Woodchuck hepatocytes remain permissive for hepatitis B virus infection and mouse liver repopulation after cryopreservation

HEPATOLOGYVO1. 34, No. 4, Pt. 2, 2001

AASLD ABSTRACTS

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INCREASE IN ItVR1 QUASISPECIES HETEROGENEITY IS ASSOCIATED W I T H INCREASE IN ALT BUT NOT W I T H LIVER HISTOLOGY IN PATIENTS W I T H CHRONIC HEPATITIS C. Michelle M Martinot-Peignoux, Tarik Asselah, INSERM U481, Clichy France; Dominique Caazals-Hatem, Anatomo-Pathologie, Cfichy France; Nathalie Boyer, Service d'H4patologie, Clichy France; Claude Degott, Anatomo-Pathologie, Clichy France; Patrick Marcellin, Service d'Hdpatologie, Clichy France

INFLUENCE OF HEPATITIS B VIRUS (HBV) GENOTYPE ON THE LONGTERM OUTCOME OF CHRONIC HEPATITIS B.Jose Maria Sanchez-Tapias, Liver Unit, Institut de Malahies Digestives, Hospital Clinic, Barcelona Spain; Josep Costa, Microbiology Department, Hospital Clinic, Barcelona Spain; Antoni Mas, Liver Unit, Institut de Malahies Digestives, Hospital Clinic, Barcelona Spain; Miquel Bruguera, Liver Unit, Instimt de Malahies Digestives, Barcelona Spain; Juan Rodes, Liver Unit, Instimt de Malalties Digestives, Hospital Clinic, Barcelona Spain

Our aim was to study the kinetic of HVR1 quasispecies, in patients with chronic hepatitis C and persistently normal ALT, according to subsequent elevation of ALT. Patients and methods: 30 patients with chronic hepatitis C and persistently normal serum ALT levels (3 normal ALT values in a 6 months period) were included in the study. 27 patients had a liver biopsy at inclusion and 17 patients underwent a second liver biopsy at 3 . 3 +- 1.2 years interval. Serum HVR1 quasispecies were assessed the day of the biopsy with PCR-SSCP with specific primers for genotype 1 and 3. The characteristics studied were gender, age source and duration of infection s e r u m ALT and HCV RNA levels, HCV genotype and histologic lesions. Results: A m o n g the 30 patients, 11 patients experienced an increase in ALT levels and kept fluctuating ALT levels (Group A) and 19 patients remained with persistently normal ALT levels (Group B). Mean interval between quasispecies assessments was 3.5-+ 1 years. Median HVR1 SSCP n u m b e r of hands observed was 2.2 -+0.6 vs 2.2-+ 0.7 (us) at inclusion and 2.9-+0.8 vs 2.2-+0.6 (p=0.001) at second assessment in Group A and Group B, respectively. An increase in HVR1 SSCP band was observed in 8/11 and 3/19 patie!~ts of Group A and Group B, respectively (p=0.006). A diminution in HVR 1 SSCP bands was observed in 0/11 and 4/19 (us) of the patients of groups A and B, respectively. Increase in HVR1 quasispecies bands was significantly and independently associated with increase in serum ALT levels but was not associated with gender, age source and duration of infection, serum HCV RNA level or HCV genotype. Histologic lesions were mild in all cases remained stable and were not associated with HVR1 SSCP quasispecies profil outcome. Conclusion: Our results suggest that during the outcome of patients with chronic hepatitis C and persistently normal ALT levels, an increase in ALT levels is associated with a significant increase in HVR1 quasispecies, although histologic lesions remain mild and stable.

introduetion and aims.Ahighlyvariableoutcomeis a remarkablefeatureof chronicHBVinfection. Spontaneous or therapeuticallyinduced remissionis a relativelyfrequenteventand entails a good prognosis whereaspersistent activitymay lead to progressiveliver damage,developmentof complications and deatk Recently,by phylogeneticanalysis of viral isolates sevendifferent genotypes of HBV(designedas A to G) have been identified. The possible influence of HBVgenotypeon the long-term outcome of chronic HBV infectimlhas not been explored and recent cross-sectional studies have provided controversialdata about the relationship between the genotype of the infecting virus and the severityof liver lesions. Methods. We report here the observationsmade after prolongedfollow-upin 258 consecutivepatients with chronic hepatitis B who were infected with different HBVgenotypes.All had abnormalALTlevelsand positiveHBV-DNAin sermn. No patient had history of current or past hepatic decompensation.Patients vetth chronic hepatitis B and other conditions that mayaffectthe liver(significantalcoholintake, infectionwith HIV,HDV, or HCV)were not included. Mean age was 36 years and 81% of the patients were male. Patients were followedfor a mean of 94 months (24 to 180) and visited at 3-6 months intervalsor more often if indicated. Clinical examinationand determinationof liver function tests and HBVserum markers(HBsAg,HBV-DNA,HBeAg,anti-HBe)were carried out at eachvisit. Sixty-sevenpatients received interferon therapy. Patients with cirrhosis were regularly screened for hepatocellular carcinoma.HBV-DNAwas determinedqualitativelyby a simplifiedPCR method (sensitivitylimit 0.01pg/mL)and HBV-genotypewas determinedby a PCR-RFLPassaythat enablesidentificationof enotypes A to F. Results. GenotypeA was identified in 140 patients (52%), genotypeD in 94 5%) and genotype F in 20 (7%). There were no differences between patients infected with different genotypesin baseline demographic,biochemicalor histopathologiealfeaturesnor in the length of follow-up,number of visits or proportion of patients treatedwith interferon, but genotype A was more prevalent in HBeAg-positiveand genotype D in anti-HBe-positivesubjects. Concomitantsustained biochemical(normalALT)and virological(negativeHBV-DNA)remission lasting up to the end of follow-up was observed in I10 (43%) patients and was unrelated to treatment. Remissionoccurred at a significantlyhigher rate in patients infectedwith genotypeA thanin thoseinfectedwith genotypeD (logrank 14.2,p = 0.002) or in thoseinfectedwith genotype F (log rank 4.2, p=0.03) and genotype A was independently associated to remission by Cox regression analysis of basal features. Similar observations were made by separated analyses of HBeAg-positiveand anti-HBe-positivepopulations. Seroconversionto anti-HBewas observedin 60% of HBeAg-positivepatients and was unrelated to HBVgenotype.However,sustained remission afterseroconversionoccurredat a significantlyhigher rate in geaotypeA than in genotypeD infected patients (log rank 4.5, p= 0.03). Clearanceof HBsAgwas observedin 29 (11%) patients and occurredat a significantlyhigherrate in genotypeA than in genotypeDinfection (logrank 4.6, p=0.03) and did not occur in genotype F infection. By Cox regression analysis, genotype A infection was significantlyassociated to clearanceof HBsAg.Hepaticdecompe.sati0n (20 oases, 8%), hepatocellularcarcinoma(6 cases, 3%), liver transplantation (7 cases, 3%) and liverrelated death (13 cases, 5%) were rather uncommon events. However,liver related death was more frequent in genotypeF than in genotypeA (p=0.02) or genotypeD (p=0.002) infectedpatients. Conclusions.These observationssuggest that viral genotypeplays an important role in the long term outcomeof chronic hepatitis B causedby genotypeA, D or F of HBV.

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PAUCITY OF PLASMACYTOID DENDRITIC CELLS IN LYMPH NODES OF HEALTHY AND HEPATITIS-B INFECTED HUMAN LIVER. Jaap Kwekkeboom, Wilco Tanis, Shanta Mancham, Geert Bezemer, Dept of Hepatogastroenterology, Erasmus Medical Center Rotterdam, Rotterdam Netherlands; H e m m o Drexhage, Dept of Immunology, Erasmus Medical Center Rotterdam, Rotterdam Netherlands;Jan Ijzermans, Hugo Tilanus, Dept of Surgery, Erasmus Medical Center Rotterdam, Rotterdam Netherlands; Solko Schalm, Dept of Hepatogastroenterology, Erasmus Medical Center Rotterdam, Rotterdam Netherlands

WOODCHUCK HEPATOCYTES REMAIN PERMISSIVE FOR HEPATITIS B VIRUS INFECTION AND MOUSE LIVER REPOPULATION AFTER CRYOPRESERVATION. Maura Dandri, Martin R Burda, Heinrich Pette Inst for Exper Virology, Hamburg Germany; Eva TOr0k, Joerg M Pollok, Xavier Rogiers, Dept of Hepatobiliary Surg and Transplantation, Hamburg Germany; David Zuckerman, Heinrich Pette Inst for Exper Virology, Hamburg Germany; Heiner Greten, Dept of Medicine, Hamburg Germany; Hans Will, Heinrich Pette Inst for Exper Virology, H a m b u r g Germany; Joerg Petersen, Dept of Medicine, Univ Hosp Hamburg-Eppendorf, Hamburg Germany

Background: Several hepatotrophic viruses, like hepatitis B (hepB), are not eliminated by the host's immune system and persist in a substantial number of infected patients. Chronically infected hepB patients have deficient humoraI and cellular immune responses to the virus. In this study, we investigated whether the persistence of hepB infection is related to a disorder in migration (and concomitant maturation) of dendritic ceils (DC), which are the major Antigen-Presenting-Cells (APC), into human liver-lymph draining lymph nodes (LN). For this purpose we compared numbers of immature mydoid DC (MDC), mature MDC, and plasmacytoid DC (PDC) in liver- and inguinal LN. PDC are not only APC, bat also the principal type I Interferon producers. Patients and Methods: Liver-LN were collected from the hepatoduodenal ligament during liver transplantation from patients with chronic hepB infection (n-7), auto-immune hepatitis (n=4) and from organ donors (healthy liver; n = 10). Three HepBpatients were HBeAg+, and four were HBeAg-.Inguinal LN (n= 5)were collected during kidney transplantation. Cryostat sections were cut at three levels of the LN and immunohistochemical double-stainings were performed for T-cells (CD3) (visualized by the peroxidase substrate AEC (red) and 1. immature MDC (Langerin); 2. mature MDC (DC-Lamp); 3. PDC (CD123), visualized by the alkaline phosphatase substrate fast blue. Nmnhers of DC were quantified in the red-coloured T-cell areas by two independent observers counting 6 microscopic fields of 600 times magnification per section, after which mean numbers for every lymph node were calculated from the numbers counted in the three levels of each LN. From a few LN DC were isolated and immunophenotyped by fiow-cytometry. Results: LN of the four different groups contained similar numbers of immature and mature MDC in their paracortex (immature MDC: inguinal LN 5_+4, healthy liver LN 5_+3, auto-immune hepatitis liver LN 7+3, hepB liver LN 5-+4 per microscopic field; mature MDC: inguinal LN 28 ± 8, healthy liver LN 35-+11, auto-immune hepatitis liver LN 47-+ f 1, hepB liver LN 27-+ 10). However, health),"liver LN contained significantly less PDC as inguinal LN (4-+3 versus 17-+7; Mann-Whitney test p=0.001). In LN from livers with auto-immune hepatitis numbers of PDC were increased in comparison with healthy fiver-LN (13_+6 versus 4-+3; p=0.01), but such increase was not observed in LN from chronically hepB-infected livers (7_+4 PDC per microscopic field). The identity of the brightly-stained CD123+ cells as PDC was confirmed by flow-cytometry on isolated DC: HLA-DR+-cells with the highest CD123-expression were CD11c-. Part of myeloid DC (HLA-DR÷/CD1lc+) expressed also CD123, but at a three times lower intensity'. Conclusion: In chronic hepB patients liver-draining LN contain similar low numbers of PDC as healthy liver LN do, while in auto-immune hepatitis the numbers of PDC in liver LN are increased. The paucity of PDC in liver LN may be one of the reasons for persistence of hepatitis B virus.

Primary hepatocytes are highly required both for research and therapeutic applications. However, sources of primary- liver cells from h u m a n beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be utilized for woodchuck hepatitis B virus (WHV) infection studies and could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of the University of Wisconsin solution as a main component of the freezing medium, stepwise reduction of DMSO to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was high (70 to 80%) and 50 to 60% of thawed cells attached to the plates. The appearance of cccDNA and of WHV replicative forms a few days after in vitro infection demonstrated that thawed woodchuck hepatocytes were still susceptible to viral infection. Furthermore, transplantation of woodchuck hepatocytes in the uPA/RAG-2 mouse model of liver regeneration demonstrated that cryopreserved cells retained the ability to divide and to extensively repopulate a xenogenic liver. Notably, in vivo susceptibility to infection with WHV and proliferative capacity of frozen/thawed woodchuck hepatocytes in recipient mice were identical to those observed by transplanting fresh hepatocytes.