Research On Pathological Features And Molecular Markers Of The Complications Following OLT

Definite diagnosis is a core in the treatments to complications following OLT. Liver puncture biopsy has been an important role in diagnosis and management of most of the complications after OLT, especially in rejection. However, sometimes it was difficult to make a differential diagnosis by liver puncture biopsy, because the histopathological changes were so complicated with the overlap of two or more etiological factors. Accordingly, to identify specific pathological features of the complications is important to differential diagnosis, in addition ,the clinical features, biochemical abnormalities ,imageologic examination, and specific markers are considerable .Our aim is to summarize the spectra of pathological features of the major complications post OLT ,and to search for the possible markers.The current research project is comprised of the following three parts.Part 1 Clinicopathological Analysis of 667 Liver Puncture Biopsies Following OLTObjectives: To explore the pathological and clinical features of the major complications after OLT by retrospective analysis of 667 liver puncture biopsies.Methods : Collection of liver allograft biopsies in our department of pathology from June 2003 to June 2006. 667 liver biopsies were obtained from 415 patients with liver dysfunction .All tissues were consisted with the following: (1) successive 4 portal structures per sample microscopically; (2) integrity data of clinical and laboratory examination.Useing SPSS 11.0 for statistical analysis on the comparison between pathological, clinical features and major complications after OLT.Results: The most commonly five complications seen in 667 episodes were: acute cellular rejection(ACR) ( 210 episodes,31.5 %) , biliary complication (BC)(161 episodes, 24.1%) , preservation injury(PI)(125 episodes,18.7 %) , drug toxicity(52 episodes,7.8%), infection or recurrence of hepatitis virus B (24 episodes,3.6%) . In ACR, the indeterminate and mild episodes were seen 183 in 210(87.5%). In PI, the mild and moderate episodes were seen 116 in 125 (92.6%).The most early five complications occuring after OLT were:PI, ACR, vascular complication, CMV infection, BC. Most of ACR episodes, especially those grade as moderate and severe occured within the first three months after translplantation .The most risk period for ACR is 8～30day after OLT, and there are 104 episodes in 210( 49.5%) .The correlatetions of pathological features with five major complications after OLT showed that: ACR is close correlative with apoptotic body, karyodieresis, angiocholitis, hepatic vein inflammation, cell drop out, and eosinophils (P<0.05). PI is close correlative with cholestasis, karyodieresis, and angiocholitis (P<0.05). BC is close correlative with cholestasis, bile duct quantity, angiocholitis, portal fibrosis and hydropsia (P<0.05). Drug toxicity is close correlative with hydropic degeneration and cholestasis(P<0.05). Infection or recurrence of hepatitis virus B is close correlative with fibrosis, liver cell drop out, apoptotic body, karyodieresis, cholestasis, angiocholitis, and hydropic (P<0.05).The clinical features such as gender, age, primary diseases, operation modus, the ranging of cold and warm ischemic time, anhepatic phase, and so on , did not correlate with the five major complications (P>0.05).The fluctuation curve of biochemical indicator according to the time after OLT showed that ALT went up in ACR and PI groups before Liver puncture, but did not in BC group. AST went up at first and then descended in ACR and PI groups, but had a continuous descend in BC group.γ-GT step up greatly and then descended slowly in PI group. In BC groupγ-GT maintained a high level after step up. DBIL and TB kept on rising in BC group, but step up a little then descended in ACR and PI groups.Conclusion: The spectra of specific pathological features of the major complications after OLT is the basis of differential diagnosis. In addition, clinical imageological examination, serological evidence, and the fluctuation curve of biochemical indicator are valuable to a definite diagnose.Part 2 Significance of C4d Deposition in Liver Puncture Biopsies After OLTA recent study has shown synergistic actions between humoral and cellular rejections in liver transplantation. In a pathological study of kidney transplantation, C4d diffuse deposition in renal interstitial capillary walls seems to serve as a specific marker of rejection. The same phenomenon was also observed in cardiac transplantation. It is not clear whether such a phenomenon occurs in hepatic transplantation.Objectives: To determine the specificity of C4d in ACR and the significance in the differential diagnosis of complications after OLT by observation of C4d deposition in liver puncture biopsies .Methods : Selected 106 liver puncture biopsies from Part 1 including the complications as ACR (20 biopsies ),BC (20 biopsies ),PI (20 biopsies ), infection or recurrence of hepatitis virus B (20 biopsies ) , drug toxicity (10biopsies), ACR co-existing with PI (8 biopsies),and ACR co-existing with BC (8 biopsies).The sample of acute appendicitis was taken as negative control. C4d deposition was detected by immunohistochemical staining (using rabbit anti-human C4d antibody). The differences were analyzed usingχ2 test, P<0.05 was accepted as significant.Results: The positive ratio of C4d deposition in ACR , infection or recurrence of hepatitis virus B, and ACR co-existing with PI ware higher than that in other complications (P<0 .05). C4d deposition in different Sites showed that: in portal areas the positive ratio in ACR , Infection or recurrence of hepatitis virus B, ACR co-existing with BC ,and ACR co-existing with PI were higher than that in other complications (P<0 .05).In hepatic sinusoidal walls, the positive ratio of C4d deposition in ACR was higher than that in other complications(P<0 .05). In central veins walls, there was no significant difference in all complications.Conclusion: C4d deposition in hepatic sinusoidal walls may be served as a specific marker in the diagnosis of liver graft ACR.Some biopsies in PI would be associated with humoral immunity injury. C4d deposition in portal areas may be associated with infection.Part 3 The Role of ACE/ACE2 in the Mechanism of preservation injuryIt was found that in addition to the classic circulating renin-angiotensin system(RAS), increasing evidence supports the existence of local tissue RAS (heart, brain and kidney etc) that would be provided some new function in regulating blood flow of organs, responsing sensitively to tissue hypoxia, and promoting cellular proliferation, etc.Objectives:To explore the significance of RAS in rat liver transplantation , and the correlatetion of ACE /ACE2 with preservation injury after OLT by detection ACE and ACE2 mRNA in rat allograft.Methods: The male Wistar and Sprague-Dawley rats were allocated to five groups: mild PI group (n=15,SD→SD, cold ischemia time is 6 hours); moderate PI group (n=15,SD→SD, cold ischemia time is 15 hours);severe PI group(n=15,SD→SD, cold ischemia time is 24 hours);ACR group (n=15, Wistar→SD, no cold ischemia time);control group (n=15,SD→SD, no cold ischemia time). The observation of dynamic histologic change was performed. The mRNA of ACE and ACE2 were detected by real-time PCR.Results: ACE2 mRNA was detectable at higher level in PI groups than that in ACR group(P<0.01,P<0.05). The expression of ACE2 mRNA in PI groups showed that: the level of ACE2 mRNA in the moderate and the severe were significantly higher than that in the mild(P<0.01,P<0.05). However, the level of ACEmRNA was no significant deviation in all groups during first 48h after OLT . But the dynamic tendency of ACEmRNA was parallel to that of histologic changes.Conclusion: The expression of ACE2mRNA was close correlative with the PI grades, possibly response to hepatocellular hypoxia ,and ACE2 would be a potential marker of PI. It implied that ACE is correlative with the inflammationary injury of liver.