Ultrastructural alterations in serial liver biopsy specimens from chimpanzees experimentally infected with a human non-A, non-B hepatitis agent

Four chimpanzees experimentally infected with an agent of human non-A, non-B hepatitis were studied to determine the sequence of ultrastructural alterations in hepatocytes during infection. Three of the four types of cytoplasmic alterations previously described in association with non-A, non-B hepatitis were observed in the hepatocytes. Sponge-like cytoplasmic inclusions (designated C-I) were detected at or near the time of peak serum aminotransferase elevations in two of the four chimpanzees. Undulating membranes (designated C-II) were observed in all four chimpanzees, at the time of the first elevation of serum aminotransferase levels. Cytoplasmic tubules (designated C-III) were first observed four, eight, and twelve weeks, respectively, after inoculation in three of the chimpanzees. Four weeks after the peak of serum aminotransferase elevations, cytoplasmic alterations could no longer be detected in hepatocytes of the four chimpanzees. Intranuclear inclusions consisting of 20-27 nm granules and vermicular particles were observed in hepatocytes from preinoculation liver biopsy specimens, as well as biopsies obtained during non-A, non-B hepatitis. The number of these particles was greatest near the time of peak elevation of serum aminotransferase levels, however. Tubulo-crystalline inclusions were noted as well in the endothelial cells from both preinoculated and infected chimpanzees. Cytoplasmic alterations in hepatocytes of chimpanzees experimentally infected with an agent of non-A, non-B hepatitis appear characteristic of infection with this agent. In contrast, intranuclear particles were not specifically related to the non-A, non-B hepatitis infection.     

A cDNA clone closely associated with non-A, non-B hepatitis.

A lambda gt11 cDNA library was constructed from RNA purified from hepatitis B viral surface antigen-negative human plasma with high alanine aminotransferase activity. A cDNA clone, designated as C8-2, was isolated by immunoscreening with mixed sera from non-A, non-B hepatitis (NANBH) carrier and convalescent chimpanzees. The recombinant protein produced by C8-2 reacted specifically with sera of patients in the chronic phase of NANBH. The sequence of C8-2, 269 bp, did not hybridized with any human or chimpanzee genomic DNA, and had no homology with those of primates and viruses. The existence of this sequence in RNA of possibly infectious plasma was shown by RNA blot hybridization and by Southern blot analysis of products amplified by the polymerase chain reaction. These results strongly suggest that C8-2 is derived from the agent of this viral hepatitis.     

Assessment of increased serum aminotransferases in a managed Atlantic bottlenose dolphin (Tursiops truncatus) population

Nonspecific chronic hepatitis and increased activities of serum aminotransferases have been reported in cetaceans (dolphins, porpoises, and whales). We identified bottlenose dolphins in our current population with episodic increases in serum aminotransferases, specifically alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and we hypothesized that hematologic and serum biochemical changes in these animals may provide clues as to potential causes of liver disease in cetaceans. A retrospective case-control study involving 1,288 blood samples collected during 1998-2006 from 18 dolphins (six cases and 12 age- and sex-matched healthy controls) was conducted to compare eosinophil and platelet counts; and serum proteins, albumin, globulins, bilirubin, gamma glutamyltransferase (GGT), cholesterol, triglycerides, glucose, iron, and erythrocyte sedimentation rates. Bottlenose dolphins with increased ALT and AST activities were more likely to have higher serum globulins, bilirubin, GGT, iron, glucose, triglycerides, and cholesterol levels, greater erythrocyte sedimentation rates, and lower platelet counts compared to healthy controls. Our findings suggest that dolphins with chronic increases in aminotransferases may have a chronic hepatitis involving iron overload with similar etiologies and pathophysiology compared to terrestrial mammals. Areas for future research include predisposing metabolic risk factors; associations between iron overload and a diabetes-like condition; and a potential overlap syndrome involving autoimmune responses that may or may not be associated with viral infection.     

Spontaneous amyloidosis in twelve chimpanzees, Pan troglodytes

Spontaneous amyloidosis was diagnosed in 11 male and 1 female chimpanzees and confirmed histologically and immunohistochemically. The chimpanzees were > or = 15 years of age when first diagnosed and averaged 22.4 years of age. The average survival time after diagnosis of systemic amyloidosis was 1.86 years with a standard deviation of 4.06 years (n = 7). The chimpanzees with amyloidosis were asymptomatic except for hepatomegaly, which became more detectable with age. Significant increases in clinical chemistry values, as compared with referenced normals and established normals, of blood urea nitrogen (BUN), asparate aminotransferase (AST), gamma-glutamyltransferase (GGT), globulin, total protein, creatinine phosphokinase (CPK), sedimentation rate, and triglycerides were found in animals 7 years of age or older with amyloidosis. These serum chemistry values, while increased in chimpanzees with amyloidosis, were generally within normal limits. Immunohistochemistry for both amyloid A protein and amyloid P component-labeled extracellular amyloid in all chimpanzees with amyloidosis was determined. Amyloid was deposited primarily in the liver. Amyloidosis in the chimpanzee is a chronic, intractable, progressive, fatal disease, and appears to be similar to secondary amy loidosis in other species.     

The clinical chemistry of chimpanzees. I. Determination of aminotransferase baseline values for hepatitis studies

Viral hepatitis in chimpanzees produces negligible symptomatology, and serum aminotransferase changes may be minimal. To maximize the predictive value of these determinations, which are the only serum indicators available for non-A non-B (NANB) hepatitis infection, normal ranges for aspartate and alanine aminotransferases (AST, ALT) were examined and categorized according to age and sex. Males were found to have higher values than females, and adults higher values than juveniles. The kinetic method used and the values obtained are described. Differences in methodologies and reporting units are discussed.     

The clearance of hepatitis C virus infection in chimpanzees may not necessarily correlate with the appearance of acquired immunity

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-gamma)-producing CD4(+) and CD8(+) T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-gamma, IFN-alpha, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.     

Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.     

Hepatitis A

Hepatitis A is a disease of worldwide distribution which occurs in endemic and epidemic form and is transmitted primarily by person-to-person contact through the fecal-oral route. Common source epidemics due to contamination of food are relatively common, and water-borne epidemics have been described less frequently. The presumed etiologic agent of hepatitis A has now been visualized by immune electron microscopic (IEM) techniques in early acute-illness-phase stools of humans with hepatitis A as well as in chimpanzees experimentally infected with material known to contain hepatitis A virus. In addition, several new serologic tests for the detection of antibody against hepatitis A virus have been described. These include complement fixation and immune adherence techniques. Current data suggest that hepatitis A is caused by a single viral agent lacking the morphologic heterogeneity of hepatitis B viral components and that there may be relative antigenic homogeneity between strains of virus recovered from various parts of the world. Serologic studies to date also indicate that hepatitis A virus is not a major contributing cause in post-transfusion hepatitis.     

Correlation between serum enzymes and serum unsaturated vitamin B12 binding proteins in primary liver carcinoma

The serum unsaturated vitamin B12-binding capacity (UBBC), unsaturated transcobalamin (UTC) I, UTC II, UTC III levels, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase activities and bilirubin concentration were estimated in 61 patients with liver diseases (31 with hepatoma, 30 with viral hepatitis). The levels of serum cobalamin, UTC I, UTC III, UBBC, alanine and aspartate aminotransferases, and bilirubin were raised in both hepatoma and viral hepatitis patients. Serum UTC II was reduced in both conditions. Alkaline phosphatase activity was significantly increased in hepatoma. Four significant correlations were observed among these parameters in the hepatoma patients while only one significant correlation was observed in viral hepatitis.     

Viral Persistence, Antibody to E1 and E2, and Hypervariable Region 1 Sequence Stability in Hepatitis C Virus-Inoculated Chimpanzees

The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.     

A case report of hepatocellular carcinoma and focal nodular hyperplasia with a myelolipoma in two chimpanzees and a review of spontaneous hepatobiliary tumors in non-human primates

Spontaneous hepatobiliary tumors in non-human primates are uncommon. Here we report a case of hepatic carcinoma and a case of hepatic focal nodular hyperplasia (FNH) and myelolipoma in two captive chimpanzees. A 16-year-old male chimpanzee (4X0392) died after an 8-month history of hepatic amyloidosis and low-grade anemia. Necropsy findings included a hepatic neoplasm with highly pleomorphic hepatocytes arranged into irregular thickened trabeculae. The diagnosis was high-grade hepatocellular carcinoma. A second male chimpanzee (4X0080), 23 years of age, died suddenly of heart failure secondary to cardiomyopathy. An incidental finding at necropsy was a liver mass characterized by multinodularity, prominent fibrous septa, and biliary hyperplasia. These features were consistent with FNH. While 4X0392 had no history of experimental viral exposure, 4X0080 was vaccinated with inactivated hepatitis B virus, an attenuated hepatitis A virus, and was experimentally infected with hepatitis C virus and human immunodeficiency virus. A survey of the literature revealed 68 reported cases of hepatobiliary tumors in non-human primates, including 12 hepatocellular adenomas, eight cholangiocellular adenomas/cystadenomas, 22 hepatocellular carcinomas, seven cholangiocarcinomas, and seven gallbladder adenocarcinomas. The majority of reported cases have been in prosimians and Old World monkeys. Hepatic neoplasia is rare in chimpanzees. Only four hepatic neoplasms have been reported in chimpanzees, three of which were associated with viral hepatitis. FNH has not been previously described in any non-human primate.     

Lymphocyte and neutrophil dysfunction associated with hepatitis B virus and hepatitis non-A, non-B virus infection in the chimpanzee.

Chimpanzees were examined for the effect of viral hepatitis infections on specific and nonspecific immune response mechanisms. The data suggest that infection with either hepatitis B virus or hepatitis non-A, non-B virus may result in suppression of cellular immune response components. Mitogen-induced lymphocyte proliferation was lower in virus-infected chimpanzees than in naive animals. Neutrophils from virus infected animals exhibited decreased or altered chemiluminescence kinetics.     

乙型肝炎患者凝血功能与胱抑素C 联合检测的临床意义

目的:探讨乙型肝炎患者凝血功能与血清胱抑素C联合检测的临床诊断价值。方法:收集260例乙型肝炎患者为实验组及健康者70例为对照组,采用全自动凝血分析仪进行活化部分凝血酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(FIB)的测定,采用全自动生化分析仪进行血清胱抑素C、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、血清γ谷氨酰转肽酶(GGT)的检测。结果:除急性肝炎组外,其他各组乙型肝炎患者的APTT、PT值均高于对照组(P<0.05)。重型肝炎和肝炎肝硬化组FIB值均低于对照组(P<0.05);对于血清胱抑素C水平,除急性肝炎组外,其他各组值均明显高于健康对照组(P<0.05),且肝炎肝硬化组依次高于重型肝炎组和慢性肝炎组。各实验组ALT和AST水平均明显高于对照组(P<0.05),而对于GGT水平,重型肝炎组和肝炎肝硬化组明显高于对照组(P<0.05)。结论:联合检测APTT、PT、FIB凝血功能指标与血清胱抑素C水平,对临床判断乙型肝炎患者病变程度及预后具有重要意义。     

Targeting viral infection by microRNA inhibition

An inhibitor of microRNA-122 reduces viral load in chimpanzees that are chronically infected with hepatitis C virus, suggesting that such an approach might have therapeutic potential in humans.     

Hepatic transcriptome analysis of hepatitis C virus infection in chimpanzees defines unique gene expression patterns associated with viral clearance

Hepatitis C virus infection leads to a high rate of chronicity. Mechanisms of viral clearance and persistence are still poorly understood. In this study, hepatic gene expression analysis was performed to identify any molecular signature associated with the outcome of hepatitis C virus (HCV) infection in chimpanzees. Acutely HCV-infected chimpanzees with self-limited infection or progression to chronicity were studied. Interferon stimulated genes were induced irrespective of the outcome of infection. Early induction of a set of genes associated with cell proliferation and immune activation was associated with subsequent viral clearance. Specifically, two of the genes: interleukin binding factor 3 (ILF3) and cytotoxic granule-associated RNA binding protein (TIA1), associated with robust T-cell response, were highly induced early in chimpanzees with self-limited infection. Up-regulation of genes associated with CD8+ T cell response was evident only during the clearance phase of the acute self-limited infection. The induction of these genes may represent an initial response of cellular injury and proliferation that successfully translates to a "danger signal" leading to induction of adaptive immunity to control viral infection. This primary difference in hepatic gene expression between self-limited and chronic infections supports the concept that successful activation of HCV-specific T-cell response is critical in clearance of acute HCV infection.     

CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection

Although the CD4(+)- and CD8(+)-T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8(+) cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8(+)-T-cell response.     

Repetitive 5-azacytidine treatments of Fao cells induce a stable and strong expression of gamma-glutamyl transpeptidase.

The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.     

Detection of TTV in peripheral blood cells from patients with altered ALT and AST levels

This work analyzes the prevalence of TTV DNA in peripheral blood cells from patients with hepatic alterations and healthy blood donors and measures levels of sodium, potassium, urea, creatinine, phosphatase alkaline, total and direct bilirubin, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in certain randomly selected patients. DNA samples from 111 individuals were evaluated. They were divided into two groups, "A" (study) and "B" (control), including 54 patients with liver enzyme alterations (ALT/AST) presenting non-B-non-C hepatitis and 57 blood donors, respectively. TTV DNA was determined by nested PCR. Certain products of the second-round PCR were sequenced. Serum biochemical assay was performed and disclosed TTV in 31.48% (17/54) of patients in group A and 5.26% (3/57) in the control group B. TTV prevalence was significantly higher in patients with liver disease than in healthy donors. In group A, sodium, potassium, urea, creatinine, phosphatase alkaline, total and direct bilirubin, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed in certain randomly selected patients and no significant difference in biochemical levels (p>0.05) was found when TTV infected and noninfected individuals were compared. Knowledge related to TTV has rapidly increased, but many fundamental aspects remain unclear. This led us to question the role of TTV and doubt remains as to whether or not it is just a commensal virus. Further studies are necessary to confirm and extend these findings.     

Escape mutations alter proteasome processing of major histocompatibility complex class I-restricted epitopes in persistent hepatitis C virus infection

Mutations in hepatitis C virus (HCV) genomes facilitate escape from virus-specific CD8+ T lymphocytes in persistently infected chimpanzees. Our previous studies demonstrated that many of the amino acid substitutions in HCV epitopes prevented T-cell receptor recognition or binding to class I major histocompatibility complex molecules. Here we report that mutations within HCV epitopes also cause their destruction by changing the pattern of proteasome digestion. This mechanism of immune evasion provides further evidence of the potency of CD8+ T-cell selection pressure against HCV and should be considered when evaluating the significance of mutations in viral genomes from persistently infected chimpanzees and humans.