Irving Millman

ANTIBODY TO HEPATITIS-B CORE ANTIGEN IN PATIENTS WITH PRIMARY HEPATIC CARCINOMA

Antibody to hepatitis-B core antigen (anti-HBc) was assayed in the serum of patients with primary hepatic carcinoma (P.H.C.) and controls from Hong Kong, West Africa, and the United States. In each region the prevalence of anti-HBc was higher in P.H.C. patients than in controls, ranging from 70 to 95% in the patients and from 20 to 68% in the controls from Asia and Africa; 24% of P.H.C. patients and 4% of controls from the U.S. had anti-HBc. These data support the hypothesis that chronic infection with hepatitis-B virus is aetiologically related to P.H.C., especially in Asia and Africa, although other factors must also be involved.

Hepatitis-B virus in bedbugs (Cimex hemipterus) from Senegal.

Bedbugs of the species Cimex hemipterus (F) were collected on four separate occasions from the bedding in the huts of village dwellers in Senegal, West Africa. Hepatitis-B surface antigen (HBSAg) was detected in unengorged nymph and adult bedbugs in each of the first three collections. 3 of 28 such specimens were HBSAg(+) in the first collection and 3 of 17 specimens were positive in the second collection. In the third, 6 of 9 were HBSAg(+) when the bed occupant was known to be HBSAg(+). 2 of these 6 positive insects did not contain human serum proteins. Bedbugs in the fourth collection were captured and kept alive without a blood meal for 30 days. 3 of 89 of these samples were HBSAg(+). These are the highest field infection-rates of hepatitis-B virus reported in any insect species. The bedbug must be considered a potential vector of hepatitis-B virus.

AUSTRALIA ANTIGEN, HEPATITIS VIRUS AND DOWNS SYNDROME*

Australia antigen, originally detected in the serum of an Australian aborigine,l is now known to be associated with viral hepatitis.z-s A series of studies have been completed to test the hypothesis that Australia antigen (abbreviated Au( 1) ) is, or is located on, a virus which causes hepatitis in some people infected with it. The evidence in support of this hypothesis, which is given in detail e l~ewhere ,~~ 6, 7 can be summarized as follows: ( 1 ) Australia antigen is associated with acute hepatitis. (2) Au( 1) is associated with chronic hepatitis. ( 3 ) Isolated Au( 1) has the appearance of a virus (200 A in diameter) under the electron microscope.* (4) Using fluorescent anti-Au( 1 ) , Australia antigen has been identified in the nuclei of the liver cells of patients with hepatitis and Au(1) in the peripheral blood; this is generally not seen in appropriate cont r o l ~ . ~ , lo (5) Australia antigen present in the blood of donors can be transmitted by transfusion to patients.11-13 The amount of Australia antigen which develops in recipients of blood is greater than that transfused. Some of the patients to whom the Au( 1) is transmitted will develop hepatitis. Other patients receiving Australia antigen by transfusion may develop antibodies against it or show no reaction. These findings, in addition to others cited in the referenced publications, provide support for the virus hypothesis, but additional evidence will be needed before it can be concluded that Australia antigen is, in fact, a virus; for example, growth on tissue culture and transmission to experimental animals of purified Au( 1) should be accomplished. These studies are in progress in our laboratory and are very promising. It is necessary to treat blood containing Australia antigen and isolated Au(1) as if the antigen is a virus, since it is possible to contract hepatitis while working with it and administration of serum containing Australia antigen by transfusion or needle injection can cause the disease in patients who receive it. Historically, some of our earliest studies on Australia antigen have been done with Down’s syndrome patients.l4-l6 We first found that Australia antigen was common in leukemia.’ We hypothesized that the presence of the antigen might indicate a susceptibility to leukemia and, if this were so, the antigen would be common in patients who had an increased risk of developing leukemia. It was known that Down’s syndrome patients are at high risk of developing leukemia, and a series of studies was initiated.2JJ7 It was soon found that the

Radioimmunoprecipitation Assay for Australia Antigen, Antibody, and Antigen-Antibody Complexes

Summary Purified Australia antigen [Au(1)], conjugated with 125I, was reacted with and quantitatively precipitated by human anti-Au(1) in the presence of heterologous antihuman IgG and “dilute” ammonium sulfate. The sensitivity of the devised radioim-munoprecipitation assay procedure proved to be up to 1000 times more sensitive for the detection of antigen and up to 20,000 times more sensitive for the detection of antibody when compared with either immunodiffusion or complement fixation assays. This method is extremely valuable not only in the quantitative detection of antigen and antibody but also in the delineation of naturally occurring antigen-antibody complexes.

Ergasteric hepatitis: endemic hepatitis associated with Australia antigen in a research laboratory.

Abstract Endemic hepatitis occurred among the personnel of a research laboratory working with human blood and tissues in the study of Australia antigen (Au(1)). All individuals were tested for seru...

Specificities of Human Antibodies to Australia Antigen

Summary Nine different human anti-Australia antigen antisera were compared to each other using population and immunologic methods. Six of the antisera fell into the same category (Philadelphia I) and included specificities detected by the previously described anti-Au(1) antisera. There were three antisera, each of which contained specificities different from those in the anti-Au(1) category. They are to be examined to see whether they identify forms of hepatitis or other conditions different from those detected by anti-Au(1).

The use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels.

Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). 125I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 microliter or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, we observed two bands of HbsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck patients hepatitis virus surface antigen (WHsAg).

Lymphocyte Transformation and Hepatitis I. Impairment of Thymidine Incorporation and DNA Polymerase Activity

Summary Lymphocytes from patients with viral hepatitis were found to be hyporesponsive to the replicating stimulus of phytohemagglutinin (PHA) in vitro. This was shown by an impairment both in the induction of DNA polymerase activity and thymidine incorporation into the cellular DNA by PHA-stimulated lymphocytes. The deficit is transient and occurs with the onset of clinical symptoms.

The production of antibodies to Australia antigen in mouse ascites fluid.

Summary Antibodies to the Australia antigen were shown to be present in the ascitic fluid of mice hyperimmunized with Australia antigen-Freunds adjuvant mixtures. As much as 100 ml can be removed from individual mice if they are restimulated by intraperitoneal injections of adjuvant-saline mixtures. The antibodies contained in these ascitic fluids were shown to be both of the gamma G and gamma M types. The specificities of the antibodies contained in the mouse ascitic fluid were shown to be similar but not identical to human antibody to Australia antigen.

Detection of Australia antigen in human tissue culture preparations.

Summary An attempt was made to propagate Australia antigen. Au(1), in tissue culture. The approach to the study was twofold: (a) the culturing of fresh biopsied tissues from patients with hepatitis and Au(1) in their blood; and (b) the addition of serum, plasma, and extracts of biopsied liver from patients with Au(1) in their blood to established cell lines and primary cells from human fetal tissues in tissue culture. Positive results were obtained only with the first approach. Two liver cultures out of 23 specimens from patients with Au(1) in their blood produced either intranuclear fluorescent granules after staining with fluorescent coupled rabbit anti-Au(1) antiserum or Au(1) in the tissue culture fluids as determined by a sensitive radio-immunoprecipitation assay technique. Fluorescent intranuclear granulation appeared during the second and sixth passage of one culture of liver. It is unlikely that this could be explained by carry-over of Au(1) from the initial biopsy specimen. Cultures of sternal bone marrow, testis, jejunal loop, and lymphocytes from patients who had Au(1) in their blood were uniformly negative for fluorescent granules as well as Au(1) by radioimmuno-precipitation assay. The results indicate a strong possibility that Au(1) replicates in tissue culture.