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Biomedical subjects

S M Feinstone

Publications and source records attributed to S M Feinstone.

18 recordsLinked to original sources

Non-A, non-B hepatitis: ultrastructural evidence for two agents in experimentally infected chimpanzees.

Two different ultrastructural alterations were observed in liver cells of chimpanzees inoculated with plasma derived from two different patients with non-A, non-B hepatitis. During the acute phase of illness in one group of four chimpanzees, peculiar tubular structures, composed of two unit membranes with electron-opaque material in between, were observed in the cytoplasm of hepatocytes. In contrast, these structures were never detected in the liver cells of the second group of five chimpanzees that received the second inoculum, However, nuclear changes, usually associated with aggregates of 20- to 27-nanometer particles, were found in hepatocytes of the latter animals. Although these particles resembled viruses, they were not as uniform as small virus particles often appear. In five other chimpanzees inoculated with non-A, non-B hepatitis material not known to be related to the first two inocula, cytoplasmic structures were found in four, and nuclear structures were found in the remaining one. Thus, all 14 chimpanzees inoculated with transmissible non-A, non-B hepatitis agents could be classified as having either nuclear or cytoplasmic changes. These observations add support to epidemiologic data suggesting that there may be more than one agent of non-A, non-B hepatitis.

Animals

Circulating immune complexes in non-A, non-B hepatitis. Possible masking of viral antigen.

Serial serum samples from 22 patients with transfusion-associated non-A, non-B hepatitis and 2 chimpanzees with the experimentally induced disease were tested for circulating immune complexes by Raji-cell radioimmunoassay. 13 patients (59%) and 1 chimpanzee had circulating immune complexes immediately before, coincident with, or during the return to normal of raised aminotransferase activity. 7 of the 10 patients with chronic non-A, non-B hepatitis had detectable complexes at levels which waxed and waned in parallel with changes in serum aminotransferase activity. Immune complexes may contain and mask viral antigens, and their presence may explain the failure of conventional immunological techniques to detect virus antigens.

Acute Disease

Enzyme-linked immunosorbent assay for detection of hepatitis A antigen in stool and antibody to hepatitis A antigen in sera: comparison with solid-phase radioimmunoassay, immune electron microscopy, and immune adherence hemagglutination assay.

Previously described techniques for detection of hepatitis A antigen (HA Ag) and antibody (anti-HA) have required purified HA Ag and expensive equipment. Herein is described an enzyme-linked immunosorbent assay (ELISA) for specific detection of HA Ag in human stool filtrates and of anti-HA in sera by using selected HA Ag-containing human stool filtrates as the antigen source. Because human stools often react nonspecifically in serological tests for HA Ag, blocking with preexposure and hyperimmune anti-HA sera from a chimpanzee inoculated with hepatitis A virus was used to confirm specific detection of HA Ag. The sensitivity of ELISA was found to be comparable to that of solid-phase radioimmunoassay (SPRIA) and immune electron microscopy (IEM). Of 37 acute-phase stools collected from nine patients, 16 were positive for HA Ag by ELISA. In 13 of these, HA Ag particles were found by IEM, and an additional 3 stools negative by ELISA contained HA Ag particles by IEM. Eight control stools were negative by both ELISA and IEM. Anti-HA was measured in sera by demonstrating its ability to block binding of the enzyme conjugate to HA Ag in a stool without detectable nonspecificity. This test (blocking ELISA) was as sensitive and specific as blocking SPIRA, IEM, and immune adherence hemagglutination and, like SPRIA and IEM, detected early-developing antibody. The ELISA is simple to perform and requires only a minimum of equipment. It is useful for screening stools for HA Ag and for monitoring HA Ag during purification, as well as for detecting early and late anti-HA in sera.

Acute Disease

Localization of hepatitis A antigen in liver tissue by peroxidase-conjugated antibody method: light and electron microscopic studies.

Hepatitis A antigen (HAAG) was localized in liver tissue from marmosets inoculated with human hepatitis A virus (HAV) by light and electron microscopy by using a peroxidase-conjugated antibody method. The fine granular peroxidase staining was scattered throughout the cytoplasm of liver cells when viewed with the light microscope. The distribution of HAAg-positive cells was focal. Virus-like particles, 24 to 27 nm in diameter, were observed in the cytoplasm of hepatocytes and smaller cells, resembling Kupffer cells, by standard thin-section electron microscopy (thin section EM). By immunoperoxidase electron microscopy (immunoperoxidase EM), HAAg was detected on the particles, which were aggregated within cytoplasmic vesicles of the hepatocyte. The surrounding membrane of the vesicles was also HAAg- positive. Similar HAAg particles were observed in the cytoplasm of smaller cells adjacent to hepatocytes as well. Thus, immunoperoxidase EM revealed that the 24- to 27-nm virus-like particles in the cytoplasm of liver cells obtained from marmosets were infected with HAV contained HAAg.

Animals

Non-A, non-B post-transfusion hepatitis.

To clarify the role of hepatitis-A virus (H.A.V.) in the aetiology of post-transfusion hepatitis unrelated to hepatitis-B virus, we have tested and titred pre-transfusion and convalescent serum samples from 32 patients for antibody to hepatitis-A antigen (anti-HA) by quantitative immune adherence haemagglutination. 12 patients had no detectable anti-HA in either pre-transfusion or late convalescent serum; the other 20 had anti-HA in pretransfusion serum and no significant chance in titre during convalescence. This study excludes H.A.V. as the agent responsible for these cases of post-transfusion hepatitis and supports the existence of "non-A, non-B" hepatitis virus(es).

Antibodies, Viral

Multiple hepatitis viruses in multiple attacks of acute viral hepatitis.

We evaluated the causes of 30 episodes of acute viral hepatitis in 13 patients who had multiple attacks. Two (seven per cent) of 30 bouts were caused by hepatitis A virus, and 12 (40%) by hepatitis B virus. No patient, however, had more than one attack with the serologic characteristics of Type A or Type B disease. Thus, there were 16 bouts (53 per cent) not attributable to either of the two recognized hepatitis viruses. None of these "non-A, non-B" episodes, evaluated for infectious mononucleosis and cytomegalovirus infection, could be ascribed to either. From this evidence, therefore, it appears that the clinical syndrome of viral hepatitis is produced not only by the two viruses (hepatitis A virus and hepatitis B virus) recognized since the 1940's but also, in all probability, by two non-A, non-B agents.

Acute Disease

Detection of hepatitis A antigen by immunofluorescence.

Hepatitis A antigen (HA Ag) was demonstrated by immunofluorescence (IF) in liver biopsies from chimpanzees with experimental hepatitis A virus infection. Blocking experiments with paired sera from patients with hepatitis types A, B, or non-A, non-B, as well as with purified HA Ag, showed that the fluorescence was specific for HA Ag. HA Ag could be demonstrated only in biopsies from chimpanzees inoculated with hepatitis A virus. In two of four chimpanzees biopsied weekly, HA Ag could be detected by IF before stool shedding of HA Ag, elevation in serum alanine aminotransferase (SGPT), or histopathological evidence of liver disease was seen. The HA Ag was detected for 4 to 5 weeks; the last IF-positive biopsy was obtained after SGPT activity had returned to normal. In the two other chimpanzees, HA Ag could be detected only in the biopsy taken at the time of SGPT elevation. In the early IF-positive biopsies, HA Ag was diffusely distributed in the cytoplasm of many cells, but it later accumulated in a focal distribution in the cytoplasm of a few of the hepatocytes and Kupffer cells. This cytoplasmic distribution agrees with previous electron microscopic data.

Alanine Transaminase

Transmission of non-A, non-B hepatitis.

In studies conducted in the early 1950s, sera from six asymptomatic blood donors, implicated in the transmission of viral hepatitis, were inoculated into 10 to 20 volunteers each. Five of these "implicated" donor sera transmitted clinically apparent hepatitis to the recipients. The stored serum samples from these studies have been reanalyzed using serologic markers for hepatitis B virus and hepatitis A virus infection. Two of the donor sera were hepatitis B surface antigen (HBsAg)-positive, and both transmitted hepatitis B virus infection to all susceptible recipients, half of whom showing clinical symptoms. The remaining three infectious donors were HBs-Ag-negative, yet were icterogenic to 10% to 47% of recipients. Testing of serum samples from these recipients with hepatitis showed no evidence of hepatitis B virus or hepatitis A virus infection. This study and other recent evidence suggest that there is a third type of human viral hepatitis--non-A, non-B hepatitis--which is due to a transmissible agent and may well be associated with a chronic carrier state.

Adult

Clinical and serological analysis of transfusion-associated hepatitis.

Of 108 prospectively followed, multiply transfused, open-heart-surgery patients, 12 (11%) developed hepatitis. Patients received only volunteer donor blood tested for hepatitis-B surface antigen (HBsAg) prior to transfusion by counterelectrophoresis (C.E.P.). 4 of the 12 patients developed hepatitis-B-virus infection. Subsequent testing of donor serums by solid-phase radioimmunoassay (R.I.A.) revealed that an R.I.A.-positive, C.E.P.-negative blood unit was transfused to 3 of the 4 type-B hepatitis cases, but to none of the remaining 104 patients; 3 hepatitis-B cases could probably have been prevented by prescreening of donors by solid-phase R.I.A. 8 hepatitis cases were serologically unrelated to the hepatitis-B virus, the hepatitis-A virus, the cytomegalovirus, or the Epstein-Barr virus. Had R.I.A.-positive donors been excluded, 8 of the 9 residual hepatitis cases (89%) would have represented "non-A, non-B" hepatitis. The existence of previously unrecognised human hepatitis virus(es) is probable.

Adult

Transfusion-associated hepatitis not due to viral hepatitis type A or B.

Twenty-two patients who had an episode of transfusion-associated hepatitis not positive for hepatitis B antigen were examined for development of antibody to heaptitis A and B antigens, cytomegalovirus and Epstein-Barr virus. Antibody response to the 27-nm virus-like hepatitis A antigen was measured by immune electron microscopy. In none of the 22 patients studied did serologic evidence of infection with hepatitis A virus develop during the study period. Nine of the 22 patients had antibody responses to cytomegalovirus, but it was difficult to relate these seroconversions to their hepatitis. In addition, all 22 patients had pre-existing antibody to the Epstein-Barr virus. It seems likely that at least a proportion of such antigen-negative transfusion-associated hepatitis is caused by other infectious agents, not yet identified.

Alanine Transaminase

Faecal shedding of hepatitis-A antigen.

Serial stool specimens from two individuals with experimental hepatitis-A infection were examined and the shedding pattern of hepatitis-A antigen (HAAg) was determined by immune electron microscopy. HAAg particles were detected at least 5 days before the development of abnormal transaminase levels and jaundice, but not later than the day of peak transaminase levels. The pattern of early faecal shedding of HAAg particles correlated well with the early infectivity of faeces and accorded with the suggestion that HAAg is involved in the aetiology of hepatitis-A infection.

Alanine Transaminase

Experimental infection of chimpanzees with hepatitis A virus.

The susceptibility of chimpanzees to viral hepatitis type A was examined with immine electron microscopy. Of four seronegative infant chimpanzees, two were inoculated with a hepatitis A acute-phase stool filtrate rich in 27 nm virus-like hepatitis A antigen (HA Ag) particles, and two were inoculated with an HA Ag-negative preinfection stool filtrate. One of each pair of chimpanzees was inoculated intravenously, the other orally. One month later both chimpanzees that had received the HA Ag-positive filtrate developed biochemical, histologic, and clinical evidence of acute viral hepatitis. HA Ag particle (27 nm) were detected in their stools by immune electron microscopy; particle shedding followed a pattern similar to that in human volunteers. Immune electron microscopy also showed that antibody HA Ag had developed in the convalescent-phase sera of the infected chimpanzees. Control animals remained free of illness at this time but did develop hepatitis three to five weeks after exposure to the two infected chimpanzee-. The infectious inoculum was titrated in two additional seronegative chimpanzees. It was concluded that hepatitis a can be successfully transmitted to seronegative chimpanzees. Moreover, these studies provide further evidence that the 27-nm virus-like HA Ag particle is the etiologic agent of viral hepatitis type A.

Animals

Relationship of hepatitis A antigen to viral hepatitis.

Progress in research on hepatitis type A has begun to accelerate because of the recent discovery of an antigen associated specifically with hepatitis type A infection and the development of tests for antibody to the antigen. Hepatitis A antigen is associated with 27 nm virus-like particles found in the liver and stool of animals experimentally infected with hepatitis type A and in the stool of humans experimentally or naturally infected with the virus. The density of the particulate antigen when isolated from the liver is 1.34, but antigen particles with densities ranging from 1.32 to 1.40 have been detected in stool. However, antigens from the liver and from the stool appear to be antigenically related. Using immune electron microscopy as a serologic tool for detecting antibody to hepatitis A antigen, we detected antibody in convalescent sera from 100 per cent of patients experimentally or naturally infected with hepatitis type A. In contrast, patients with hepatitis type B or non-B hepatitis not epidemiologically compatible with a diagnosis of hepatitis type A did not have a serologic response to hepatitis A antigen. Antibody was found in approximately 50 per cent of normal individuals tested; the frequency was directly related to age. By the use of immune electron microscopy for the detection of hepatitis A antigen and antibody, the temporal relationship of antigen, antibody and liver damage was determined in experimentally infected humans and chimpanzees. On the basis of serologic comparisons, hepatitis type A does not appear to be related to experimental hepatitis caused by the GB agent of Deinhardt, nor is the hepatitis A antigen serologically related to the fecal antigen of Cross.

Adolescent

Review of infectivity studies in nonhuman primates with virus-like particles associated with MS-1 hepatitis.

Using the technique of immune electron microscopy we have conducted hepatitis A infectivity studies in marmoset monkeys and chimpanzees. Marmosets inoculated with human serum containing the MS-1 strain of hepatitis A virus have developed hepatitis and seroconverted to 27 nm virus-like particles isolated from stools of humans in the early acute stages of hepatitis. Similar results have been observed through several marmoset subpassages, and the virus-like particles have been recovered from the liver of animals in the acute phase of hepatitis. Chimpanzees inoculated with stool filtrates containing the virus-like particles develop hepatitis with concomitant excretion of the particles in early acute phase stools and subsequent development of serum antibody to the particles. These studies provide evidence that the above particles constitute the virus of hepatitis A of the MS-1 prototype.

Acute Disease

Serologic and animal inoculation studies of a communal outbreak of viral hepatitis, type A.

Sera from individuals in an outbreak of viral hepatitis in a multifamily household, probably spread by contaminated food, were studied for antibodies to hepatitis A virus (anti-HAV), and selected acute phase sera were inoculated into marmosets. Significant rises in anti-HAV titers between acute and convalescent sera occurred in all of 15 individuals in the outbreak who experienced serum enzyme elevations and in one of 14 individuals whose serum enzyme levels remained normal. The remaining 13 individuals in the latter group had antibody levels in both early and late sera compatible with residual immunity from prior HAV infections and correlating with resistance to reinfection. Groups of marmosets were infected with acute phase sera from two of the cases; in both instances the inoculated sera contained substantial levels of anti-HAV. The marmosets developed specific anti-HAV seroconversions as well as enzyme elevations.

Alanine Transaminase