferase (ast) ratio, sex, and the presence of diabetes mellitus were independent predictors of nafld. using these variables, a formula was derived using a linear regression model: nafld index (nafldi) = 8×alt/ast ratio +bmi (+3, if female; +2, if diabetes mellitus).nafldi had an area under receiver-operating curve of 0.812 (95% confidence interval, 0.801-0.824). at a value <31.0, nafldi ruled out nafld with a sensitivity of 89.0% and a negative likelihood ratio of 0.22, and at a value >36.0, nafldi detected nafld with a specificity of 91.2% and a positive likelihood ratio of 5.42. in the validation cohort, the predictive power of nafldi was maintained at similar levels.aim: since nash could progress to liver cirrhosis and hepatocellular carcinoma, it is important to correctly diagnose between nash and simple steatosis (ss). the aim of this study was to determine the prevalence of nash among nafld patients and to clarify differences in clinical features between nash and ss. subjects and methods: thirty-one patients with nafld showing abnormalities in serum transaminase (ast and /or alt >40 iu/l) were enrolled (sex: male 11, female 20; mean age: 49.5 yrs, mean body mass index: 29.3), after obtaining informed consent. differential diagnosis between nash and ss was performed histologically according to the matteoni classification and clinical features were compared. results: among the patients with nafld, 81% and 19% were diagnosed with ss and nash, respectively. no significant differences in the sex, mean age and bmi were seen between nash and ss groups. the levels of ast, alt, homeostasis model assessment-insulin resistance (homa-ir) and hyaluronic acid were significantly elevated in nash patients compared to ss patients. no significant differences in serum levels of adiponectin, as well as the rates of occurrence of diabetes, hypertension and hyperlipidemia were observed between the two groups. conclusion: the prevalence of nash in nafld patients was about 20%. nash patients showed higher levels of serum transaminase, homa-ir and hyaluronic acid, compared to ss patients. a large-scale biochemical study is required to accurately diagnose nash patients and confirm these results. conclusion: nafldi was a simple, efficient screening tool for nafld that could be utilized for selecting individuals for liver ultrasonography and for determining the need for lifestyle modifications.pe432 aim: this study was conducted to evaluate the hepatoprotective effects of the centella asiatica extract in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury in rats. methods: sprague dawley rats were treated with alcohol extract of centella asiatica orally in two doses (20 and 40 mg/kg/day) for 3 months along with intraperitoneal injection of mptp (1 ml/kg). biochemical parameters such as serum total protein, albumin and marker enzymes were estimated. histopathological studies of liver were also carried out to confirm the biochemical changes.results & discussion: mptp -induced hepatotoxic effects were evident by a significant (p < 0.05) increase in the serum marker enzymes and a decrease in the total serum protein and albumin. administration of extract of centella asiatica effectively inhibited these changes in a dose-dependent manner; maximum effect was with 40 mg/kg. histopathological examination of liver tissue corroborated well with the biochemical changes. hepatic steatosis, hydropic degeneration and necrosis were observed in mptp-treated group, while there was a significant reduction in these changes in the treatment group. conclusion: centella asiatica extract exhibited hepatoprotective action against mptp induced liver injury. further optimization of tuberculosis chemotherapy requires a comprehensive evaluation of the effects of antitubercular drugs on metabolic processes in organism.wistar albino male rats, body weight (b.w.) of 160-200 g, were divided into three groups: group i received pyrazinamide per os at a dose of 1000 mg/kg b.w./day, whereas group ii received a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; the control group was composed of intact animals. the contents of free amino acids were determined using an amino acid analyzer -881 (czech republic). the study of the effects of pyrazinamide administered in different doses on the liver contents of free amino acids showed the largest number of changes at a dose of 1000 mg/kg b.w./day. the content of free amino acids at the level of 16 amino acids and total sum of amino acids significantly differed from controls. part of these changes could be regarded as compensatory answer of organism to this drug action. further pyrazinamide dose increasing caused exhaustion of liver adaptive possibilities. the study of the influence of pyrazinamide on liver contents of free amino acids allows to fully estimate the effects of this substance on metabolic processes in this organ. moreover, the effect of pyrazinamide on the majority of free amino acids in the liver is dose-dependent. background: taiwan is an endemic area of hbv and hcv infection, chemotherapy for lymphoma patients who has been hbv infection, may induce serious clinical sequela due to reactivation of hbv. this study want to clarify the difference of the chemotherapy induced hepatitis between hbv and hcv carrier in lymphoma patients. methods: from july, 2003 to july 2006, 537 non-hepatocellular carcinoma patients were enrolled, 49 (12.5%) cases were lymphoma, in these lymphoma patients, 24(48.9%) cases have been hbv infected, and 22 (44.9%) cases have no hbv or hcv infected. 3 (0.6%) cases have been infected with hcv. all patients received chemotherapy with the regimen of chop or r-chop. liver function , viral markers, hbv dna, hcv rna were checked before and after chemotherapy. results: hepatitis happened in 16 (32.7%) lymphoma patients, 11 (45.8%) cases were in hbv infected patients, 3 (12%) cases were in non-hbv infected patients, 2 cases of hcv infected patients suffered from hepatitis. hbv infected hepatitis patients hbv dna elevated more than 2 log as before chemotherapy. non of the hcv infected patient has elevated of hcv rna after chemotherapy. the mortality rate in hbv infected patient is 33%. no mortality in hcv infected patients after chemotherapy. conclusions: 1.high rate of hbv reactivation and mortality in chemotherapeutic lymphoma patients who has been hbv infected. screening of hbv viral markers among candidates for cancer chemotherapy is mandatory, especially in lymphoma patients. large number and prospect study for chemotherapy induced hepatitis in hcv carrier are needed. background: excessive drinking leads to social, psychological, physical and other problems. this study investigated the epidemiology of ald and analyses the associated risk factors. methods: from 6,043 residents 3,815 blood samples were collected. alcohol consumption and the impact of alcohol on liver function, blood lipids, blood pressure and bmi and mcv have been evaluated. results: the drinking rate and average daily alcohol intake was 35.0% and 36.97±48.76g respectively. the total alcohol intake was 297.90±506.52kg and the average drinking age was 19.21±11.34 years. the average -gt, ast, alt, mcv, chol, tg, ldl-c, hdl-c and bp increased gradually with increase in alcohol intake. the population ald prevalence was 3.98%. the prevalence of ald among the drinking population and the alcoholic population was 11.76% and 44.17% respectively. conclusion: chol, -gt, ast, alt, and mcv were highly correlated with daily alcohol intake which closely related to the occurrence of ald. n. tanaka 1 , w. okiyama 1 , t. aoyama 1 background: alcoholic liver disease (ald) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. polyenephosphatidylcholine (ppc), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons. however, its precise mechanism remains uncertain. we examined the effects of ppc on ald using peroxisome proliferator-activated receptor (ppara)-null mice treated with an ethanol-containing diet, which showed pathological features similar to human ald. methods: male ppara-null mice were pair-fed a lieber-decarli control or 4% ethanol-containing diet with or without ppc at a clinically comparable dose (30 mg/kg/day) for 6 months. o. parkash 1 , a. almas 1 , s.h. ali shah 1 , w. jafri 1 , s. hamid 1 , j. akhtar 1 1 aga khan university hospital karachi 1.hdv-hbv co-infection presents mostly as moderate to advanced liver disease. background: liver injury due to dengue infection is not uncommon. acute liver injury is a severe complicating factor in dengue, predisposing to life-threatening hemorrhage, dic and encephalopathy. results: there were no significant differences of demographic features and laboratory parameters such as peak serum alt, total bilirubin and creatinine between 2 groups. however, peak ast was higher in superinfected group than control group (median: 2,000 iu/l vs 731 iu/l, p=0.035,). additionally, the peak serum albumin levels, prothrombin time and platelet counts were lower in superinfected group than control group (median: 3.0 mg/dl vs 3.3 mg/dl, p=0.02, 51.8 % vs 87.2 %, p=0.027 and 103 x 10 3 /mm 3 vs 165 x 10 3 /mm 3 , p<0.001, respectively). of superinfected group, 9 patients were followed over 6 months after resolution of aha. interestingly, serum hbv-dna levels decreased significantly over 3 months following resolution of aha, then rebounded subsequently (median: -1.89, -1.85, -0.38, 0.58 and 1.10 log10 copies/ml at 1, 3, 6, 12 and 24 months, respectively).methods: the overlapping fragments of hev isolate swgx40 were amplified with reverse-transcription nested polymerase chain reaction (rt-npcr) and the 5' and 3' ends of viral genome were amplified with rapid amplification of cdna ends (race). the pcr products were cloned and sequenced. the phylogenetic analysis of swgx40 was performed.result: the genome of swgx40 consisted of 7,233 nucleotides, excluding the poly (a) tail of 36 residues. the genome contained three open-reading frames (orfs), orf-1 encoding 1705 amino acids, orf-2 encoding 674 amino acids and orf-3 encoding 114 amino acids. the full-length genomic sequencing showed that swgx40 strain shared similarity with all known hev genotype 1, 2 and 3 isolates by 73.4% to 76.5%, and with an identity of 83.1% to 91.2% among genotype 4 hev isolates, and a high nucleotide identity as 94% with chinese guangxi human strain lz-105.conclusions: acute hav super-infection may suppress hbv-dna replication in chronic hbv carriers and chronic hepatitis b, although the suppressive effect did not seem to sustain longer than 3 months. conclusion: the swine hev strain swgx40 was phylogenetically close to the human hev strain lz-105, both from the same region in south china. therefore it was concluded that hev sub-genotype 4b might have existed in south china at least for 6 years and now it was prevalent both in local human and swine, which also strongly supported the zoonosis hypothesis of hepatitis e. associated with splenic volume were damping index (r=0.213, p=0.022) and blood flow of portal vein (r=-0.314, p=0.001). conclusions: the splenomegaly in portal hypertension was more frequent in non-alcoholic groups, and associated with a damping index and a blood flow of portal vein. the measurement of blood flow of portal vein, damping index, and splenic volume by a doppler sonography can be helpful to predict the varices.z.q. zhang 1 , j. cao 1 , w. lu 1 , l.g. shi 1 1 objective: to appraise the clinical efficacy of simple non-invasive models of ast-to-alt ratio (aar), ast-to-platelet ratio index (apri), spleen-to-platelet ratio index (spri), age-platelet index (api), age-spleen-to-platelet ratio index (aspri) for predicting hepatitis b associated cirrhosis. methods: 138 patients and 32 patients were diagnosed pathologically as non-cirrhosis and cirrhosis, respectively. the simple non-invasive models were calculated as described originally. spss 13.0 was used for statistical analyses.background: to reveal the microrna (mirna) expression profile of the hepatic fibrosis inducing cells, rat hepatic stellate cells (hscs), during in vitro activation. results: the areas under roc curve of aar, apri, spri, api, aspri for predicting the cirrhosis were 0.81, 0.62, 0.71, 0.73, 0.74, respectively which were larger than those under the diagnosis reference line (p 0.000, 0.033, 0.000, 0.000, 0.000, respectively).methods: the hscs were isolated from male sd rats by in situ perfusion and density-gradient centrifugation. the quiescent and activated hscs, which were harvested at day 2 and 14, respectively, were then subjected to immunocytochemical staining (desmin and -sma), oil red o staining and quantitative rt-pcr (desmin, -sma, albumin, cd31, cd68 and cytokeratin-19). after extraction and labeling, the hy3-labeled cellular rna samples and hy5-labeled reference pool rna samples were mixed pair-wise and hybridized to the lna mercury microarray. differentially expressed mirnas were filtered and randomly verified by stem-loop rt followed by quantitative pcr.conclusion: all of the simple non-invasive models of aar, apri, spri, api, aspri can be used for predicting hepatitis b associated cirrhosis; and aar has the most practical efficacy for predicting hepatitis b associated cirrhosis. results: both the purity and the total activation of hscs were validated. global analysis of the mirna expression profile based on quiescent and activated hscs demonstrated 21 differentially expressed mirnas. among these, 12 mirnas were up-regulated more than 2-fold in activated hscs as compared to that in quiescent hscs, while 9 mirnas were less than the threshold level (0.5-fold) during the hsc activation. furthermore, the expression of mir-16, 15b, 122, 138, 143 and 140 had been proved. background/aims: only limited patients with chronic hepatitis b virus (hbv) infection will develop liver cirrhosis, and no effective methods to precisely predict ones who will develop cirrhosis. we try to establish a model to predict the patients with the risk of cirrhosis development basing on a clinical epidemiological factor survey. z.q. zhang 1 , w.y. bao 1 , w. lu 1 , l.g. shi 1 methods: cirrhosis patients with hbv markers (case group) and asymptomatic hbsag carriers (control group) were recruited and inquired by researchers with a specific designed questionnaire including 98 items. a multivariate logistic regression analysis were conducted to establish a predictive model, in which two third patients selected randomly as model sample and another 1/3 patients as validating sample, key factors screened out as variables. the predictive performance of the model was evaluated. objective: to explore the practical significance of the peripheral blood corpuscle counts for prediction of hepatitis b associated cirrhosis. methods: 122 and 31 male patients with chronic hepatitis b were pathologically diagnosed as non-cirrhosis and cirrhosis. peripheral blood corpuscle counts were measured by coulter ac•t diff hematology analyzer. results: red blood cell (rbc), platelet (plt), neutrophil (n) counts in cirrhosis were significantly lower than those in non-cirrhosis; and lymphocyte, mid-cell counts were similar to those in non-cirrhosis. the areas under the roc curves of rbc, plt, n counts for prediction of cirrhosis were 0.24, 0.32, 0.34 respectively; according the optimal cut-off determined by the roc curves, the sensitivity, specificity, positive predictive value, negative predictive value, accuracy of rbc, plt, n counts for prediction of cirrhosis were 0.55-0. method: pbmcs of 8 active chb patients under pyg-interferon treatment were analyzed for their th17, treg and pdc by flow cytometry. they were determined by cd4/il-17 for th-17, cd4/cd25/foxp3 for treg and cd123/cd86/cd14-for pdc. pbmc were collected every 4 weekly during the treatment until end of therapy (week 48) and every 12 weekly until the end of follow-up (week 72). alt was quantified at every time of the pbmc collection. ifn-gamma release cells were analyzed by elispot to hbv-core and s ag.background: alpha fetoprotein (afp) is a well-recognized tumor marker for hcc; elevated level of afp is found in at least 70% of hcc. other liver diseases such as cirrhosis and chronic hepatitis are also related with an elevated level of afp. the regulation of afp gene expression has been relatively less studied although the gene has been suggested to play a role in hcc development. in this study, we tried to identify genetic variations in afp gene and analyze its effect on serum afp level and possible hcc progression.results: in parallel with decline in alt for the first 12 weeks, we found decline in both pdc (r=0.71, p=0.03) and elispot (r=0.65, p=0.03 for hbv-core ag; r=0.48, p=0.05 for hbv-s ag). although there is trend that th-17 decline with treg decline, but they are not statistically significant differences in the same period. there is no significant difference between the svp and non-svp patients.methods: direct dna sequencing was carried out to sequence afp promoter and 500 bp upstream and downstream of afp coding regions in dna samples isolated from 99 hcc subjects and 105 controls respectively. for each samples serum afp levels were determined using commercially available elisa kits. conclusions: under pegyintron treatment, pdc, ifn-gamma change in the same trend of alt during 12 weeks of treatment. it implied that pdc take regulatory effects on ifn-gamma releasing cells and be very tightly related to alt. there wasn't a significant difference in both treg and th-17, which implies that treg and th17 might be of important cells in keeping the stability of the immune system.results: a total of 30 snps were detected in the afp genomic region analyzed, including 29 known snps and one novel snp. among the identified snps, the c>g nucleotide change in the position -250 bp upstream of afp transcriptional start site showed a significant association with hcc (p < 0.05) and a decrease in afp gene expression level. conclusion: our preliminary results indicated a possible association between serum afp expression and -205g allele. the identified snp is located in afp promoter region with possible binding sites for known transcription factors, such as tfiid, coup, apf and nfiii. -30°tail-suspension (ts) rats were used as the model to simulate the physiological effects of weightlessness. thirty-two wistar male rats were randomly divided into 4 groups: control for 7d (7d con), 14d con, ts for 7 d (ts7d) and ts14d. histopathological changes of testicle of the rat were observed by he stain. localization and expression of ar and hsp70 in testicle of rat were observed comparatively by means of immunohistochemistry, and the density of ar and hsp70 immunoreactivety in four groups were compared. results: signal molecules mrna level are shown in the table 1 (** p<0.01, * p<0.05). tnfa, il1, il6 and il8 were higher in group 1, 2 and 4 than those in group 5. ifna was higher in group 5 than that in other groups. there are no significant difference in infc, il2, and il4. there was a positive correlation between tnf and myd88 in group 2, tnf and nfkb in group1 and 2. results: obvious pathological lesions presented in testicle of ts7d and ts14d rat. germinal epithelium irregularity and malformed spermatozoa were found in seminiferous tubules. degeneration and necrosis of germinal epithelium appeared in testicle of ts7d and ts14d rat. ar immunoreactive cell density in the ts7d and ts14d groups were significantly decreased compared with the in-phase normal control groups ( p < 0. 01). while hsp70 immunoreactive cell density in the ts7d and ts14d rats were significantly increased than those of control rats( p < 0. 01), and in testicular interstice or extracellular there were very strong ehsp70 immunoreactive positive staining signals. the results indicate that ground simulated weightlessness induced by 7d-14d tail-suspension in rats can lead to the serious injury , depressed expression of ar and enhanced expression of hsp70 in testicle. despite the absence of any serologic marker of hbv recurrence, however, it remains unknown whether there is occult reinfection in the liver graft. we aimed to detect and quantify the presence of intrahepatic hbv dna in the liver grafts of patients who remain seronegative for hbsag for more than 1 year after liver transplantation. materials and methods: liver biopsy and blood samples were obtained from 31 patients who had been receiving nucleoside analogue prophylaxis alone and remained persistently seronegative for hbsag for at least 1 year (median 44.5 months, range 13.6 to 126.4 months) after liver transplantation for chronic hepatitis b. quantitative polymerase chain reaction was performed to detect and quantify total and covalently-closed circular (ccc) hbv dna in the liver (lowest detection limit, 10 copies/ml), serum and pbmc. direct sequencing was used for hbv quasispecies screening. results: liver biopsy was performed and intrahepatic hbv dna as measured by quantitative real-time pcr was detectable in 26 of 31 recipients. donors anti-hbc status before liver transplant was significantly related to the presence of intrahepatic hbv dna in the recipient's study biopsy (p=0.038). donor intrahepatic hbv cccdna levels correlated with recipient post-liver transplant intrahepatic hbv cccdna levels (p=0.004). hepatitis b virus sequencing results and phylogenetic analysis revealed that hbv reinfection in two recipients were of donor origin, four recipients were of recipient origin and four recipients were of both donor and recipient origins. conclusions: our findings demonstrate the presence of occult hbv reinfection with persistence of hbv dna in liver allografts despite long term nucleoside analogue prophylaxis after liver transplantation, suggesting the need to continue indefinite antiviral therapy. the use of liver grafts from anti-hbc-positive donors might increase the risk of occult hbv reinfection. both donor and recipient hbv dna could contribute to occult hbv reinfection in liver transplant recipients. aim: to construct one noninvasive assessment model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with chronic hepatitis b(chb). methods: we have retrospective analyzed 137 consecutive patients with chronic hepatitis b who underwent percutaneous liver biopsy. we calculated sensitivity, specificity, positive predictive value(ppv), and negative predictive value(npv) of an apri 1.5 in combination with different hyaluronic acid(ha) cut-off points medium (acm). we stimulated pbmcs or dcs under hcm and acm, and evaluated the function. results: after adding the stimulants under hcm, the cd83 and cd86 expression of dcs from cirrhotic patients (lc) were lower than those from healthy contorols (hc). in both hc and lc, the cd83 and cd86 expression of dcs stimulated under acm was lower than that under hcm. the il-12 production in acm was lower than that in hcm. the expression of cd98, which is related to amino acid transport, was not different between hcm and acm. however, dcs cultured in acm expressed lower levels of phospho-p70 s6k than those cultured in hcm. finally, we ascertained that the ifn gamma production by pbmcs was significantly decreased under acm.conclusions: an imbalance in plasma amino acids of advanced cirrhotic patients suppresses the maturation of dcs via mtor/s6k signaling pathway.