Rapid inactivation of infectious pathogens by chlorhexidine-coated gloves.

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 virions. 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 fluid-borne 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.

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages.

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.

A serum factor for macrophage activation after in vitro dodecylglycerol treatment of mouse lymphocytes.

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.

Hepatitis B virus and primary hepatocellular carcinoma: treatment of HBV carriers with Phyllanthus amarus.

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.

Enhanced metabolic activation of chemical hepatocarcinogens in woodchucks infected with hepatitis B virus.

The metabolism of chemical carcinogens was investigated in liver preparations from 28 captive woodchucks (Marmota monax). Of these, 23 were naturally infected with the woodchuck hepatitis virus (WHV), and eight also had primary hepatocellular carcinoma (PHC). Twenty-nine parameters were investigated in liver subcellular fractions, including cross-reactivity with HBsAg, and biochemical parameters, such as gamma-glutamyl transpeptidase, cytochrome P-450 and microsomal monooxygenases (aryl hydrocarbon hydroxylase, ethoxycoumarin and ethoxyresorufin deethylases, aminopyrine and dimethylnitrosamine demethylases, and testosterone 7 alpha-, 16 alpha- and 6 beta-hydroxylases), uridine 5'-diphosphoglucuronosyl transferase, GSH and related enzymes (peroxidase, reductase and S-transferase), as well as other cytosolic enzyme activities (glucose 6-phosphate and 6-phosphogluconate dehydrogenases, NADPH- and NADH-dependent diaphorases, and DT diaphorase). In addition, liver preparations were used in order to quantify the metabolic activation into bacterial mutagens of five procarcinogens (aflatoxin B1, the pyrolysis products Trp-P-2 and MeIQ, 2-aminofluorene and dimethylnitrosamine) and the decrease of potency of three direct-acting mutagens (sodium dichromate, ICR 191 and 4-nitroquinoline 1-oxide). WHV infection produced a significant stimulation of carcinogen metabolism, as shown by the simultaneous change in detoxification parameters (GSH depletion) and activation indices (enhancement of microsomal monooxygenases and of procarcinogen activation into mutagenic metabolites). There were no significant differences between WHV-positive samples from animals without PHC and the noncancerous tissue of PHC-bearing animals, whereas a decrease of both activation and detoxification indices was recorded in the tumorous tissue. There was a considerable interindividual variability among WHV carriers, which was tentatively ascribed to genetic factors. Pregnancy was the only known factor influencing the results in WHV carriers. However, even by excluding pregnant animals, the effects on carcinogen metabolism produced by WHV infection were still statistically significant. These results, together with previous data obtained in humans, revealed that metabolic factors may play a role in the synergism between viral hepatitis and chemical hepatocarcinogens in the etiopathogenesis of PHC.

Interaction of fucoidan from Pelvetia fastigiata with surface antigens of hepatitis B and woodchuck hepatitis viruses.

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.

Hepatitis B virus and hepatocellular carcinoma--treatment of HBV carriers with Phyllanthus amarus.

Extracts of Phyllanthus amarus inhibit the DNA polymerase of HBV and related viruses. Woodchuck carriers of woodchuck hepatitis virus (WHV) were treated intraperitoneally with P. amarus extract. Three of four animals which had been recently infected lost the virus. Animals infected for about 3 months or more had a decrease in virus levels. Human carriers of HBV were treated orally for 1 month. About 60% of the carriers lost HBV, which did not return during the observation period. Fractions containing active principles are now being isolated and characterized.

Vertical transmission of woodchuck hepatitis virus.

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.

The hybrid EIA test: a specific and sensitive assay for the detection of woodchuck antibody to hepatitis surface antigen (anti-WHs).

'Ausria II' polystyrene beads (Abbott Labs, N. Chicago) are reacted with woodchuck 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 woodchuck 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.

Presence of antibodies to the polymerase gene product(s) of hepatitis B and woodchuck hepatitis virus in natural and experimental infections.

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.

Metabolism of mutagens and carcinogens in woodchuck liver and its relationship with hepatitis virus infection.

Thirty-six wild-caught woodchucks (Marmota monax) were characterized according to sex, weight, trapping locality, liver pathology, and serum or hepatic markers of woodchuck hepatitis virus. Liver subcellular fractions were assayed for microsomal cytochromes P-450, aryl hydrocarbon hydroxylase, glutathione, cytosolic enzymes involved in its metabolism (glutathione S-transferase, glutathione peroxidase, and glutathione reductase), in the hexose monophosphate shunt (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), NADH- and NADPH-dependent diaphorases, and DT diaphorase. Moreover, liver postmitochondrial fractions were assayed for their ability to activate procarcinogens [i.e., a tryptophan pyrolysate product, aflatoxin B1, 2-aminofluorene, and trans-7,8-dihydrobenzo(a)pyrene] to mutagenic metabolites in the Ames reversion test and to decrease the activity of direct-acting mutagens [i.e., 4-nitroquinoline N-oxide, 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine X 2HCl, and sodium dichromate]. A considerable interindividual variability in metabolism was observed among the examined woodchucks. Some of the investigated parameters were more elevated in virus carriers, especially in those suffering from chronic active hepatitis, but only a few of the recorded differences (i.e., oxidized glutathione reductase and NADPH-dependent diaphorase) were statistically significant. The comparison of the monitored activities in woodchucks and in other rodent species (rat and mouse) led to the conclusion that the liver metabolism of mutagens and carcinogens in woodchucks is more oriented in the sense of activation, while detoxification mechanisms are more efficient in rats and mice.

Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies.

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.

Tree squirrel hepatitis B virus: antigenic and structural characterization.

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.

A newly identified hepatitis B type virus in tree squirrels.

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.

Human ferritins present in the sera of nude mice transplanted with human neuroblastoma or hepatocellular carcinoma.

Fifteen nude mice were inoculated with a human neuroblastoma cell line and 14 with a human primary hepatocellular carcinoma cell line. Human ferritins were detected in the sera of the mice which developed tumors. Of 14 mice bearing human neuroblastoma, 12 had human liver-type ferritin (8 to 52 ng/ml) in their sera, and three of these also had HeLa-type ferritin (acidic ferritin) (29 to 40 ng/ml). Of 10 nude mice bearing human primary hepatocellular carcinoma, eight had human liver-type ferritin (10 to 820 ng/ml), and one of these had HeLa-type ferritin at a level of 43 ng/ml. Since the ferritins in the sera of these mice were produced by the human tumor cells, these observations support the hypothesis that the elevated ferritins often found in the serum of patients with cancer are, in part, derived from their tumors.

Woodchuck hepatitis virus: experimental infection and natural occurrence.

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.

Hepatitis B surface antigen and polymerized albumin binding activity in sheep serum.

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.

Immunological cross-reactivities of woodchuck and hepatitis B viral antigens.

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.