Genetic control of mouse cytomegalovirus-induced myocarditis.

Mouse cytomegalovirus (MCMV) infection of mice induced myocarditis, characterized by a mononuclear cell infiltrate with associated necrosis of myofibres. Myocarditis was observed in parallel with viral inclusion-bearing cells in the heart during the acute phase of the infection. Myocarditis also persisted after the acute phase when viral antigens were no longer detectable by immunoperoxidase histochemistry and infectious virus could not be cultivated from various organs. The influence of host genetic factors on the development of cytomegalovirus-induced myocarditis was investigated using H-2 congenic and recombinant inbred mouse strains. Analysis of congenic variants with C57BL/10 and BALB/c backgrounds and the A/J strain revealed that genes linked to the H-2 complex influenced susceptibility to peak levels of MCMV-induced myocarditis seen 7 and 10 days post-infection. In addition, non-H-2 genes of the BALB/c background were important in determining the severity of myocarditis. Analysis of the strain distribution pattern of the CXB recombinant inbred series did not disclose the identity of the BALB/c non-H-2-linked allele conferring susceptibility to MCMV-induced myocarditis. The level of myocarditis seen in the F1 hybrid between the high-responder BALB/c and low-responder C57BL/6 strains suggested dominant inheritance. The amount of viral replication in the major target organs did not correlate with the severity of myocarditis. In conclusion, at least two genes, one mapping to the H-2 complex and another non-H-2-linked gene, influenced the development of myocarditis in MCMV-infected mice.

Autoantibodies to isolated human hepatocyte plasma membranes in liver disease. I. Specificity for chronic active hepatitis.

Circulating autoantibodies reacting with human hepatocyte plasma membranes (HHPM) were quantitated in acute and chronic liver disease using an enzyme-linked immunosorbent assay (ELISA). Anti-HHPM were found most frequently in patients with chronic active hepatitis (CAH), a disease postulated to result from autoimmune processes directed at organ-specific antigens on the surface of hepatocytes. The high incidence of anti-HHPM in CAH (75%) contrasted significantly with all other groups assayed, including primary biliary cirrhosis (44%), alcoholic liver disease (21%), acute viral hepatitis (17%), and chronic persistent hepatitis (8%). The titers of anti-HHPM in CAH were significantly greater than in other liver disease and control groups. Anti-HHPM quantitated by ELISA correlated with hepatocellular membrane staining by indirect immunofluorescence. Autoantibodies to HHPM were found with an equivalent frequency in three etiological subgroups of CAH: autoimmune CAH, hepatitis B virus (HBV)-related CAH, and CAH associated with excess alcohol consumption. Anti-HHPM of the IgG and IgA isotypes were found in the highest frequency. There was a trend for patients with a histologically more severe disease to have higher titers of anti-HHPM. Immunoblots of SDS-PAGE-separated HHPM showed antibodies to react with a number of polypeptides, some of which appeared human specific. These data suggest that isolated HHPM are a source of relevant hepatocellular membrane antigens. Further studies of the different antigenic specificities of anti-HHPM are required to define which of these may be of pathogenetic importance in chronic active hepatitis.

Multiple autoantibodies following cytomegalovirus infection: virus distribution and specificity of autoantibodies.

Multiple autoantibodies were found in the sera of BALB/c, C57BL/10 and C3H mice following mouse cytomegalovirus (MCMV) infection. The complex pattern of intra-organ, intratissue and intracellular reactivity observed by immunoperoxidase histochemistry suggested that many autoantibodies of varying specificities were elicited. This evidence from immunoperoxidase histochemistry was confirmed by immunoblot, where autoantibodies binding to polypeptides of a variety of sizes and tissues of origin were observed. In addition to these central findings, the tissue and organ distribution of MCMV in three mouse strains of differing genetic resistance were described. No correlation was found between the distribution of virus and the tissue and organ specificity of the autoantibodies produced following infection. Inflammatory responses accompanied MCMV infection in a number of tissues. In BALB/c mice, myocarditis and salivary gland inflammation were evident at Day 56 post-infection in the absence of MCMV, but in the presence of autoantibodies to cardiac muscle and salivary duct epithelium. This model for virus-induced autoimmunity can be applied to studies of the relationship between virus infection, autoimmunity and disease.

The relationship between liver-specific lipoprotein and the hepatocyte plasma membrane.

Liver-specific lipoprotein (LSP) has been the subject of intense investigation as a candidate target antigen in chronic active hepatitis. Fundamental to the interest in LSP has been the belief that it is an antigen complex of hepatocyte plasma membrane origin. In this study the physical, biochemical and antigenic relationships between LSP and isolated hepatocyte plasma membrane (HPM) were investigated. Electron microscopic examination of LSP showed it to be devoid of plasmalemma sheets that were abundant in HPM. The plasma membrane marker enzyme 5'-nucleotidase was enriched 11-fold in HPM relative to liver homogenate, whereas the enzyme activity in LSP was 17% of that found in liver homogenate and only 1.5% of that found in HPM. The antigenic relationship between LSP and HPM was assessed using sera from rabbits immunized with either mouse LSP or mouse HPM. By filtration ELISA, antibody to LSP reacted poorly with entrapped HPM, relative to antibody to mouse HPM. Antisera to LSP and HPM were both effectively absorbed by the immunizing antigen, however antibody to LSP was not absorbed with HPM, and minimal cross-absorption of HPM antibody with LSP was found. By immunoblot of SDS-PAGE separated LSP and HPM, it was shown that antigenic cross-reactivity between LSP and HPM at the polypeptide level was rare. By immunofluorescence, antibody to LSP failed to react with the surface of viable mouse hepatocytes, whereas antibody to HPM showed linear fluorescence. The data show that the two preparations, LSP and HPM, are dissimilar antigen complexes. HPM may be a more appropriate preparation for the study of autoimmune liver disease than LSP.

An enzyme-linked immunosorbent assay for the detection of hepatocyte plasma membrane antibodies.

An enzyme-linked immunosorbent assay (ELISA) using plates coated with hepatocyte plasma membranes (HPM) was developed for the measurement of antibodies directed at hepatocyte surface antigens. Precoating ELISA plates with poly-L-lysine (PLL) provided firm attachment for the adsorption of HPM. The use of HPM, in preference to whole hepatocytes, excludes pathologically irrelevant cytoplasmic antigens. In addition, there is no necessity for glutaraldehyde fixation which is commonly used in cellular assays to maintain cellular integrity and which may result in loss or alteration in antigenic specificities. The assay was used to study loss of tolerance to mouse HPM in mice immunized with rat HPM. Three mouse strains were immunized, each strain developed antibodies to rat HPM and autoantibodies to mouse HPM with autoantibody levels reaching a peak 6-10 weeks after commencement of immunization. The correlation between ELISA and indirect immunofluorescence for the measurement of HPM autoantibodies was 0.79 (P less than 0.001) within the serum titration range of 1:25 to 1:200. Antibody to control kidney plasma membrane (KPM) was also measured by ELISA, after elimination of endogenous alkaline phosphatase activity using levamisole. Immunization with rat HPM elicited organ-non-specific autoantibodies to KPM, but these were at lower levels than autoantibodies to HPM.

Autoantibody to liver-specific lipoprotein in the mouse: regulation by naturally occurring autoantigen specific suppressor cells.

Indirect and direct evidence is presented that normal, non-immune mice have cells which suppress antibody production to murine liver specific lipoprotein (LSP) autoantigens with little or no effect on the response to a closely related foreign antigen complex, rabbit LSP. Suppressor control mechanisms were suggested in low responder BALB/c mice which produced LSP autoantibody only after exposure to low dose X-irradiation or treatment with cyclophosphamide. Adoptive transfer experiments in X-irradiated BALB/c mice and untreated C57BL/6 mice showed that cells from normal mouse spleen prevented LSP autoantibody production when put into the circulation of mice prior to immunization with foreign rabbit LSP. This suppressor activity was destroyed by treatment with antisera to T cells and by low dose X-irradiation. The normal spleen cells were ineffective as suppressors if given after primary immunization with rabbit LSP had commenced. It is suggested that the adoptive transfer of normal spleen cells prior to immunization with foreign LSP supplements homeostatic mechanisms in favour of tolerance to LSP autoantigen. It is argued that the LSP autoantibody response in the mouse is a unique model for the study of autoantigen specific naturally occurring suppression.

Thymus-dependence of autoantibody responses to liver specific lipoprotein in the mouse.

Proposed mechanisms for the induction of autoantibodies to liver specific lipoprotein (LSP) assume that the autoantibody response is T-dependent. This hypothesis was tested in the athymic nude mouse. Athymic homozygote (nu/nu) nude mice and appropriate control mouse strains were immunized with rabbit or human LSP and infected with murine cytomegalovirus (MCMV) in an attempt to induce autoantibodies to LSP. Antibodies to LSP were measured by passive haemagglutination and by an enzyme linked immunosorbent assay. Nude mice did not produce antibodies to either foreign LSP species or autoantibodies to mouse LSP when immunized with either rabbit or human LSP. Control heterozygote (nu/+) mice and C57BL/6J mice produced antibody to foreign LSP and autoantibody to mouse LSP when immunized with xenogeneic LSP. Athymic nu/nu mice also failed to produce autoantibody to mouse LSP following infection with MCMV, in contrast to control nu/+ and C57BL/6J mice which produced LSP autoantibody after infection with MCMV. It is concluded that the autoantibody response to LSP is T-dependent.

Autoantibodies to liver-specific lipoprotein following hepatitis induced by mouse cytomegalovirus.

Autoantibodies to liver specific lipoprotein (LSP) are produced following acute non-fatal hepatitis in murine cytomegalovirus (MCMV) infected mice. Both C57B1 and BALB/c mice produced a transient LSP autoantibody response demonstrated by passive haemagglutination and enzyme linked immunosorbent assay. C57Bl mice produced both IgM and IgG LSP autoantibody and BALB/c mice produced only IgM autoantibody. The autoantibody was species non-specific, reacting with both mouse and rabbit LSP. Plasma containing LSP autoantibody reacted with the surface of normal mouse hepatocytes by immunofluorescence. This model provides an opportunity for study of LSP autoantibody production during viral hepatitis.

Autoantibody responses to liver-specific lipoprotein in mice.

Autoantibody to the hepatocyte membrane antigen, liver-specific lipoprotein (LSP) was induced in mice by immunization with LSP-containing protein preparations from human, rat, rabbit and mouse liver and also with purified allogeneic LSP. Each of the strains of mice used (C57B1, BALB/c, C3H) showed the capacity to produce high titre autoantibody to LSP. Autoantibody to LSP demonstrated by passive haemagglutination was absorbed by normal mouse hepatocytes but not by kidney or spleen cells and reacted with the cell membrane of normal mouse hepatocytes by immunofluorescence. The liver was examined histologically in all mice and where inflammation was found it was attributable to the Freund's complete adjuvant used in immunization rather than liver protein immunogen. The demonstration of high titre autoantibody to LSP in mice without associated hepatitis contrasts with chronic hepatitis in man and experimental chronic hepatitis in rabbits where autoantibodies to LSP have been implicated in the pathogenesis of the disease.

Biologic and immunologic studies on a murine model of regional lymph node metastasis.

Some biologic, hematologic, and immunologic aspects of the growth and metastasis of the MC-2 fibrosarcoma indicated its suitability as a model for the study of lymphogenous metastasis. The tumor was maintained in syngeneic female BALB/c mice by the serial sc passage of 10(5) viable tumor cells. It metastasized macroscopically in all mice to regional lymph nodes (RLN) and to the lungs. Both forward and retrograde node-to-node metastases were found. Tumor growth and metastasis were associated with splenomegaly, thymus atrophy, cachexia, neutrophilia, lymphopenia, and anemia. Tumor excision at various times after inoculation showed that all mice whose tumors were excised when there was histologic evidence of metastasis in all RLN (day 13; mean of tumor wt, 122 mg) died subsequently from metastases, whereas no animals died whose tumors were excised on or before day 8 (mean of tumor wt, 15 mg). The onset of metastasis was seen in some RLN on day 8. All survivors were immune to challenge with 10(5) viable tumor cells, which demonstrated the immunogenicity of the tumor. Concomitant tumor immunity could be demonstrated prior to the onset of metastasis (days 6 and 7) but not early (days 0--2) or late (days 15, 19, and 20) in primary-site tumor growth. The early immune response to the tumor demonstrable as concomitant tumor immunity appeared to be abrogated by the progressive growth and metastasis of the neoplasm. Tumor cells passaged in adult thymectomized, X-irradiated, syngeneic recipients produced larger RLN metastases and smaller primary tumors than those passaged in control mice.

Epidemiology of chronic hepatitis in Perth, Western Australia.

A survey was made on 50 patients with active chronic hepatitis (ACH) seen in Perth, Western Australia. The aetiology varied: three cases followed metabolic disease, 8 drugs or alcohol, 12 were due to hepatitis B and no cause in 27. There was a male preponderance in the first three groups and a female preponderance in the idiopathic group. The drug dependent group had a greater mean age than the other groups. Autoantibodies were present in 40 of the cases--the most frequent were antismooth muscle antibody in 24 cases and antinuclear factor in 13 cases. Six patients (12%) improved and are well following discontinuation of therapy. Seven (14%) have died. The rest (74%) remain on treatment.

Dissociation of correlates of cellular immunity in man: functional heterogeneity within the antigen-reactive cell population?

Cellular immune responsiveness (CMI) to ubiquitous bacterial antigens was assessed in serial peripheral blood samples collected from normal subjects, employing three in vitro correlates of CMI in parallel. Delayed cutaneous hypersensitivity (DCH) was also assessed on a number of occasions in these subjects. These experiments showed that the reactivity of peripheral blood leukocytes from normal individuals as measured in each of the in vitro tests fluctuated from day to day, and that the reactivity of cells functioning in each of the tests fluctuated independently of one another. In contrast, DCH reactivity in these individuals was not subject to such fluctuations. The results are discussed in terms of the periodic appearance of functionally distinct subpopulations of T lymphocytes in the peripheral circulation.