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Objective:Gloves containing chlorhexidine gluconate in an instant-release matrix on their inner surface (CHG gloves) were tested to determine their ability to rapidly inactivate infectious pathogens that may permeate or leak through the latex surface.Design:CHG gloves were exposed for 1 to 10 minutes to blood or media containing infectious pathogens (e.g., bacteria, fungi, parasites, and viruses) as well as to lymphocytes and macrophages that are known to be the primary carriers of human immunodeficiency virus (HIV). Inactivation of pathogens was determined either by in vitro assay or in vivo infectivity. Stressed control and CHG glove fingers were submerged in a viral pool (retrovirus or bacteriophage) and after a set time, the glove interiors were checked for presence of permeated viz-ions.Results:CHG gloves rapidly inactivate all the pathogens tested including retrovirus and hepatitis B virus (90% to 100%). In the stressed glove fingers, live virus was detected in 26% of the control group but not in any of the CHG group.Conclusions:The use of CHG gloves may reduce the risk of exposure to infectious fluidborne pathogens should the integrity of the latex barrier be compromised by overt failure or by permeation of viruses. Rapid destruction of lymphocytes and macrophages may facilitate inactivation of HIV associated with these cells. Tests have shown that CHG coating does not alter physical properties of the glove, and, furthermore, CHG gloves do not show potential for dermal irritation or sensitization.

Alkylglycerols, inflammation products of cancerous tissues, are potent macrophage activating agents. A brief in vitro treatment (30 min) of mouse peritoneal cells with a low concentration (50 ng/mL) of dodecylglycerol (DDG) in 10% foetal calf serum supplemented RPMI-1640 medium (FCS medium) activates macrophages for Fc-receptor mediated ingestion activity. A serum factor(s) was shown to be required for the activation of macrophages. When non-adherent cells were treated with rac-sn-1(3)-dodecylglycerol (DDG) in a serum free-0.1% egg albumin supplemented RPMI medium (EA medium) for 30 min and cultured in FCS medium for 2 h, the resultant conditioned medium contained a signal factor able to activate macrophages (macrophage activating factor). A conditioned medium prepared with electrophoresed serum alpha 2-globulin fraction in EA medium markedly enhanced activation of macrophages. Incubation of DDG-treated non-adherent cell ghosts in EA medium containing alpha 2-globulin also produced the macrophage activating signal factor. Therefore, it is concluded that a serum factor in alpha 2-globulin fraction is processed by pre-existing functions or enzymes of DDG-treated non-adherent cell membrane to yield a macrophage activating signal factor.

A viricide capable of eliminating hepatitis B virus (HBV) from chronic carriers should, theoretically, decrease the risk of primary hepatocellular carcinoma. Extracts of Phyllanthus amarus have been shown to inhibit the DNA polymerase of HBV and woodchuck hepatitis virus (WHV) in vitro. Three of four recently infected WHV carriers treated i.p. with P. amarus extract lost WHV, animals infected for greater than or equal to 3 months showed a decrease in virus levels. Preliminary results in human carriers treated orally with P. amarus for 1 month indicated that approximately 60% of the carriers lost HBV during the observation period.

Woodchuck sera were tested for antigens and antibodies with tests which detect human hepatitis virus antigens and antibodies. Data on 264 woodchuck sera are presented.

SummaryRadioactive (3H) uridine was incorporated into RNA isolated from the HBsAg of a chimpanzee carrier. HBsAg was purified by precipitation as an immune complex with the IgG fraction of chimpanzee anti-HBs. RNA was extracted from the washed complex with buffered phenol precipitated with alcohol and four nucleotides were identified by thin layer chromatography after alkali degradation.We acknowledge with appreciation the time and assistance of Dr. Manfred E. Bayer for the electron microscopic examination of the purified HBsAg—anti HBs complexes.Addendum. After this work was prepared for publication we became aware of the possibility that radioactive uridine could have been incorporated into uridine diphosphoglucose and not RNA. We have since repeated the procedure for the isolation of nucleic acid described in methods but included two additional steps. (1) The chimpanzee plasma was treated with 25 μg/ml of RNAse (4000 units/mg, Worthington, Freehold, NJ) and incubated for 15 min at 37°. A control contai...

One newborn and 24 fetal woodchuck litters from a woodchuck hepatitis virus (WHV) endemic population were examined for serological or hepatic evidence of WHV. In 18 of 24 fetal litters, there was detectable WHV DNA in the livers, either at explant culture or tissue extract. Most of those WHV DNA-positive liver extracts, which were examined by Southern blot, showed integration of WHV. However, WHV DNA replicative forms without integration were demonstrated in livers of two litters from late gestation. Woodchuck hepatitis surface antigen was detected in the sera of two other fetal litters from the late gestation period. WHV DNA was demonstrated in sera of three litters at different stages of ontogeny.

Hepatitis B surface antigen (HBsAg, Australia antigen) was detected by radioimmunoassay in freeze-thaw lysates of washed platelets from HBsAg carriers. Incubation of platelets from an HBsAg-negative person with medium containing HBsAg resulted in the platelets becoming positive for HBsAg. We suggest that platelets may phagocytize HBsAg-coated particles, and this may be an important transport mechanism for the hepatitis B agent.

DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R-HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyl-transferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this single-stranded gap.

Tree squirrel hepatitis B virus (THBV)-associated particles isolated from the livers of naturally infected animals share one or more antigenic determinants with hepatitis B surface antigen in solid-phase immunoassays. Characterization of THBV-associated polypeptides by sodium dodecyl sulfate/polyacrylamide gel electrophoresis reproducibly demonstrated major components with apparent sizes of 15.5 and 17 kDa. Peptide mapping of these components shows that they are related to the peptide maps of the major surface antigen polypeptides associated with hepatitis B virus and like viruses. Sodium dodecyl sulfate/polyacrylamide gel analysis also demonstrated discrete bands at 14.5, 19, 20, and 35 kDa. Upon blotting of THBV-associated polypeptides with sera containing antibodies to hepatitis B core antigen or hepatitis B X antigen, only the 35-kDa band became detectable, suggesting that this component is core related. These results establish the presence of both surface and core antigen-related polypeptides associated with purified THBV and better define the relationship of THBV to the family of hepatitis B virus and like viruses.

Antibodies against synthetic peptides derived from the polymerase gene of the hepatitis B virus (HBV) were present in 80% of renal dialysis patients infected with HBV and in woodchucks infected with woodchuck hepatitis virus (WHV). Polymerase antibody (anti-pol) appeared as the earliest marker of both HBV and WHV infections in approximately half of the individuals tested, suggesting that these antibodies were generated following early viral replication in the liver during the incubation period and prior to the appearance of virus in the blood. Many HBV- or WHV-infected individuals negative for surface antigen throughout infection also had anti-pol, but anti-pol appeared only after anti-surface, anti-core and/or anti-e. The presence of anti-pol did not correlate with other serologic markers of HBV or WHV infection, nor did it correlate with histologically confirmed hepatitis in woodchucks. However, there was a significant correlation between the presence of anti-pol and elevated liver enzyme levels in the sera of renal dialysis patients. In several cases, anti-pol was the sole marker of infection, suggesting that underlying infection and low levels of virus replication were present. Most individuals with anti-pol had antibodies to one of the three synthetic peptides, suggesting it may be immunodominant in natural infections. In human populations, groups with a high frequency of HBV infection have a high frequency of polymerase antibodies, and groups with a low frequency of HBV infection have a low frequency of polymerase antibodies. A standard assay for the detection of polymerase antibodies is described, and possible clinical applications are discussed.

The automated complement fixation system presented in this paper differs from those described by Studievic et al. (1971) and Vargues and Henley (1974). In the one developed here only one sampler is used with a wheel presenting a double row of cups. This system can assay 60 samples per h and can be modified to double this number. Our unit requires an incubation period of 15 min at 37°C and we have never observed any contamination of the samples during the assay run. Anti-HBc antibodies could be detected in all HBs Ag carriers and acute hepatitis B patients; chronic renal hemodialysis patients with a past history of hepatitis who were currently HBs Ag-negative still possessed anti-HBc antibodies for at least one year after the acute phase. There exists a group of people who are HBs Ag and anti-HBs negative but carriers of anti-HBc antibodies. 5.4% of the blood donors, patients and staff members of our hemodialysis unit are in this category. It is difficult to conclude that all sera containing only anti-HBc are infectious. Nevertheless, anti-HBc must be connected with either an acute replication of the hepatitis B virus or past infection. Therefore, it seems proper to consider sera containing only anti-HBc as possibly hazardous and these sera should not be used for transfusion. We propose the inclusion of a systematic screening for anti-HBc in blood banks; the automated complement fixation test is especially suitable for this purpose. The systematic detection of HBs Ag alone has not resulted in the elimination of transfusion hepatitis. Exclusion of positive anti-HBc blood may lower the frequency of infections.

During July and August of 1973, 9,198 mosquitoes were collected in the Republic of Senegal. Eight species of mosquitoes were found in the collections: Culex thalassius, Culex pipiens quinquefasciatus, Culex trigripes, Culex phillipi, Aedes irritans, Aedes aegypti, Anopheles gambiae, and Mansonia sp. Specimens were sorted by biological condition; those obviously engorged were designated as (E), females with swollen abdomens not conspicuously blooded were considered gravid (G), and those with normal or shrunken abdomens were considered neither blooded nor gravid (U). Representative samples of each species were tested by solid phase radioimmunoassay for hepatitis B surface antigen (HBSAg, Australia antigen). A total of 12 mosquitoes were found to be HBSAg positive out of 1,658 individuals tested. These were: 9 Culex thalassius, 1 (E), 5 (G), 3 (U); 2 Culex pipiens quinquefasciatus, 1 (E), 1 (U); and Aedes irritans, 1 (U).

Virus-associated particles have been isolated from the livers of three common gray tree squirrels (Sciurus carolinensis pennsylvanicus) that have histological evidence of hepatitis. Two of these livers were also positive by orcein staining, suggesting the presence of surface antigen in the cytoplasm of hepatocytes. Fractionation of these particles by CsCl density equilibrium gradient centrifugation and assay of the fractions for surface antigen, core antigen, and DNA polymerase activities demonstrate the presence of all three at an approximate density peak of 1.27. Electron microscopic examination of purified virus preparations showed spherical particles with a mean diameter of 25 nm. Initial characterization of the DNA polymerase product by gel electrophoresis showed a single DNase I sensitive band, migrating slightly faster than the woodchuck hepatitis virus DNA polymerase product. The presence of apparently cross-reacting antibodies was demonstrated by purified hepatitis B surface and/or core antigens binding to some squirrel sera in solid phase assays. Infected tree squirrels appear to lack detectable antigen in their sera. These results suggest that the tree squirrels studied are chronic carriers of a hepatitis B type virus. The host-virus interaction described herein may be useful in understanding the chronic carrier state associated with hepatitis B in man.

Of 24 patients with post-transfusion hepatitis studied prospectively at Philadelphia General Hospital, 12 had received at least 1 unit of Australia-antigen (Au)-containing blood, and 12 had received only blood in which the antigen was not detected. By immunodiffusion, 7 of the 12 Au recipients and none of the recipients of negative blood developed detectable serum-Au, and 1 developed antibody. By radioimmunoassay these figures became 8 and 9, respectively. The two groups had similar symptoms and signs; 3 of the Au recipients and 4 of the Au-negative recipients became jaundiced. Median incubation period in Au cases was 51 days (range 17-132), and in Au-negative cases 62·5 days (range 42-94). This was not a significant difference, nor was there any difference in the time to reach maximum S.G.P.T. or the duration of S.G.P.T. elevation. The maximum S.G.P.T. level was similar in the two groups, as was the maximum serum-bilirubin and prothrombintime, and no histological distinction could be made. The median peak serum-alkaline-phosphatase in the Au recipients was 41 international units (I.U.), and in the Au-negative blood recipients 113·5 I.U. With this sole exception, these findings indicate that there are no distinguishing features of hepatitis in recipients of Au vs. Au-negative blood with regard to incubation period, symptoms, physical findings, clinical course, laboratory findings, and severity of disease.

Summary1. By following a carefully standardized procedure highly immunogenic particles uniformly were obtained by ultra-centrifugation from ground extracts of the H37Ra strain of Mycobacterium tuberculosis var. hominis. These particles, which ranged in size from 20-200 mμ, were just as effective as the living whole cells of this strain in protecting mice against a severe tuberculous infection. 2. The sediment comprised of the particles prepared from H37Ra cells was red in color and therefore has been termed the “red fraction.” The immunogenic activity of this fraction was almost eliminated when the particles were diluted 5 times or more with 0.25 M sucrose buffer, autoclaved at 126°C for 15 minutes, or filtered through a Berkefeld filter. However, lyophilization in 0.25 M sucrose buffer significantly reduced their immunogenic activity while lyophilization in 0.88 M sucrose buffer did not. Sonic oscillation appeared to have no significant effect on the activity of the particles. When the cells were washed ...

SummaryPlasma, frozen and thawed lymphocytes, and purified Australia antigen fractions from various patients suffering from viral hepatitis were tested for an effect on PHA-stimulated lymphocytes isolated from normal individuals. The addition of these materials to lymphocyte cultures produced no significant change from that of PHA alone or PHA plus control materials from normal (nonhepatitis) patients.

SummaryVarious mycobacterial cells were converted to protoplasts by treatment with a mycobacteriophage concentrate. The technic involved production of sufficient adapted phage by growth either in the 607 or H37Ra strains of tubercle bacilli. Further concentration was necessary in order to obtain a sufficiently high phage bacteria ratio. This was accomplished by lyophilization. The significance of the reported findings is discussed.

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated 32P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85°C but was stable at 56°C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.

SummaryThe presence of DNA polymerase in normal human serum and its possible association with Australia antigen was investigated. DNA polymerase activity was determined in 135 samples of normal serum. Only 3 samples revealed appreciable amounts of activity. The addition of a variety of polynucleotide templates did not stimulate polymerase activity.Australia antigen is a particle associated with hepatitis and is reported to contain RNA. A DNA polymerase, “reverse transcriptase” might be required for replication of this RNA. We found DNA polymerase activity in only one of five preparations of purified Australia antigen. A similar activity was also found to be present in serum from certain normal volunteers. In either case, polymerase activity does not require nor is it stimulated by added polynucleotides suggesting that the enzyme may already be associated with a template such as RNA or DNA. A comparison between the amount of DNA polymerase activity in serum and the presence of Australia antigen in three se...

SummaryPurified 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.

There is considerable data to support the hypothesis that Australia antigen is an infectious agent that causes hepatitis in man. (a) Association with acute viral hepatitis. (b) Association with chronic hepatitis. (c) Virus-like appearance under the electron microscope (200-A particles). (d) Transmission of Au(1) from man to man. (e) Transmission and passage of partially purified Au(1) to an animal host (infant African green monkey). (f) Localization [with fluorescent anti-Au(1)] of Au(1) in the nuclei of liver cells of patients with hepatitis and/or Au(1) in their blood. (g) Distribution of Au(1) in institutions, disease groups, and populations is consistent with the distribution of an infectious agent. (h) RNA identified in Au(1) particles isolated from blood. (i) Apparent replication of Australia antigen in tissue cultures of human liver cells. There is also considerable evidence that Australia antigen has many of the characteristics of a serum protein polymorphism. Since neither of these hypotheses has been rejected they can be combined to make a third hypothesis, namely, that Australia antigen is an infectious agent which causes hepatitis in some people infected with it and that it has the characteristics of an (inhertied) serum protein polymorphism. We propose calling agents of this postulated class "Icrons."

OUR suspicion that the Australia antigen associated with acute and chronic hepatitis is a virus and is the cause of the disease has been supported by examination of material from affected patients. Au(1) was first detected in the serum of an Australian aborigine1, and the geographic distribution, disease association, genetics and physical and chemical characteristics of this unusual antigen have been described2–4. One of the most startling findings in the investigation of Au(1) was the appearance of purified fractions when viewed with the electron microscope, as observed by Bayer et al.5. The fractions contained uniformly round particles measuring approximately 200 A in diameter with cores in some particles and what appeared to be surface subunits (Fig. 1). Antibody to Au(1) has occurred in transfused patients who have received multiple transfusions and was produced in rabbits by hyperimmunization with human sera containing Au(1). Both the human and rabbit antisera gave identical precipitin reactions6.

We have studied the localization of Australia antigen, a particulate substance associated with hepatitis, by means of the fluorescent antibody technique. Preparations were made from 61 liver biopsy specimens taken from patients with infectious hepatitis, serum hepatitis, and a variety of other diseases. When tested with fluorescein-conjugated rabbit anti-Au(1) antisera all 26 patients who had Au(1) in their serum had specific fluorescence in their liver cells. The fluorescence appeared in three forms: as discrete particles within the nucleus, diffuse fluorescence of the entire nucleus, and fluorescence of the nuclear rim. Occasionally there were also fluorescent particles in the cytoplasm. Other specimens were tested with the fluorescent antibody including a variety of human tissues, buffy coat smears, peripheral lymphocyte cultures, and cells obtained from bile and duodenal drainage. Among these specimens, fluorescence was found in the cytoplasm of a few cells in the bone marrow of two patients with hepatitis and Au(1) in their serum, and in the liver, spleen, mesentery, and testis of one patient with leukemia, chronic hepatitis, and Au(1) in his serum. We have shown that the presence of fluorescent particles in the liver cells is strongly associated with the presence of Au(1) in the serum and the diagnosis of viral hepatitis. We believe that this study adds support to the hypothesis that Australia antigen is an antigenic determinant of a virus capable of causing hepatitis.

SummaryBy grinding cells of Mycobacterium tuberculosis var. hominis H37Ra using a ball mill and by differential centrifugation four particulate fractions and one supernatant fraction were obtained. Aconitase, catalase, fumarase, α ketoglutaric dehydrogenase, isocitric dehydrogenase and malic dehydrogenase were located almost entirely in the supernatant fraction, while cytochrome oxidase, DPNH oxidase, and succinic dehydrogenase were found to be associated mainly with the two largest particulate fractions.

SummaryLymphocytes 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.

SummaryAn 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 bon...

SummaryA 7.7% incidence of antibodies directed against Gm (a), Gm (b) and Gm (f) antigens of IgG was noted in 298 adolescent drug addicts, as compared to 0.9% in a nonaddicted control group. Isoimmunization as a stimulus for Gm antibody production in addicts was indirectly excluded. A significant association between anti-Au and anti-Gm in the sera of drug addicts was found, which suggests that the hepatitis agent associated with Australia antigen may be responsible for the induction of Gm antibodies in this group.The authors thank Dr. Parvez Lalezari for the red blood cell antibody determinations. This work was supported, in part, by the American Cancer Society Institutional Research Grant IN-28L, by USPHS Grants CA-06551, CA-06927, RR-05539 and Contract No. 70-2234B from the National Institutes of Health, and by an appropriation from the Commonwealth of Pennsylvania.

Antibody to hepatitis-B core antigen (anti-HB c ) 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-HB c 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-HB c . 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.

Purified and concentrated preparations of Australia antigen had no stimulating effect on leukocytes of human subjects under study when tested either on DNA-polymerase activity, 3H-thymidine uptake or chromosomal alterations. Moreover, in patients with chronic hepatitis and cirrhosis of the liver no correlation between antigenemia and chromosome aberrations in blood leukocyte cultures could be detected. On the other hand, a serum obtained from a virus hepatitis patient with Australia antigen in the blood was found to stimulate leukocyte cultures from one patient with Down's syndrome and antigenemia, one mentally retarded patient and three normal donors. This stimulating agent is obviously not associated with Australia antigen.

An aqueous extract of the plant Phyllanthus niruri inhibits endogenous DNA polymerase of hepatitis B virus and binds to the surface antigen of hepatitis B virus in vitro. The extract also inhibits woodchuck hepatitis virus (WHV) DNA polymerase and binds to the surface antigen of WHV in vitro. The extract, nontoxic to mice, was tested for antiviral activity in woodchucks (Marmota monax). In a trial using six long-term WHV-carrier woodchucks, five treated animals showed a faster decrease in woodchuck hepatitis virus surface antigen titer compared to one untreated control. In animals recently infected with WHV, the extract was effective when administered i.p. in three out of four animals in reducing and within 3-6 weeks eliminating both the surface antigen titer and DNA polymerase activity in serum. The treatment was discontinued after 10 weeks, and the treated animals have remained free of detectable markers of WHV for more than 45 weeks. In contrast, three untreated controls remained positive for both markers for WHV. One of the controls died after 8 weeks; the other two controls have remained positive for WHV markers for more than 45 weeks. In a third trial with long-term carriers, test animals treated subcutaneously with the extract for 12 weeks did not respond; but on switching the mode of administration to i.p., two out of the five animals showed a significant decrease in woodchuck hepatitis virus surface antigen titer compared to controls.

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 (HB s Ag) was detected in unengorged nymph and adult bedbugs in each of the first three collections. 3 of 28 such specimens were HB s Ag(+) in the first collection and 3 of 17 specimens were positive in the second collection. In the third, 6 of 9 were HB s Ag(+) when the bed occupant was known to be HB s Ag(+). 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 HB s Ag(+). 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.

‘Ausria II’ polystyrene beads (Abbott Labs, N. Chicago) are reacted with wood-chuck serum positive for WHsAg in a dilution predetermined by titration. This modified bead is used in a blocking assay to detect the presence of antibody to the surface antigen of woodchuck hepatitis virus (anti-WHs). Serum containing wood-chuck anti-WHs and commercial horseradish peroxidase (HRP) labeled anti-HBs are sequentially added. A drop in optical density at 492 nm of 50% or more due to the blocking of HRP conjugated anti-HBs by anti-WHs compared with a control (negative woodchuck serum) is a measure of anti-WHs. The ease and simplicity of converting readily available ‘Ausria II’ beads to specific reagents for detecting anti-WHs should be welcomed by investigators studying WHV. The method described is both sensitive and reproducible.

SummaryData are presented to show that both an H37Ra strain of Mycobacterium tuberculosis and a ribosomal RNA fraction from this strain are effective inhibitors of tumor growth in mice. Mice were pretreated with tubercle bacilli or RNA fraction and challenged 5 weeks later with mixtures of tumor cells and the respective pretreatment agents. Comparative data with BCG are presented. Inhibition did not appear to be due to any direct toxic effect of tubercle bacilli or ribosomal RNA fraction on tumor cells. The necessity, for an inhibitory effect, of tuberculin hypersensitivity was deemed unlikely because the ribosomal RNA fraction neither induces nor provokes tuberculin hypersensitivity. The inhibition realized with mycobacterial extract avoids the risk to the tumor-bearer of viable organisms.

Sera from sheep and other domestic animals contain a substance that gives a strongly positive test for antibody to hepatitis B virus surface antigen by the accepted radioimmunoassay procedure. We have purified this substance from sheep serum to near homogeneity by ion-exchange, affinity, and molecular exclusion chromatography and have identified it to be an IgM. We present evidence that this sheep IgM is an antibody to polymerized sheep albumin. This antibody may arise due to infection by hepatitis B virus, hepatitis B virus-like viruses, or other pathological agents and may react with hepatitis B virus surface antigen by combining with polymerized albumin bound to the hepatitis B virus receptor for this polymer.

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).

A sulfated polysaccharide isolated from Pelvetia fastigiata, a marine algae, was found to inhibit in vitro the reaction of the surface antigen of hepatitis B virus (HBsAg) or of woodchuck hepatitis virus (WHsAg) with antibody to HBsAg (anti-HBs). The polysaccharide was composed mainly of 1----2 linked L-fucose-4-sulfate with some (less than 10%) 1----3 linkages. The inhibition of the reaction of HBsAg with anti-HBs or of WHsAg with anti-HBs was found to be directly proportional to the molecular size of the polysaccharide. Comparison of its inhibitory activity with that of carrageenans and dextran sulfates showed that, in addition to the size, the configuration of the component sugar and the presence of deoxy sugar may play a role in the inhibition of reaction of HBsAg or WHsAg with anti-HBs. The fucose sulfate polymer, fucoidan, however, had no effect in vivo on woodchuck hepatitis virus in woodchuck chronic carriers.

Sera from 588 woodchucks were assayed for woodchuck hepatitis virus (WHV) markers using hepatitis B virus (HBV) reagents which have cross-reactivity with WHV markers. Twenty per cent of these woodchucks, trapped in Delaware, Maryland and Pennsylvania, had WHsAg; 50% of these had DNA polymerase. There are areas of high and low endemicity within these states. Female woodchucks may have a higher incidence of WHV markers than do males. Woodchuck hepatitis surface antigen (WHsAg) and anti-WHc often occur together but less commonly than HBsAg and anti-HBc do in human HBV infection. Experimental infection of woodchucks with WHV produced a prolonged infection (up to 40 weeks). WHsAg and DNA polymerase appeared to be more reliable indicators of infectivity than anti-WHc, woodchuck hepatitis e antigen (WHeAg) or anti-WHe. WHeAg was not detected throughout this period of infection, while anti-WHe appeared late in two of three experimentally infected animals. Four male and four female woodchucks which developed primary hepatocellular carcinoma in captivity were analyzed for WHV markers throughout their period of confinement. Seven were WHsAg and anti-WHc positive when captured. The animal that was free of WHV markers on capture converted to the WHsAg and anti-WHc positive state prior to the development of primary hepatocellular carcinoma. One primary hepatocellular carcinoma animal produced WHeAg and none anti-WHs or anti-WHe.

Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

Summary1) Various microorganisms were studied for ability to metabolize myricyl palmitate (beeswax), tubercle wax, and tributyrin. All cultures except one produced positive results varying only in rate and extent to which they attacked these substrates. A pronounced lag phase in utilization of substrate by 3 microorganisms tested was attributed to saturation of enzyme sites with endogenous substrates. 2) With the exception of Candida albicans intact cells did not show esterase activity. However, positive results were obtained with extracts which suggested that deesterification was an initial step in utilization of waxes as sources of energy for the various microorganisms tested.

SummaryNine 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).

SummaryAntibodies to the Australia antigen were shown to be present in the ascitic fluid of mice hyperimmunized with Australia antigen-Freund's 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.

SummaryMycobacterium tuberculosisvar. hominisstrains H37Rv, H37Ra and H37RaN; Mycobacterium tuberculosisvar. bovisstrain BCG-4; and Mycobacterium smegmatiswere ground in a ball mill and fractionated according to the methods outlined earlier(2). Enzymes aconitase. fumarase. DPNH oxidase, catalase. cytochrome oxidase, isocitric, alpha-ketoglutaric, malic and succinic dehydrogenase were localized in one or more fractions using the two criteria established by Alexander and Wilson(12a. b). Cytochrome oxidase. DPNH oxidase and succinic dehydrogenase were found to be particulate bound by both criteria. However, the locus of other enzymes determined by one criterion did not always coincide with that determined by the second criterion. A modified criterion was then established and this was discussed. With the new criterion most of the enzymes of the tricarboxylic acid cycle and terminal electron transport system could be localized. The particles found within the various fractions were electron opaque and labile to...

BLUMBERG and his colleagues have published data on the relation of Australia antigen, Au(1), to hepatitis1,2. While existing antisera, either of human or rabbit sources3, frequently can be used to detect this antigen in patients with hepatitis by either immunodiffusion4 or complement fixation5 it is probable that the antigen is undetectable in some patients. One possible reason for this may be the neutralization of antigen by antibody. Sera may manifest the presence of antigen at one time and antibody at another time2. In such a case, a precipitin band forms between sera from early and later bleedings from the same patient.