Frozen Liver Biopsy Research Articles

DETECTION OF HCV ANTIGENS AS A UNIQUE TOOL TO IDENTIFY HCV INFECTION OF LIVER GRAFT: RELEVANCE TO MANAGEMENT OF LIVER DISEASE.

O31 Aims: Early recurrence of HCV disease in liver graft is often difficult to demonstrate by histology. HCV antigens (HCV-Ag) can be detected by immunohistochemistry. A correlation between the number of infected hepatocytes and the pattern of histological damage was previously shown in transplanted patients. Aim of this study was to evaluate the impact of HCV-Ag detection in the management of patients transplanted for HCV related disease. Methods: HCV-Ag were detected, on frozen sections, using a 4 steps IPx technique with FITC-conjugated human anti-HCV IgG. The % of infected hepatocytes was estimated. Between 1996 and 2003 177 frozen liver biopsies were obtained from 116 HCV-RNA positive patients, transplanted, at different times from OLT. Histology was coded as: hepatitis, rejection, other (steatosis, cholangitis, cholestasis, reperfusion damage) or undefined when rejection grade II-III was excluded and different patterns coexisted. Results: Number of biopsies (no.), number of positive biopsies (no.+, %) and median % of positive hepatocytes (m%) at different time intervals are reported in the table.FigureHCV positive hepatocytes were found in 46%, 79% and 63% of biopsies at the 3 time intervals. Both the higher number of positive biopsies and the higher median number of positive cells were observed when hepatitis was identified by histology. This was particularly true in the acute phase of reinfection (21-180 days from OLT). In patients with serial biopsies an increase was usually observed when the first biopsy was taken very early and a decrease (spontaneous or treatment induced) when the second was after 6 months. Quantitative HCV viremia at the time of biopsy was available in 56 cases. Result was under the cut off sensibility in 9 cases, all negative for tissue HCV-Ag. A correlation was found (r=0.45) in the whole series but wide discrepancies were observed in individual patients. Detection of HCV-Ag had no impact on clinical management until 20 days from OLT: no antiviral treatment was started, rejection was treated in all positive cases. Similarly, after six months, rejection was treated independently of HCV-Ag positivity, antiviral treatment was not avoided in HCV-Ag negative patients. The high number of infected hepatocytes prompted a final diagnosis of hepatitis, confirmed by follow up analysis, in 8 further patients between 21 and 180 days and interferon treatment was started in 3 cases. In the same period rejection was treated in 2 patients with more than 50% HCV-Ag positive hepatocytes, but follow up analysis turned diagnosis to hepatitis. Conclusions: HCV-Ag detection is a unique tool to directly demonstrate recurrence of HCV infection in liver grafts. We confirm a relationship between the pattern of liver damage and the number of infected hepatocytes in the first 6 months after OLT: during this period, when multiple pathogenic mechanisms more frequently coexist, its relevance for clinical decision making seems particularly high.

Role of replicative senescence in the progression of fibrosis in hepatitis C virus (HCV) recurrence after liver transplantation.

Although hepatitis C virus (HCV) recurrence is almost universal after orthotopic liver transplantation (OLT), the impact of viral infection on liver graft is highly variable and difficult to predict. Because of the possible relationship between replicative senescence (RS) and the accelerated development of liver fibrosis, we aimed to assess the potential role of RS in the severity of HCV-related chronic hepatitis recurrence after OLT. One hundred three liver biopsies from 56 patients receiving transplants for HCV-related cirrhosis were studied, including 30 revascularization biopsies and 52 and 21 biopsies performed during and beyond the first year of OLT, respectively. The presence of senescent cells in liver grafts was assessed by the senescence-associated beta-galactosidase (SA-beta-Gal) staining method. Chronic hepatitis was defined by fibrosis stage and necrotico-inflammatory activity grade using the METAVIR score. A total of 34 of the 103 (33%) frozen liver biopsies displayed SA-beta-Gal-positive cells, including 6 (20%) of the revascularization biopsies, 14 (34%) of the biopsies performed within the first year, and 10 (46%) of the biopsies performed beyond 1 year of follow-up. The presence of senescent cells in revascularization biopsies was significantly associated with the degree of ischemic necrosis at time of OLT (P = 0.01) and hepatitis C recurrence in the first year after OLT (P = 0.05). Furthermore, the presence of RS in the biopsy performed within the first year was associated with further development of fibrosis (P = 0.05). These data show that RS has a significant impact upon the course of liver transplantation, especially in the long-term progression of fibrosis observed in HCV-infected patients.

A study of some hepatic immunological markers, iron load and virus genotype in chronic hepatitis C

Background/Aims Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype. Methods Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, β 2m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases. Results A negative correlation was found between the number of CD8 + cells and fibrosis. CD8 + cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and β 2m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68 + cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients. Conclusions In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.

A study of some hepatic immunological markers, iron load and virus genotype in chronic hepatitis C

<h2>Abstract</h2><h3>Background/Aims</h3> Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype. <h3>Methods</h3> Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, β₂m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases. <h3>Results</h3> A negative correlation was found between the number of CD8⁺ cells and fibrosis. CD8⁺ cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and β₂m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68⁺ cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients. <h3>Conclusions</h3> In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.

Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation.

Treatment of acute cellular rejection (ACR) is associated with increased viral load, more severe histologic recurrence, and diminished patient and graft survival after liver transplantation for hepatitis C virus (HCV). Recurrence of HCV may be difficult to distinguish histologically from ACR. Because the immunologic mechanisms of ACR and HCV recurrence are likely to differ, we hypothesized that ACR is associated with the expression of a specific subset of immune activation genes that may serve as a diagnostic indicator of ACR and provide mechanistic insight into the pathophysiology of ACR and recurrence of HCV. The goal of the study was to study intragraft gene expression patterns in ACR and during recurrence of HCV in HCV-infected recipients. High-density microarrays were used to determine relative intragraft gene expression in two groups of HCV-infected liver transplant recipients: four with steroid responsive ACR by Banff criteria and four age- and gender-matched HCV-infected recipients with similar necroinflammatory activity but without histological criteria for rejection (no cholangitis or endotheliitis). Immunosuppression was similar in both groups. Other etiologies of graft dysfunction were excluded by ultrasound, cholangiography, and cultures. High-quality total RNA was extracted from snap frozen liver biopsies, reverse transcribed, labeled with biotin, and fragmented according to established protocol. Twenty-five genes were relatively overexpressed, and 15 were relatively underexpressed by > or = twofold in the ACR when compared with the HCV group. ACR was most notably associated with the relative overexpression of genes associated with major histocompatibility complex I and II, insulin-like growth factor-1 expression, apoptosis induction, and T-cell activation. In HCV-infected liver transplant recipients, ACR is associated with an intragraft gene expression profile that is distinct from that seen during recurrence of HCV. These experiments provide evidence that alloimmunity, as indicated by expression of T-cell activation and apoptosis-inducing genes, is less important in recurrence of HCV than in ACR. Further studies are required to determine whether gene expression profiles, either intragraft or in serum, can be used for the diagnosis and differentiation of ACR from recurrence of HCV.

Lack of Association Between Hepatitis C Viral RNA in Serum and Liver and Histologic Gradings: A Study on Irish Anti-D-Treated Patients

In this study the authors applied a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect hepatitis C virus (HCV) RNA in 15 frozen liver biopsy samples from anti-D-treated patients. They also correlated the presence or absence of HCV RNA in the serum and liver of each patient with their histologic gradings. RNA was extracted from 36 frozen liver biopsy samples. These included 15 liver biopsy samples from patients infected with HCV through contamination of anti-D blood products. Three of these 15 anti-D-treated patients were receiving alpha-interferon treatment at the time of liver biopsy. Nine frozen liver biopsy samples from patients with a history of intravenous drug abuse were included as positive controls. HCV-negative frozen liver biopsy samples from 12 noninfected patients were used as negative controls. RNA was also extracted from six frozen skin biopsy specimens to check for cross-contamination of samples. Eleven of 15 anti-D-treated patients were HCV RNA positive by RT-PCR, with 100% correlation between HCV RNA in the serum and liver. The nine frozen liver biopsy samples from the intravenous drug abuse patients (positive controls) were also RT-PCR positive for HCV RNA. The 12 noninfected samples and the negative control biopsy samples were negative for HCV. Twenty-seven percent of the recombinant immunoblot assay-positive patients were serum and liver HCV RNA negative. HCV-positive patients receiving alpha-interferon therapy at the time of biopsy had cleared the virus from the serum and the liver. There was no correlation between the presence or absence of serum and liver HCV RNA with the histologic grading. This lack of correlation shows clearly the importance of histopathologic evaluation of liver biopsy samples in monitoring HCV-associated liver disease progression. In addition, this finding indicates that one cannot rely only on the presence or absence of HCV RNA in either serum or liver tissue as a parameter in monitoring HCV-associated liver disease progression in this unique cohort of anti-D-treated patients.

The antigenic heterogeneity of the bile duct epithelium in alcoholic liver disease. VA Cooperative Study Group 275.

The chronic alcoholic patient is usually immunosuppressed, but the significance of this phenomenon in terms of bile duct injury is unclear. The immunoreactivity of the bile duct cells was examined in a series of 69 frozen liver biopsy specimens obtained from patients with alcoholic liver disease, comprising 29 cases of cirrhosis, 26 of alcoholic hepatitis, 10 cases of alcoholic fatty liver, and 4 specimens from normal livers. Liver diseases such as primary biliary cirrhosis and human hepatic allograft rejection, known to have an autoimmune basis, share the characteristic feature of damage to the bile duct epithelial cells. In both instances the damage seems to be immune mediated, but the nature of the antigens involved is not established. We used the avidin-biotin-peroxidase complex method to test in alcoholic liver disease for the expression of a battery of surface antigen markers that have been incriminated in tissue injury and are usually present in lymphoid cells but also expressed by epithelium. In this study we investigated the expression of the following molecules: HLA class I (ABC) and class II (HLA-DR, HLA-DP, HLA-DQ), CD29, CD45RA, CD45RO, CD56, interleukin 1 (IL-I), IL-2, IL-4, interferon (IFN-gamma), tumor necrosis factor beta, and transforming growth factor beta1 (TGF-beta1). The bile duct epithelial cells strongly expressed HLA-ABC in all cases, CD56 in 47 of 55, IL-4 in 15 of 41, TGF-beta1 in 14 of 25, and CD29 in 4 of 25 cases. The other markers including IFN-gamma, HLA-DR, HLA-DP, and HLA-DQ were not expressed by bile duct cells. The expression of HLA class I agrees with previous observations while the absence of class II expression does not. The expression by the bile duct epithelium of CD56 confirms our own previous report. A new observation is the finding of molecules such as IL-4, TGF-beta1, and CD29 strongly expressed in the bile ducts cells. The presence of these molecules, taken together with the lack of IFN-gamma expression, contradicts previous speculations that attributed to IFN-gamma a role in the induction of major histocompatibility antigens and adhesion molecules in immune-mediated alcoholic liver disease.

The Antigenic Heterogeneity of the Bile DuctEpithelium in Alcoholic Liver Disease

Abstract The chronic alcoholic patient is usually immunosuppressed, but the significance of this phenomenon in terms of bile duct injury is unclear. The immunoreactivity of the bile duct cells was examined in a series of 69 frozen liver biopsy specimens obtained from patients with alcoholic liver disease, comprising 29 cases of cirrhosis, 26 of alcoholic hepatitis, 10 cases of alcoholic fatty liver, and 4 specimens from normal livers. Liver diseases such as primary biliary cirrhosis and human hepatic allograft rejection, known to have an autoimmune basis, share the characteristic feature of damage to the bile duct epithelial cells. In both instances the damage seems to be immune mediated, but the nature of the antigens involved is not established. We used the avidin-biotin-peroxidase complex method to test in alcoholic liver disease for the expression of a battery of surface antigen markers that have been incriminated in tissue injury and are usually present in lymphoid cells but also expressed by epithelium. In this study we investigated the expression of the following molecules: HLA class I (ABC) and class II (HLA-DR, HLA-DP, HLA-DQ), CD29, CD45RA, CD45RO, CD56, interleukin 1 (IL-I), IL-2, IL-4, interferon (IFN-γ), tumor necrosis factor β, and transforming growth factor β1 (TGF-β1). The bile duct epithelial cells strongly expressed HLA-ABC in all cases, CD56 in 47 of 55, IL-4 in 15 of 41, TGF-β1 in 14 of 25, and CD29 in 4 of 25 cases. The other markers including IFN-γ, HLA-DR, HLA-DP, and HLA-DQ were not expressed by bile duct cells. The expression of HLA class I agrees with previous observations while the absence of class II expression does not. The expression by the bile duct epithelium of CD56 confirms our own previous report. A new observation is the finding of molecules such as IL-4, TGF-β1, and CD29 strongly expressed in the bile ducts cells. The presence of these molecules, taken together with the lack of IFN-γ expression, contradicts previous speculations that attributed to IFN-γ a role in the induction of major histocompatibility antigens and adhesion molecules in immune-mediated alcoholic liver disease.

Biliary expression of heat shock protein: a non-specific feature of chronic cholestatic liver diseases.

To analyse the expression of heat shock protein (HSP) 60 in biliary epithelium in auto-immune liver conditions and also in chronic cholestatic and other liver diseases. Hepatic expression of HSP-60 in frozen liver biopsy specimens from patients with primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), auto-immune hepatitis (AIH), obstructive jaundice (LDO), alcoholic liver disease (ALD), and from normal controls was studied by immunohistochemistry using the APAAP technique and confocal laser scanning microscopy. Increased expression of HSP-60 was demonstrated in the biliary epithelium of patients with PBC, LDO and, to a lesser extent, with PSC. Focal, weaker, biliary epithelial expression of HSP-60 was observed in AIH, ALD and normal liver tissue. Increased expression was also seen on Kupffer cells in LDO and in hepatocytes in areas of piecemeal necrosis in AIH. Enhanced biliary expression of HSP-60 is a common feature of chronic biliary disease irrespective of aetiology and is not specific to auto-immune diseases.

Polymerase chain reaction for the early diagnosis of cytomegalovirus hepatitis in liver transplant patients

Background: In liver transplant (LTX) patients, cytomegalovirus (CMV) hepatitis as a cause of graft dysfunction occurs in 15–25% of the patients. Polymerase chain reaction (PCR), applied to liver biopsy specimens, may increase the ability to detect CMV DNA at a local site. In this study, PCR was used to compare its relation to the development of clinical CMV hepatitis. Study design: Nested polymerase chain reaction (nPCR), derived from a conserved region of the CMV major immediate-early gene, was used to examine 141 frozen liver biopsies from 61 LTX patients for the presence of CMV DNA. 134 biopsies were obtained from 54 patients with pathological liver function tests within four months after transplantation. The remaining seven patient biopsies were derived from the one-year investigation after LTX and served as controls. The results were compared to virus isolation, antigen detection by immunohistology and in situ hybridization for CMV DNA of the biopsy specimens. Histological examination was performed to verify a diagnosis of viral hepatitis. Results: CMV DNA was amplified in 11% ( 15 134 ) of the biopsies, corresponding to 20% ( 11 54 ) of the patients. Virus isolation revealed CMV in 5% ( 7 134 ) of the samples. None of the nPCR-negative biopsies was virus culture positive. CMV genomes were detected by nPCR more frequently than CMV hepatitis was diagnosed by using the combination of CMV-specific histopathology and/or immunohistology and/or CMV-positive virus isolation ( p < 0.01). However, when this comparison was performed within individual patients, the difference was not significant ( p > 0.05). If the results of in situ hybridization were included in the diagnostic criteria of CMV hepatitis, the nPCR was comparable to these, both at the biopsy and the patient levels ( p > 0.1 and p > 0.05, respectively). For the diagnosis of CMV hepatitis the negative predictive value of CMV-nPCR was 1.0. The positive predictive value ranged from 0.55 to 0.82 depending on the criteria of CMV hepatitis. The nPCR also detected signs of CMV infection in the liver graft earlier than virus isolation, 11 versus 21 days, respectively, after transplantation. Conclusion: The frequency of CMV DNA positivity, measured by nPCR, was similar to that revealed by other combined methods. We suggest that the combined findings of histological cholangitis and/or lobulitis together with nPCR for CMV DNA can be used as a diagnostic criterion for initiation of antiviral treatment against CMV hepatitis.

Hepatocellular expression of HLA-A, B, C molecules predicts primary response to interferon in patients with chronic hepatitis C.

Primary response rate to alpha-interferon (IFN) is about 50% in patients with chronic hepatitis C. Criteria for predicting a positive primary response are lacking. HLA-A,B,C molecule expression is known to be stimulated by viral infections. In 36 consecutive interferon-treated anti-HCV positive patients with an available frozen liver biopsy sample, the predictive value of liver HLA-A,B,C expression, and of histologic, clinical, and biochemical parameters was evaluated. Response to treatment was defined by normalization of transaminases, and disappearance of serum HCV-RNA within 3 months. According to these criteria, 17 patients were classified as nonresponders and 19 were classified as responders. The pattern of HLA-A,B,C hepatocellular positivity varied from normal (negative or occasional faint staining of hepatocellular membranes) to diffuse, strong "honeycomb" positivity. The highest scores of positivity were found in nonresponder patients. The discriminant capacity of HLA-A,B,C scores of positivity was compared with clinical, biochemical and histologic parameters by discriminant analysis. HLA-A,B,C expression was found to be the main discriminant parameter, in addition to alkaline phosphate (ALP) and gamma-glutamyl-transpeptidase (GGT) which added little additional information. The higher hepatocellular expression of class I MHC molecules in nonresponder cases may reflect a different viral effect on hepatocytes, which is induced by different HCV genotypes or levels of viremia. From a clinical point of view, the pretreatment HLA-A,B,C pattern of positivity represents a powerful tool in the selection of patients for interferon treatment.

Down-regulation of intercellular adhesion molecule 1 on hepatocytes in viral chronic hepatitis treated with interferon alfa-2b

Background: Intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 are expressed markedly on hepatocytes in viral chronic hepatitis. The aim of this study was to determine whether treatment induces changes in the intrahepatic expression of adhesion molecules. Methods: The expression patterns of these molecules were studied in liver tissue from 12 patients with chronic hepatitis B and from 14 patients with chronic hepatitis C before and after treatment with interferon alfa-2b. Immunoperoxidase staining was performed in frozen liver biopsy sections. Results: Partial or complete responses to therapy were achieved in 7 (58%) of 12 patients with chronic hepatitis B and in 10 (71%) of 14 patients with chronic hepatitis C. In the first biopsy specimen, hepatocytes showed a marked expression of intercellular adhesion molecule 1. In the second biopsy specimen obtained from responders, hepatocytes showed no reactivity in 4 patients with chronic hepatitis B and in 5 with chronic hepatitis C. However, its expression did not significantly change among nonresponders. A similar pattern of reactivity for lymphocyte function-associated antigen 3 was observed. Conclusions: Beneficial effect of interferon alfa-2b appears to be associated with a down-regulation of adhesion molecules on hepatocytes, suggesting that this novel immunomodulatory action of interferon could be important to induce a therapeutic response in viral chronic hepatitis.

Cell-mediated hepatic injury in alcoholic liver disease

Background: The mechanism responsible for the initiation and perpetuation of alcoholic liver disease (ALD) remains poorly understood. This investigation attempted to elucidate the role of cell-mediated immune phemonena in the pathogenesis of ethanol-induced liver injury. Methods: Frozen liver biopsy specimens from 144 patients with moderate to severe ALD were examined by the avidin-biotin immunoperoxidase technique for the expression of antigenic markers of T and B lymphocytes, natural killer cells, and class I and II MHC molecules in the tissue. Results: Expression of CD3 by lymphocytes correlated significantly with regenerating nodules, intralobular inflammation, central sclerosis, and abnormalities of Kupffer cells. B cells were rarely present, and natural killer cells were absent. CD3+ lymphocytes expressed either CD4 or CD8 surface molecules. Enhanced class I MHC expression correlated significantly with portal inflammation, limiting plate erosion, vascular abnormalities, and hemosiderosis. Expression of class II MHC molecules correlated significantly with necrosis, bile stasis, and Mallory bodies. Conclusions: The distribution and persistence of CD4+ and CD8+ cells in actively advancing ALD, the enhanced MHC expression on hepatocytes, and their relationship to alcoholic hyalin and necrosis lend support to the hypothesis that a cytotoxic T lymphocytehepatocyte interaction plays a role, perhaps via lymphokine production, in the genesis or perpetuation of ALD.

Detection of hepatitis B virus DNA in the liver of children with chronic hepatitis B by in situ hybridization and its relation to other viral markers.

The aim of the study was to detect hepatitis B virus (HBV) DNA by in situ hybridization (ISH) with a 35S-labeled radioactive probe in frozen liver biopsy tissue sections of 63 hepatitis B virus surface antigen (HBsAg)-positive children. The results were compared to other markers of viral replication. HBV DNA was detected in 48 children. Of the 15 negative cases, four had hepatitis B envelope antigen (HBeAg), 10 anti-HBe, and one neither HBeAg nor anti-HBe. Free HBV DNA in serum and liver was positive in one patient. Forty of the positive children were HBeAg- and six anti-HBe-positive; two were negative for both. Of 45 36 had HBV DNA in serum. In 38 of 47 HBV DNA and in 31 of 42 HBcAg could be detected in the liver. The HBV DNA signals were located mainly over the cytoplasm of hepatocytes. The distribution of HBV DNA in the tissue was classified as homogeneous, inhomogeneous with focal patches, and focal. It is concluded that in situ hybridization is a reliable method for detection of HBV DNA in liver tissue of children with chronic hepatitis B. The technique, which can be applied to small amounts of liver tissue, provides informations about the distribution of replicative viral sequences, complementing laboratory data, liver histochemistry, and histology.

Detection of Hepatitis B Virus DNA in the Liver of Children with Chronic Hepatitis B by In Situ Hybridization and Its Relation to Other Viral Markers

SummaryThe aim of the study was to detect hepatitis B virus (HBV) DNA by in situ hybridization (ISH) with a 35S‐labeled radioactive probe in frozen liver biopsy tissue sections of 63 hepatitis B virus surface antigen (HBsAg)‐positive children. The results were compared to other markers of viral replication. HBV DNA was detected in 48 children. Of the 15 negative cases, four had hepatitis B envelope antigen (HBeAg), 10 anti‐HBe, and one neither HBeAg nor anti‐HBe. Free HBV DNA in serum and liver was positive in one patient. Forty of the positive children were HBeAg‐ and six anti‐HBe‐positive; two were negative for both. Of 45 36 had HBV DNA in serum. In 38 of 47 HBV DNA and in 31 of 42 HBeAg could be detected in the liver. The HBV DNA signals were located mainly over the cytoplasm of hepatocytes. The distribution of HBV DNA in the tissue was classified as homogeneous, inhomogeneous with focal patches, and focal. It is concluded that in situ hybridization is a reliable method for detection of HBV DNA in liver tissue of children with chronic hepatitis B. The technique, which can be applied to small amounts of liver tissue, provides informations about the distribution of replicative viral sequences, complementing laboratory data, liver histochemistry, and histology.

Intrahepatic up-regulated expression of extracellular matrix protein receptors in chronic active hepatitis type B

The aim of the present study was to investigate the differences in expression of β1 integrins between normal liver and the inflamed livers of patients with chronic active hepatitis B. Immunohistochemical staining with monoclonal antibodies that specifically recognize the common β1 chain and five different α subunits has been performed in frozen liver biopsy sections from 10 patients with chronic active hepatitis B and from 4 patients with normal livers. The major findings of our study were de novo expression in liver with chronic active hepatitis B of α2 and α3 subunits on both periportal hepatocytes and on lobular hepatocytes in close proximity to lymphocyte infiltrates. These results indicate the existence of an up-regulatory process in the expression of β1 integrins, especially the α2 and α3 subunits, in the inflamed liver tissue from patients with chronic active hepatitis B, suggesting that these integrins could play an important role in the development of liver fibrosis and in regulating intrahepatic lymphocyte migration.

Relationship between HBcAg in serum and liver and HBV replication in patients with HBsAg-positive chronic liver disease.

The expression of hepatitis B core antigen (HBcAg) in serum and in hepatocytes was evaluated in relation to HBV replication. Fifty chronic HBsAg carriers with histological evidence of liver disease were studied, including 24 HBeAg-positive patients, 2 HBeAg/anti-HBe-negative patients, and 24 anti-HBe-positive cases, two of them with evidence of delta agent infection. Serum HBV-DNA was evaluated in all patients and related to HBcAg examined at the same time in frozen liver biopsies by immunofluorescence and to HBcAg detected in the corresponding serum by a recently developed radioimmunoassay. HBV-DNA was present in serum in 20 (83%) HBeAg-positive patients, all positive for serum HBeAg, whereas liver core antigen was detected in 14 (73%) of 19 cases. Among HBeAg-negative patients, 50% showed the presence of circulating DNA viral sequences, and HBcAg was identified in five of 26 (19%) cases in serum and in six of 24 (25%) in the liver respectively. In 15 patients, liver fragments permitted examination in parallel by immunofluorescence for HBcAg and molecular hybridization for viral DNA in liver cells. A DNA pattern characteristic of viral replication was found in cases with evidence of active virion production, independently from HBeAg and anti-HBe, and in these patients HBcAg was present both in serum and in hepatocytes. In two cases with free HBV-DNA, without evidence of replicative activity, core antigen was not detected in the liver, but in one patient HBcAg was found in the serum. A similar finding was also noted in another patient, in whom the hybridization pattern was consistent with integration of viral genome into high-molecular-weight cellular DNA. Whether serum HBcAg detected in these patients without HBV-DNA in serum reflects the presence of defective viral particles or of core antigen released as a viral protein remains to be determined.

Detection by immunofluorescence of a new "core-like" Ag/Ab system in liver and serum of patients with NANB hepatitis.

Using direct immunofluorescence, a nuclear antigen was found in liver of chronic hepatitis patients with circulating NANBe Ag or anti-NANBe, and selected sera from either group were used as source of conjugates. The new Ag/Ab system was designated NANBc Ag and anti-NANBc since it behaved like the core Ag of HBV . NANBc Ag was detected in coded frozen liver biopsies from patients with chronic persistent 15/25 (60%) or active 27/50 (54%) hepatitis and cryptogenic cirrhosis 16/30 (53.3%) devoid of HBV markers. Only 2/30 alcoholic cirrhosis cases (7%) used as controls were positive (p less than or equal to 0.001). The homologous anti-NANBc antibody was always detectable by indirect immunofluorescence in the patients' serum when NANBc Ag was found in the liver. It was also found in 11/135 (8%) additional cases without any other NANB marker. A correlation was observed between coded detection of the NANBc Ag/Ab system by immunofluorescence and demonstration of NANBe Ag or anti-NANBe by immunodiffusion. In acute post-transfusion NANB hepatitis, anti-NANBc was first detectable 14 days after transfusion and persisted as long as ALT remained elevated, or longer. IgM anti-NANBc present at onset became associated with an increasing proportion of IgG after the 28th day. The prevalence of anti-NANBc in sporadic NANB hepatitis (11/50 = 22%) was significantly lower (p less than or equal to 0.001) than in cases with parenteral exposure such as post-transfusion, occupational or drug addict hepatitis (47/72 = 65%). Immunofluorescent tests for NANBc Ag and Ab are promising assays for the serological diagnosis of NANB hepatitis.