Detection of HBs antigen in routine paraffin embedded liver tissue by enzyme-labelled antibody technique

HB surface antigen (HBs Ag) was detected using the enzyme-labelled antibody technique on routinely processed liver biopsy material fixed in Bouin's fixative and embedded in paraffin. Of 85 examined specimens, 45 cases were HBs Ag positive by both the immunofluorescent test and the enzyme labelled antibody technique. The remaining 40 cases were negative by both techniques. The specificity of HBs Ag detected by the enzyme-labelled antibody technique was confirmed by the blocking test using guinea pig specific HBs antibody. The results indicate that the enzyme-labelled antibody technique may be useful for detecting HBs Ag on routine paraffin sections.</p

Tissue localization of C1q in HBs antigen positive liver disease patients by direct immunofluorescent technique

Tissue localization of a subcomponent of the first component of complement (CLq) was examined in one postmortem case of HBs antigen (HBs Ag) positive hepatocellular carcinoma and in six cases of chronic hepatitis from liver biopsy specimens. The direct immunofluorescent method was used after fixation with 2% para-formaldehyde in concentrated ammonium sulfate. CLq localization was found in collagen fibers and the cytoplasm of fibroblasts in the connective tissues of specimens examined. The localization was particularly marked in the region of the fundal glands of the gastric wall. Apart from collagen fibers, other sites of localization included the surface membrane of lymphocytes, especially those cells of the mesenteric lymph nodes. In HBs Ag positive specimens, immune deposit-like substances appeared localized intra-hepatically and in the renal glomeruli. Since C3 and C4 were identified concomitantly, it indicates that these substances were indeed immune diposits. Despite the finding that C3 and C4 were identified together in the hepatic cell cytoplasm, C1q itself was not demonstrated in all hepatic cell cytoplasms.</p

Cyclin A2-CDK2 regulates embryonic gene activation in 1-cell mouse embryos

AbstractRecruitment of maternal mRNA in mice appears essential for embryonic gene activation (EGA) that is initiated in the 1-cell stage. The identity of which recruited mRNAs is responsible, however, is not known. We report here that recruitment of cyclin A2 mRNA may be critical for EGA. Cyclin A2 protein accumulates in pronuclei between 6 and 12 h after fertilization, the time when EGA is initiated. This cyclin A2 may be generated from maternally recruited cyclin A2 mRNA because its accumulation was inhibited by 3′-deoxyadenosine, which inhibits mRNA polyadenylation. When CDK2 activity or pronuclear accumulation of cyclin A2 was inhibited with CDK2 inhibitors or by microinjected siRNAs, respectively, DNA replication was not inhibited but the increase of transcriptional activity was prevented. In addition, microinjection of recombinant cyclin A2-CDK2 protein increased transcriptional activity. Cyclin A2-CDK2 is activated following egg activation, because an increase in phosphorylation of retinoblastoma protein was observed using antibodies that recognize site-specific phosphorylation catalyzed by this kinase and treatment with a CDK2 inhibitor or microinjection with cyclin A2 siRNAs prevented the increase in retinoblastoma protein phosphorylation. These results suggest that recruitment of maternal cyclin A2 mRNA following egg activation is linked to EGA

Detection of serum blocking factors and antibodies to the albumin receptor on HBsAG particles in healthy persons and patients with liver diseases.

An enzyme-linked immunosorbent assay (ELISA) for the detection of serum blocking factors (BF), or antibodies to the albumin receptor on HBsAg particles, was developed, and its clinical usefulness was examined in healthy persons and patients with liver diseases. Thirteen of 80 anti-HBs-positive female (16.3%) had BF, but all 25 male anti-HBs-positive, 41 female and 32 male anti-HBs-negative subjects were negative for BF. The activity of BF in BF-positive cases was not associated with the positive reciprocal hemagglutination titer of anti-HBs. For a neutralization test of BF, the BFs from 5 cases were absorbed with IgG-immunobeads. It was determined that these IgG-BFs were antibodies to the albumin receptors on HBsAg particles. No significance between positive-BF and abnormal S-GPT levels was recognized. These results suggest that the present test for the detection of BF, or anti-albumin receptor antibody, different from anti-HBs, might be useful for diagnosis of hepatitis B and as a marker for HB virus.</p

Detection of liver HBc antigen and its antibody in sera from viral hepatitis by the immunofluorescent complement technique

Hepatitis B core antigen (HBc Ag) and hepatitis B surface antigen (HBs Ag) were detected in the liver tissue of a patient with chronic aggressive hepatitis by the immunofluorescent complement technique. The presence of anti-HBc was examined by the same method in 67 human sera previously tested for HBs Ag, anti-HBs and s-GPT levels. HBc Ag was localized mainly in the nucleus and sometimes in the cytoplasm of the hepatic cells. HBs Ag was found only in the cytoplasm. The focal area of HBc Ag positive hepatic cells seemed to correspond to the HBs Ag positive cells. Double staining demonstrated the simultaneous presence of HBs Ag and HBc Ag in individual cells. Anti-HBc positive serum was found in 46 (68.7%) cases. Forty-eight (71.6%) indicated a combination of HBs Ag and anti-HBc.</p

Detection and characterization of antibody to liver cell membrane in sera from patients with chronic active liver diseases.

Sera from 84 patients with chronic liver disease [CLD] (74 chronic active) and from 53 blood donors were tested immunochemically for anti-liver cell membrane antibody (LMAb). LMAb to rat liver tested by an indirect immunofluorescent technique was positive in 53.3% of CLD patients with positive HB surface antibody (HBsAb) and 40.0% of HBsAb positive blood donors. Pepsin digestion of the sera indicated that the binding between liver cell membrane and IgG was at the Fc site on the immunoglobulin. The sera with positive LMAb from HBsAb positive blood donors had elevated Clq-binding activity (Clq-BA). The LMAb to rat liver was a macro-molecular IgG (19-22S IgG) when assayed by Sephadex G-200 column chromatography and 5-40% sucrose density gradient ultracentrifugation. The results suggest that the LMAb in serum from a patient with chronic active liver disease may be an immune complex which consists of various antigens such as HB virus and its antibodies in serum.</p

Detection of tissue T-cell in patients with chronic active hepatitis using fragmented sheep red blood cell (SRBC) membrane.

Fragmented sheep red blood cell (SRBC) membrane was used for detection of T-cells in liver biopsy specimens from patients with chronic active hepatitis. SRBC was separated with Lymphoprep, sonicated, then filtered through a 3 mu Millipore-membrane as a fragmented SRBC reagent. Tissue T-cells were stained by an indirect immunofluorescent technique using SRBC reagent and fluorescein isothiocyanate (FITC)-labelled rabbit anti-SRBC. Positively staining lymphocytes were present in portal tracts and in areas of piecemeal necrosis. There also seemed to be a positive correlation between the number of positively staining lymphocytes and the activity of chronic hepatitis; numerous lymphocytes being stained in areas of severe piecemeal necrosis. Our findings suggest that the fragmented SRBC technique for detection of T-cells is reliable and reproducible, that it could be used as a clinical routine method, and that it is useful for further elucidating the nature of host immune reactions on tissue levels.

Tissue immune complexes demonstrated in the liver of patients with chronic aggressive hepatitis using FITC-labelled human Clq.

Immune complexes in liver specimens from 10 patients with chronic liver diseases [2 with chronic persistent hepatitis (CPH), 3 with chronic aggressive hepatitis (CAH) of moderate activity, 3 with CAH of severe activity, and 2 with liver cirrhosis] were examined by a technique of direct immunofluorescence using FITC-labelled human purified Clq (FITC-Clq). FITC-Clq bound to the nuclei of all cells in liver tissue. After DNase treatment, positive nuclei were absent, but positive staining with FITC-Clq remained in amorphous deposits and hepatic cell membranes in the areas of piecemeal necrosis of four CAH patients. Since FITC-Clq could not be demonstrated in the liver tissue of CPH and liver cirrhosis which contained no piecemeal necrosis, positive fluorescence in the liver of CAH patients was thought to indicate immune complexes bound to FITC-Clq. The fact that these positive substances, however, were few in number, may be the result of physiological mechanisms of immune clearance which rapidly eliminate immune complexes from the body.</p

Existence of serum HBe antigen and expression of liver HB surface and core antigens in hepatitis type B patients.

A study of 52 liver biopsies (47 hepatitis type B and 5 asymptomatic carriers) was performed to clarify the roles of HBe antigen (HBeAg), HB surface antigen (HBsAg) and HB core antigen (HBcAg). In this study, the Gudat classification was modified so as to classify the patterns of HB antigens into six reaction types including: type O (negative for both liver HBsAg and liver HBcAg), type III-A (characterized by a spotty HBsAg pattern) and type III-B (characterized from a sub-lobular to lobular HBsAg localization pattern). This classification enabled accurate prediction of the prognosis of hepatitis. Patients with positive serum HBeAg had either minimal hepatitis with mild clinical features or chronic aggressive hepatitis with severe clinical features. Ten patients negative for both HBeAg and HBeAb were all positive for liver HBcAg. In all 3 patients on corticosteroid administrations liver tissue was markedly positive for HBcAg and serum was usually positive for HBeAb.</p