Electron microscopic observations on hepatitis B antigen-associated particles in the sera of patients with various liver diseases

To analyze the appearance of three forms of hepatitis B antigen-associated particles (HB Ag particles) and antigen-antibody (Ag-Ab) complexes in the sera of patients with various liver diseases, electron microscopic observations with the combinations of a variety of immunological assays were made at first on the HB Ag and Ab mixed in vitro in various ratios, and then on the samples from the sera of each patient. The number of patients observed were 64 in total, which consisted of various types of hepatitis, Hodgkin's disease, Down's syndrome and an asymptomatic carrier. For the detection of HB Ag-Ab complexes a modified method of ALMEIDA was used, and for the isolation of large HB Ag particles (Dane particles) DANE'S method was employed. Electron microscopy proved to be a useful method for detecting HB Ag and the Ag.Ab complexes when the ratio of HB Ag to Ab was in the equivalence. Large aggregates of Ag-Ab complexes were frequently observed in the attacks of acute hepatitis and the recrudescences-of chronic aggressive hepatitis. The aggregates were also observed in fulminant hepatitis but the ratio of HB Ag to Ab was different from each other among 3 cases examined. The large HB Ag particles were not observed in more than half of the cases in the attacks of acute hepatitis, but appeared in the major. ity of cases in chronic aggressive hepatitis, even massively during the period with transiently elevated levels of serum glutamic pyruvic transaminase. A few large particles were also found in sera of an asymptomatic carrier, Hodgkin's disease, and Down's syndrome.</p

Immunohistochemical characterization of the lymphocyte and the immunoglobulin-containing cell in the epithelium and the lamina propria of normal human intestines.

In order to clarify difference of the mucosal immunity in various sites of normal large and small intestines, we studied the population of lymphocyte subsets and immunoglobulin (Ig)-containing cells in situ in biopsy specimens taken from various sites (ascending colon, sigmoid colon and rectum) of the large intestine and from the duodenum using an immunohistochemical method. Monoclonal antibodies against pan-T (Leu 1), cytotoxic/suppressor T (Leu2a), helper/inducer T (Leu3a), suppressor T (Leu15) and natural killer/K (Leu7) cells, and polyclonal antibodies to human IgG, IgA and IgM were used. In the duodenum, intraepithelial lymphocytes (IELs) were more prominent than in the large intestine. Immunoelectron microscopic observation revealed that some Leu2a+ IELs possessed pseudopods extending into intestinal epithelial cells, indicating that some IELs belong to the cytotoxic T cell subset. Leu7+ IELs were scarcely observed and Leu7+/Leu1+ ratio was higher in the large intestine than in the duodenum. Furthermore, the number of Leu7+ cells were more in the distal than the proximal colon. In the lamina propria Ig-containing cells tended to be fewer in the rectum than in the duodenum and the proximal colon. Our findings may suggest the variation of local immune responses and the difference of assigned immunological functions among the various sites of the intestines.</p

An Analysis of Risk Factors for Developing Hepatocellular Carcinoma in a Group of Hepatitis C Patients with Stage 3 Fibrosis following Interferon Therapy

The risk of Hepatocellular carcinoma (HCC) is high in HCV-infected patients who have biochemically and histologically active chronic hepatitis. To observe the long prognosis of Chronic Hepatitis C (CHC) patients with stage 3 fibrosis (F3), 55 CHC patients after initial Interferon (IFN) therapy were followed up for up to 12 years (average 9.8 ± 2.3 years). According to the annual average alanine aminotransferase (ALT) levels, patients were grouped into, low (ALT ≦ 30 IU/l); moderate (ALT >30 <80 IU/l) and high (ALT ≧ 80 IU/l) ALT groups. Eleven patients were re-treated with IFN. During the follow-up period of 12 years, HCC developed in 26 patients with an average annual incidence of 3.9%. Biochemical responders to initial IFN therapy (n = 8) and those re-treated with IFN (n = 10), except 1, did not develop HCC. Cox regression analysis to evaluate risk factors for HCC occurrence, found development of Liver Cirrhosis within 3 years of initial IFN therapy(P = 0.05) and the 3 year annual average ALT post initial IFN therapy (P = 0.033) to be significant. The 12 year annual average ALT was also found to be significantly related to HCC occurrence (P = 0.016), on univariate analysis. Patients belonging to the continuously low ALT group (ALT ≦ 30 IU/l for ≧3 years), did not develop HCC or receive IFN re-treatment. In CHC patients with F3, after initial IFN therapy, keeping ALT continuously low, below 30 IU/l for 3 years or more seems important. Continuing treatment with anti-inflammatory drugs along with subsequent IFN re-treatment may prevent or delay HCC even in elderly patients

Tubular structures in the hepatic nuclei of a patient with delta agent.

Liver biopsy specimens obtained from a 31-year-old female with delta-positive hepatitis were studied by routine electron microscopy. In several nuclei of hepatocytes, there were filamentous or microtubular structures 15 to 20 nm in diameter, in the vicinity of which, round particles, probably cross sections of tubular ones, were seen. In these nuclei, irregular granules approximately 20 to 30 nm in diameter were also found in clusters. However, cores of Dane particles were not found in such hepatocytes. These intranuclear microtubular structures may be associated with delta agent.</p

Localization of hepatitis C virus RNA in human liver biopsies by in situ hybridization using thymine-thymine dimerized oligo DNA probes: improved method.

To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.</p

Immunoelectron Microscopic Localization of MHC Class 1 and 2 Antigens on Bile Duct Epithelial Cells in Patients with Primary Biliary Cirrhosis

We studied the distribution of class 1 and class 2 major histocompatibility complex (MHC) antigens on bile duct epithelial cells in liver from patients with primary biliary cirrhosis (PBC) by an immunohistochemical method using monoclonal antibodies to HLA-ABC products and HLA-D subregion products (HLA-DR, -DP, -DQ). By light microscopy, the expression of MHC class 1 antigens (HLA-ABC antigens) was enhanced in PBC compared with controls. While negligible staining of MHC class 2 antigens was detected on the bile duct in controls, de novo expression of MHC class 2 antigens, as well as the coexpression of HLA-DR, HLA-DQ, and HLA-DP antigens on the bile duct epithelial cells, was observed in PBC. By electron microscopy, HLA-ABC and HLA-DR antigens were present preferentially along the basolateral domain of the cell surface of the bile duct epithelial cells and on the membrane of the endoplasmic reticulum in the cytoplasm, suggesting that these MHC antigens are synthesized by the bile duct epithelial cells in PBC. The distribution of these MHC antigens on the basolateral surface of the bile duct epithelial cells, where they are easily accessible to immunocytes, supports the idea that MHC-restricted cytotoxic T lymphocytes are involved in the bile duct injury in PBC. </p

Non-B hepatocellular carcinoma: influence of age, sex, alcohol, family clustering, blood transfusion and chronic liver disease.

In 144 cases of hepatocellular carcinoma (HCC), 166 cases of cirrhosis without HCC and 142 cases of chronic hepatitis, we examined HBsAg, anti-HBs and anti-HBc in sera and compared the following factors between hepatitis B virus marker-negative and -positive patients: age, sex, alcohol consumption, family clustering of liver diseases, and histories of blood transfusion and post-transfusion hepatitis. Results of this study demonstrated several distinct differences in clinical backgrounds between non-B (negative for HBsAg, anti-HBs and anti-HBc) and B (positive for HBsAg) patients with HCC. Non-B patients were significantly older, had a lower frequency of familial tendencies for liver diseases, and more frequently had cancers other than HCC in their families. Some of these differences were also observed between non-B and B patients with cirrhosis and chronic hepatitis. Among patients with chronic hepatitis, the non-B patients had received blood transfusion or had post-transfusion hepatitis more frequently than the B patients. However, this difference was not apparent in patients with liver cirrhosis or HCC, suggesting that progression of non-A, non-B post-transfusion hepatitis to cirrhosis and HCC may not be as frequent as progression to chronic hepatitis.</p

Determination of antibody to hepatitis B core antigen by radioimmunoassay in chronic liver disease.

Antibody to hepatitis B core antigen (anti-HBc) was measured by radioimmunoassay using CORAB (Abbott Laboratories) in 10 cases of chronic persistent hepatitis (CPH), 46 cases of chronic aggressive hepatitis (CAH), 33 cases of liver cirrhosis (LC) and 53 cases of hepatocellular carcinoma (HCC) in relation to hepatitis B surface antigen (HBsAg) and its antibody (anti-HBs). Ninety-eight point four percent of patients with HBsAg and 93.8% of patients with anti-HBs were positive for anti-HBc and the titers of anti-HBc in patients with HBsAg were significantly higher than those with anti-HBs. Thirty-five point five percent of patients negative for either HBsAg or anti-HBs were positive for anti-HBc. The titers of anti-HBc in patients with CPH, CAH and LC were relatively low, whereas 7 (46.8%) of the HCC patients negative for either HBsAg or anti-HBc had high titers of anti-HBc. The significance of the presence of anti-HBc alone is discussed.</p

Hepatitis B virus associated particles in the bile canaliculus.

The liver biopsy specimen from a patient with hepatitis B surface antigen was pbserved by electron microscopy. Dane particles, uncoated core particles and tubular forms were demonstrated in hepatocytes. Dane particles and tubular forms, approximately 25nm in diameter, were also found in the bile canaliculi. These findings suggest that hepatitis B virus and associated particles are released from hepatocytes into the bile duct

Intrafamilial clustering of genotypes of hepatitis C virus RNA.

Hepatitis C virus (HCV)-RNA in the blood was measured by polymerase chain reaction (PCR) in 37 subjects from eight families in which 2 or more persons tested seropositive for antibodies against C100-3 or CP9. HCV-RNA was positive in 17 of 37 subjects. Two or more HCV-RNA-positive subjects were observed in six of the families. Intrafamilial HCV infection was studied by determining the HCV-RNA type (I, II, III or IV) by PCR using type-specific primers. In two families, all of the subjects showed type III infection, and in three other families, all of the subjects showed type II infection, with different types of HCV infections being observed in only one family. The HCV type was uniform in all but one. These findings suggest a possibility of intrafamilial infection between husbands and wives and between members of the same household.</p