Phenotypic and functional characteristics of lymphocytes isolated from liver biopsy specimens from patients with active liver disease.

Liver-derived lymphocytes were isolated from 73 liver biopsy specimens obtained from patients with chronic active liver disease and from six samples of normal liver. Mean absolute numbers (+/- S.E.M) of liver-derived lymphocytes recovered from needle biopsy specimens by mild enzymatic digestion of the liver tissue varied from 0.7 +/- 0.3 x 10(3)/mm3 in allografts being rejected to 8.9 +/- 0.9 X 10(3)/mm3 in chronic non-A, non-B hepatitis. By two-color flow cytometry, T lymphocytes (CD3+) were the major liver-derived lymphocyte population in all biopsy specimens. The mean CD4/CD8 ratio (0.6 +/- 0.2) was similar for liver-derived lymphocytes obtained from samples of normal or diseased liver. However, activated (human leukocyte antigen DR+) T cells were significantly (p less than 0.05) increased in liver-derived lymphocytes obtained from liver disease specimens than they were in samples from normal livers. Natural killer cells were less numerous than T cells in specimens obtained from diseased livers, with the mean natural killer/T cell ratio ranging from a low of 0.1 in allograft rejection to a high of 0.8 +/- 0.3 in primary sclerosing cholangitis. Liver-derived lymphocytes isolated from diseased liver contained significantly fewer (p less than 0.05) CD3-CD56+ or CD56+CD16-natural killer cells than did those obtained from normal liver samples. Natural killer activity was consistently detectable in liver-derived lymphocytes obtained from specimens of normal or diseased livers. Moreover, natural killer activity in the liver did not differ significantly from that in either normal or patient peripheral blood.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocellular expression of lymphocyte function-associated antigen 3 in chronic hepatitis.

T lymphocyte-mediated cytolytic immune reactions are considered a major cause of hepatocyte injury in chronic viral and autoimmune hepatitis. To further investigate local immune responses, we studied the expression of lymphocyte antigens and cell-cell interaction molecules known to be involved in effector-target cell interactions by light and electron microscopy in liver biopsy specimens from patients with chronic viral and autoimmune hepatitis. CD8+ lymphocytes were found to be the predominant population of cells in the inflammatory infiltrate in chronic hepatitis B and non-A, non-B hepatitis. In contrast, CD4+ cells constituted a comparably higher proportion of cells and were more numerous than CD8+ cells in chronic autoimmune hepatitis. In both viral and autoimmune hepatitis, a substantial portion of lymphocytes expressed activation antigens such as T11/3 (CD2R) and IL-2-R (CD25). Lymphocyte function-associated antigen-3 (CD58), which mediates lymphocyte adhesion and activation and is the natural ligand of the CD2/T11 lymphocyte surface receptor, could be demonstrated on endothelial cells and hepatocytes. Hepatocellular lymphocyte function-associated antigen-3 expression in chronic hepatitis showed membranous and cytoplasmic staining of hepatocytes and had a positive correlation with the degree of inflammatory activity. These results suggest that effector-target interactions between hepatocytes and lymphocytes mediated by the lymphocyte function-associated antigen-3/CD2 pathway play a role in chronic inflammatory liver disease. Possible functional consequences of this interaction include enhancement of antigen-specific immune reactions and antigen-independent mechanisms of T cell activation, which may contribute considerably to the degree of inflammatory activity and tissue damage in chronic hepatitis.     

Acute liver biopsy lesions in early autoimmune ("lupoid") chronic active hepatitis

We studied two female patients with autoimmune ("lupoid") chronic active hepatitis whose liver biopsies at initial presentation showed the unusual features of an acute hepatitis. Centrilobular hepatocyte swelling and multinucleation, acidophilic degeneration, cholestasis, mild fatty change and bile duct damage in one case resembled lesions of non-A, non-B hepatitis. Lobular and portal infiltrates of plasma cells with piecemeal necrosis suggested transition to chronicity as well as an autoimmune component. This was additionally supported by the presence of hypergammaglobulinemia and auto-antibodies in both patients. We conclude that liver biopsy features in the acute presentation of lupoid hepatitis may be difficult to distinguish from those seen in acute hepatitis due to virus or drugs.     

Role of hepatitis C virus in non-B chronic liver disease.

To assess the contribution of the recently identified hepatitis C virus to chronic liver diseases of unknown cause and chronic hepatitis attributed by exclusion to non-A, non-B hepatitis, we tested for antibody to hepatitis C in hepatitis B surface antigen-negative patients with a spectrum of chronic liver diseases. Antibody to hepatitis C virus, a marker of hepatitis C infection, was detected with a first-generation radioimmunoassay at the following frequencies in the following patient groups: 69% of transfusion-associated non-A, non-B hepatitis; 53% of non-transfusion-associated non-A, non-B hepatitis; 26% of hepatitis B surface antigen-negative hepatocellular carcinoma; 8% of cryptogenic cirrhosis; 5% to 7% of autoimmune chronic liver diseases; 19% of patients with miscellaneous types of chronic liver disease; and 0.67% of healthy controls. Among non-transfusion-associated cases, 81% with a history of intravenous drug use but only 18% with occupational exposure as health workers had antibody to hepatitis C virus. Among cases of hepatocellular carcinoma, 63% of Japanese patients but only 11% of American patients had evidence of hepatitis C infection. Comparison in a subgroup of 79 serum samples of a second-generation radioimmunoassay with the first-generation assay demonstrated a 12% increase in antibody frequency from 30% to 42%. We conclude that hepatitis C plays a substantial role in transfusion-associated and non-transfusion-associated non-A, non-B hepatitis as well as in hepatocellular carcinoma, especially in Japan, a limited role in cryptogenic cirrhosis, and essentially no role in autoimmune chronic liver diseases. Application of more sensitive immunoassays will increase the frequency of antibody seropositivity in all subgroups, but relative distinctions among risk groups are likely to remain.     

Acute liver biopsy lesions in early autoimmune (“lupoid”) chronic active hepatitis

ABSTRACT— We studied two female patients with autoimmune (“lupoid”) chronic active hepatitis whose liver biopsies at initial presentation showed the unusual features of an acute hepatitis. Centrilobular hepatocyte swelling and multinucleation, acidophilic degeneration, cholestasis, mild fatty change and bile duct damage in one case resembled lesions of non-A, non-B hepatitis. Lobular and portal infiltrates of plasma cells with piecemeal necrosis suggested transition to chronicity as well as an autoimmune component. This was additionally supported by the presence of hypergammaglobulinemia and auto-antibodies in both patients. We conclude that liver biopsy features in the acute presentation of lupoid hepatitis may be difficult to distinguish from those seen in acute hepatitis due to virus or drugs.     

The chronic sequelae of non-A, non-B hepatitis.

Twenty-six of 388 patients (6.7%) followed prospectively after open-heart surgery developed non-A, non-B hepatitis. Of these 26, 12 had an elevated (often fluctuating) serum alanine aminotransferase (SGPT) for greater than 1 year. Liver biopsy, done in eight of 12, showed chronic active hepatitis in six and chronic persistent hepatitis in two; one patient with chronic active hepatitis had early cirrhosis. Anicteric patients with peak SGPT greater then 300 IU/L were at greatest risk of developing chronic hepatitis. Chronic non-A, non-B hepatitis was symptomatically mild and unaccompanied by physical signs or laboratory evidence of autoimmune disease or severe chronic liver disease. In all 12 patients there was spontaneous improvement in serum transaminase over a period of 1 to 3 years, and four patients had sustained normalization of SGPT. Thus chronic active hepatitis is a common sequela of acute non-A, non-B hepatitis but may have a better prognosis than chronic active hepatitis of other causes.     

Histological improvement of chronic hepatitis B, and non-A, non-B with interferon treatment: application of a numerical scoring system for evaluating sequential morphologic changes

In order to assess the sequential changes of liver pathology after interferon therapy, 70 liver biopsy specimens, collected from 23 HBeAg-positive patients with chronic hepatitis B and 12 patients with chronic non-A, non-B hepatitis, were studied using a numerical scoring system proposed by Knodell et al. The biopsy specimens were obtained immediately prior to the administration of interferon and within one week following the termination of interferon therapy. Three patients with chronic hepatitis B were negative for DNA-polymerase (DNA-P) prior to the interferon administration. Ten patients (50%) lost DNA-P activity. HBeAg became negative in 8 patients (34.8%), of whom 2 seroconverted to anti-HBe. Serum alanine aminotransferase levels normalized in 9 patients with chronic hepatitis B, and non-A, non-B hepatitis. The total Histological Activity Index (HAI) scores in both patients with chronic hepatitis B, and non-A, non-B decreased significantly (P less than 0.001 and P less than 0.005, respectively) after interferon treatment. There was also a significant improvement (0.02 less than P less than 0.001) in each histological category except for fibrosis. When the changes of the HAI scores in patients with chronic hepatitis B were correlated to the outcome in the DNA-P and HBeAg/anti-HBe system after treatment, the histological improvement did not significantly correlate to the outcome of these serological parameters.     

Histological improvement of chronic hepatitis B,and non-A,non-B with interferon treatment: Application of a numerical scoring system for evaluating sequential morphologic changes

In order to assess the sequential changes of liver pathology after interferon therapy, 70 liver biopsy specimens, collected from 23 HBeAg-positive patients with chronic hepatitis B and 12 patients with chronic non-A, non-B hepatitis, were studied using a numerical scoring system proposed by Knodell et al. The biopsy specimens were obtained immediately prior to the administration of interferon and within one week following the termination of interferon therapy. Three patients with chronic hepaitits B were negative for DNA-polymerase (DNA-P) prior to the interferon administration. Ten patients (50%) lost DNA-P activity. HBeAg became negative in 8 patients (34.8%), of whom 2 seroconverted to anti-HBe. Serum alanine aminotransferase levels normalized in 9 patients with chronic hepatitis B, and non-A, non-B hepatitis. The total Histological Activity Index (HAI) scores in both patients with chronic hepatitis B, and non-A, non-B decreased significantly (P<0.001 and P<0.005, respectively) after interferon treatment. There was also a significant improvement (0.02<P<0.001) in each histological category except for fibrosis. When the changes of the HAI scores in patients with chronic hepatitis B were correlated to the outcome in the DNA-P and HBeAg/anti-HBe system after treatment, the histological improvement did not significantly correlate to the outcome of these serological parameters.     

Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients.

In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.     

Identification of T lymphocytes and subsets in liver biopsy cores of acute viral hepatitis

The lymphocytic infiltrates were examined in 10 liver biopsy cores taken from nine cases of acute viral hepatitis (AVH), using cryostat sections. The cells were identified and characterized, using the monoclonal antibodies to T lymphocytes (OKT3), T helper cells (OKT4), and T suppressor/cytotoxic cells (OKT8) in an indirect immunofluorescence technique. Of the nine cases of AVH, three were type B, one was acute type A, with underlying chronic hepatitis type B, and the remaining five were non-A, non-B, with no serological markers. One of the patients with acute non-A, non-B hepatitis was biopsied twice with a 2-month interval due to clinical relapse. Both these biopsies were included in the study. The cellular infiltrates, both lobular and portal, were graded from 1 + to 4 + and evaluated by examining the histological sections stained with hematoxylin and eosin. The approximate percentage of each set of cells staining with different antibodies was estimated in each case. The predominant cells were found to be T lymphocytes in all nine cases of AVH; of the two subsets, T suppressor/cytotoxic cells formed the majority in all except one patient who had AVH type A superimposed on chronic B hepatitis.     

Hepatitis C

The major cause of chronic post-transfusion hepatitis, the hepatitis C virus (HCV), has been identified. HCV is a single-stranded linear RNA virus with characteristics similar to the flaviviruses. A different agent, the hepatitis E virus, is associated with epidemic (enterically-transmitted) non-A, non-B hepatitis. At present, infection with HCV is recognized by the finding of anti-HCV antibodies, positive in up to 90% of patients with chronic non-A, non-B post-transfusion hepatitis. Antibodies to HCV are detected in 1% of normal volunteer blood donors and in the majority of donors implicated in post-transfusion hepatitis. HCV antibodies are also found in patients with autoimmune liver disease and hepatocellular carcinoma. Moreover, HCV infection may contribute to the pathogenesis of liver disease in alcoholic patients. The role of HCV infection in fulminant non-A, non-B hepatitis and hepatitis-associated aplastic anemia has not been elucidated as yet. Therapy of chronic non-A, non-B hepatitis with recombinant human alpha-interferon has been shown to improve or normalize aminotransferase levels in approximately 50% of patients, most of whom have evidence of HCV infection. However, relapse after cessation of treatment is common. In the future, screening blood for evidence of HCV infection may prevent most cases of non-A, non-B post-transfusion hepatitis.     

Peripheral blood lymphocyte subsets in patients with chronic hepatitis C – effects of interferon treatment

Abstract: Thirty-three patients with chronic hepatitis non-A, non-B/C were included in a randomized controlled study of recombinant alpha-2b interferon treatment 3 MU three times weekly for 36 weeks. In lysed whole blood, lymphocyte subpopulations were enumerated by flow cytometry detecting fluorescein or phycoerytrin conjugated monoclonal antibodies directed against seven different epitopes. Patients with chronic active hepatitis were significantly older than patients with chronic persistent hepatitis (p>0.05). Before treatment, the proportions of different subsets of lymphocytes were within the normal reference values and the CD4/CD8 ratio was also normal. No increased activation of T-cells was noticed. Patients over 50 years of age, however, had a significantly increased (p>0.01) proportion of HLA-DR + lymphocytes, mainly B-cells. Treatment decreased the absolute number of peripheral blood leukocytes and lymphocytes. There was also a significant decline in the proportion of CD8 + lymphocytes and NK-cells, and a significant increase in the proportion HLA-DR + cells and of the CD4/CD8 ratio. The increased proportion of HLA-DR + cells, however, did not reflect peripheral T-cell activation; instead, it was due to increasing B lymphocyte numbers.     

丙型肝炎病毒感染者外周血淋巴细胞亚群的变化及意义

目的调查丙型肝炎患者外周血淋巴细胞亚群变化及影响因素。方法利用流式细胞术检测241例丙型肝炎患者和117例健康人群外周血淋巴细胞亚群数值,通过回顾性分析,调查我国丙型肝炎人群淋巴细胞亚群变化及其相关的影响因素。结果在健康人群中,CD3＋CD4＋T细胞、CD3＋CD16＋CD56＋NKT细胞频率以及CD4/CD8比值与年龄呈显著正相关;而在慢性丙型肝炎人群中,CD3＋CD4＋T细胞与年龄呈显著正相关,CD3-CD19＋B细胞则与年龄呈显著负相关;在肝硬化人群中,只有CD3-CD16＋CD56＋NK细胞频率与年龄呈显著正相关;在15～49岁的健康人及慢性肝炎人群中,女性CD3＋CD4＋T细胞亚群频率高于男性,而在肝硬化患者中女性CD3-CD19＋B细胞频率低于男性;同时,在HCV感染的不同阶段,CD3-CD16＋CD56＋NK细胞亚群频率均较正常人显著降低,而CD3＋CD8＋T细胞频率则显著升高,在15岁以上人群,CD3-CD19＋B细胞在健康人、慢性肝炎、肝硬化人群中呈持续升高的现象。结论通过回顾性分析健康人群和HCV感染不同阶段人群淋巴细胞亚群的分布特点及其与HCV疾病进展、年龄、性别的关系,为临床科学评价丙型肝炎人群免疫状态提供重要的免疫指标。     

Further studies by immunofluorescence of the monoclonal antibodies associated with experimental non-A, non-B hepatitis in chimpanzees and their relation to D hepatitis

To further investigate the specificity of the monoclonal antibodies (48-1 and S-1) associated with non-A, non-B hepatitis, extensive immunofluorescence studies were performed on liver biopsy specimens from chimpanzees with experimental hepatitis A, B, non-A, non-B or delta, or from normal chimpanzees. Both 48-1 and S-1 antibodies reacted in the same manner with liver biopsy specimens from 47 of 50 (94%) chimpanzees with acute or chronic non-A, non-B hepatitis and 15 of 18 (83%) chimpanzees with type D hepatitis. Examinations of serial liver biopsy specimens revealed that the duration of expression of the antigen reacting with the antibodies in hepatocytes of chimpanzees infected with non-A, non-B viruses appeared to be longer than that of chimpanzees infected with the hepatitis delta-virus. By thin-section electron microscopy, the presence of the microtubular aggregates, identical to those previously described for chimpanzees with non-A, non-B hepatitis and shown by immunoelectron microscopy to react with the antibodies, was noted in hepatocytes during the acute phase of hepatitis delta-virus. The antibodies did not react with liver biopsy specimens from chimpanzees acutely or chronically infected with hepatitis B virus or hepatitis A virus, or from normal chimpanzees. The present results confirm our previous observations with the 48-1 and S-1 antibodies. Furthermore, the finding that these two antibodies were also associated with hepatitis D would support the possibility that non-A, non-B agents and the hepatitis delta-virus may have a similar nature or may elicit a similar host response.     

中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围

目的 调查中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围.方法 利用流式细胞术检测2846例乙型肝炎患者和117例健康人群外周血淋巴细胞亚群数值,调查我国健康人群和乙型肝炎人群的参考值范围.结果 调查了16～60岁健康人群和HBV感染相关的急性肝炎、慢性肝炎、重型肝炎和肝硬化人群外周血CD3+T淋巴细胞、CD3+CD...     

The role of acute hepatitis type A, B, and non-A non-B in the development of chronic active liver disease

In 19 patients followed from biopsy-verified acute viral hepatitis to chronic active liver disease and 74 patients followed to complete resolution verified by a normal liver biopsy, sera from the acute phase were studied for serologic evidence of hepatitis type A and B. Eleven of the 19 patients who developed chronic active liver disease progressed from acute hepatitis type B and 7 from acute hepatitis type non-A non-B. One patient could not be classified because the sera were exhausted. None had serological markers of actual hepatitis type A infection. Of the 74 patients with a histologically complete resolution, the acute episode could be classified as type B hepatitis in 47 and type A hepatitis in 13 patients. The remaining 14 patients were classified as having acute viral hepatitis type non-A non-B. Our findings confirm that type B and non-A non-B hepatitis may give rise to chronic liver disease, whereas type A hepatitis so far has not been demonstrated to initiate a chronic liver disease.     

Intravenous immunoglobulin prophylaxis causing liver damage in 16 of 77 patients with hypogammaglobulinemia or IgG subclass deficiency

Sixteen of 77 patients (21 percent) with common variable immunodeficiency or IgG subclass deficiency contracted non-A, non-B hepatitis in association with intravenous infusions of immunoglobulin. The hepatitis seemed to run a more severe course in these patients than in non-immunodeficient patients. Twelve patients had clinical symptoms, and five died with hepatitis being the cause of death in two and a contributing factor in three. Liver biopsy specimens showed early chronic active hepatitis and cirrhosis. In addition to increases in liver enzymes, 13 patients had increases in alkaline phosphatase levels. All but two patients who contracted hepatitis had been given 50 mg/kg per week or more of intravenous immunoglobulin. Lymphocyte counts, T/B cell ratios, and T-lymphocyte function did not differ between those in whom hepatitis developed and those in whom it did not develop. The hepatitis was associated with more than one batch of a Swedish intravenous immunoglobulin, the immunoglobulin being derived from United States sources as well as from European plasma. Three previous brief reports in the literature have also associated non-A, non-B hepatitis with the intravenous infusion of various immunoglobulins. Biologic materials given to patients, including immunoglobulin, should, whenever possible, be prepared so as to ensure absence of viruses.     

Electron microscopic evidence of non-A, non-B hepatitis markers and virus-like particles in immunocompromised humans

Characteristic pathological alterations of the liver in chimpanzees inoculated with non-A, non-B hepatitis sera have been described, but no corresponding findings have been reported in humans. Electron microscopic studies of the liver biopsy specimens of two homosexual patients with acquired immune deficiency syndrome, one without hepatitis (Patient 1) and one with chronic active hepatitis in remission (Patient 2), revealed the cytoplasmic tubular structures which are characteristic of chimpanzee non-A, non-B hepatitis. A cluster of 23 nm double-shelled particles was also seen in the cytoplasm of a hepatocyte in patient 1 who had received a blood transfusion 8 days before the biopsy. These particles were smaller than the Dane particles, Epstein-Barr virus, cytomegalovirus or herpes simplex virus, and different from hepatitis A virus particles; the antibodies to all of which are found in high concentration in acquired immune deficiency syndrome patients. These observations may reflect the morphologic findings for non-A, non-B hepatitis infection in humans.     

Low prevalence of hepatitis C antibodies in chronic liver disease in Finland.

High prevalence of hepatitis C antibodies (anti-HCV) have been found in the Middle- and Southern European countries in connection with chronic liver diseases. In a study of Finnish chronic liver disease patients no anti-HCV antibodies were found in 22 autoimmune chronic active hepatitis, in 5 chronic persistent hepatitis and in 38 alcoholic liver disease patients. 2/30 primary biliary cirrhosis patients were anti-HCV positive. As a comparison 3/9 patients with acute community acquired non-A non-B hepatitis and 28/48 i.v. drug addicts had anti-HCV antibodies. The results indicate that HCV infections in Finnish chronic hepatitis patients are rare.     

慢性乙型肝炎患者外周血NK细胞和T淋巴细胞计数变化及其临床意义

目的观察慢性乙型肝炎患者外周血NK细胞和T淋巴细胞数量的变化。方法在56例乙型肝炎肝衰竭、49例HBeAg阳性慢性乙型肝炎和41例乙型肝炎病毒携带者使用流式细胞仪检测外周血CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞和NK(CD3-CD16+CD56+)细胞占淋巴细胞的比率(%)。结果肝衰竭患者CD3+T细胞和CD3-CD16+CD56+NK细胞计数比慢性乙型肝炎患者和乙型肝炎病毒携带者显著性降低(P0.05);慢性乙型肝炎患者CD3-CD16+CD56+NK细胞计数比乙型肝炎病毒携带者显著性升高(P0.05)。结论乙型肝炎肝衰竭患者外周血T细胞和NK细胞数量减少,而HBeAg阳性慢性乙型肝炎患者外周血T细胞数量增多。