Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein.

Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein. The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals. Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells. Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro. The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies.

Comparison of the immune response to recombinant gp120 in humans and chimpanzees.

OBJECTIVE: To assess similarities and differences in antibody responses to recombinant (r) HIV-1IIIB gp120 in chimpanzees, previously protected from HIV-1 infection, and human volunteers immunized in connection with a Phase I clinical trial. METHODS: Frozen sera from humans immunized with rgp120 from HIV-1IIIB and chimpanzees immunized with the same antigen or recombinant soluble gp160 were compared in a variety of serologic assays. RESULTS: The magnitude of the antibody response to gp120 was similar in both species; however, the half-life of the antibody response to rgp120 was approximately 4.5 times longer in humans (9 weeks) than in chimpanzees (2 weeks). Antibodies to gp120 in both species were broadly cross-reactive with gp120 from diverse isolates of HIV-1 and were effective in blocking the binding of gp120 to CD4. Antibody binding to native gp120 was greater than to denatured gp120 in both species. Antibody responses to the principal neutralizing determinant (V3 domain) and virus neutralization titers were approximately 10-fold lower in humans than chimpanzees. The relative avidity of antibody binding to gp120 was higher in the sera from the immunized chimpanzees than in the immunized humans. CONCLUSIONS: While the antibody responses to rgp120 elicited in man and chimpanzees were in many ways similar, significant differences did occur. Predictions made on the basis of chimpanzee immunogenicity studies over-estimated the potency of the virus neutralizing titers and under-estimated the duration of the antibody response achieved in humans.

Adhesion mediated by intercellular adhesion molecule 1 attenuates the potency of antibodies that block HIV-1 gp160-dependent syncytium formation.

Several lines of evidence suggest that leukocyte adhesion molecules can promote HIV-1-mediated cell fusion and syncytium formation. In the present studies, the human kidney cell line, 293, was transfected with the envelope glycoprotein gene of the MN strain of HIV-1 alone or cotransfected with a cDNA encoding intercellular adhesion molecule 1 (ICAM-1). It was found that 293 cells transfected with the HIV-1MN env gene expressed the HIV-1 polyglycoprotein precursor, gp160, and the mature gp120-gp41 complex. When mixed with a CD4+ T cell line (CEM), the gp160-transfected cells mediated heterotypic cell fusion and formed multinucleate syncytia. Virus-neutralizing monoclonal antibodies to the V2 and V3 domains of gp120 were able to inhibit syncytium formation, as were monoclonal antibodies to CD4. When ICAM-1 was coexpressed with gp160, syncytium formation between the transfected kidney cells and uninfected CD$+ T cells was markedly enhanced. Inhibitors of HIV-1 infectivity (e.g., monoclonal antibodies to gp120, recombinant soluble CD4) were able to prevent syncytium formation; however, the syncytium-blocking activity of these agents was significantly attenuated in cultures in which ICAM-1 was cotransfected with gp160. These results confirm that leukocyte adhesion molecules can promote gp160-mediated syncytium formation and demonstrate, for the first time, that adhesive interactions mediated by ICAM-1 and its contrareceptor, LFA-1, attenuate the syncytium-inhibiting activity of virus-neutralizing monoclonal antibodies and soluble CD4. These findings suggest that the type and magnitude of leukocyte adhesion molecules expressed on cells may be a significant variable in in vitro HIV-1 neutralization assays.

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

A method for the separation and determination of neutral compounds in postmortem tissues.

A convenient method for the extraction of neutral drugs in necrotic tissues is described. The procedure includes the use of hot dilute acid for isolating neutrals from tissue extract residues and of internal standard to facilitate quantitative and recovery measurement of the drugs by gas chromatography. The recovery of seven neutral compounds from liver ranged from 47.0% to 89.3%. The limit of detection of these drugs were 0.5 to 1.0 mg/kg.

Phencyclidine-related deaths in Los Angeles County, 1976.

Concentrations of phencyclidine in blood and liver are presented in five fatal cases occurring in Los Angeles County in 1976. Eleven other deaths in which phencyclidine contributed to death are described; acute psychotic reactions were observed in some of these cases. Two cases involved the drowning of individuals whose swimming capabilities may have been diminished from the effects of PCP. One case is presented in which a 20-year-old male took a massive overdose of phencyclidine for suicidal purposes.

Persistence of morphine in blood in association with hepatic necrosis and renal tubular necrosis.

A case is reported in which a 26-year-old male survived in a hospital for 38 h after an intravenous heroin injection. Postmortem analysis showed 0.2 mg/100 ml morphine in the blood and 0.05 mg/100 g morphine in the liver. The persistence of morphine in the blood was attributed to the inability of damaged liver cells to take up the morphine and of the hypoxic kidney to excrete the compound.

Morphine in lymph nodes of heroin users.

During the autopsy of heroin users, the most consistent morphologic finding is the enlargement of hepatic lymph nodes. Nodes from seven heroin addicts were analyzed for morphine and a concentration range of 0.02 to 0.87 mg/100 g was found. Morphine was detected in all of the nodes examined and the concentration was generally higher than that in the blood.

Solid-phase extraction, identification, and quantitation of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid.

A procedure has been developed to extract and recover minute amounts of delta-9-carboxytetrahydrocannabinol (THC-COOH) from urine. A new non-isotopic internal standard is introduced to permit a chromatographic assay of the metabolite. The method affords a 91% recovery of 20 ng/mL of the THC-COOH acid from spiked urine with the assurance of a 3.8% coefficient of variation.

Analysis of dihydrocodeine in urine using Sep-Pak C18 cartridges for sample cleanup.

A rapid and precise method for the isolation and identification of dihydrocodeine from urine is reported. The narcotic is isolated from urine using Sep-Pak C18 cartridges for cleanup, requiring less than 30 min for preparation. Identification is performed by gas chromatography/mass spectrometry.

Antemortem conversion of codeine to morphine in man.

Forty-five drug overdose cases involving codeine were investigated. Concentrations of codeine and morphine were determined in blood, bile, liver, kidney, and urine. Ratios of codeine to morphine values for each of these specimens were compared and evidence was developed that codeine was metabolized partially into morphine in the antemortem stage. Morphine concentration was less than that of codeine in blood, liver, kidney, and urine. However, bile analyses showed that the amount of morphine exceeded that of codeine, suggesting a more active demethylation activity in the hepatic system than in the blood and other tissues studied. Controlled-in-vitro studies showed that no codeine demethylation occurred in postmortem tissues during cold storage for a period as long as 30 days.

Hydromorphone detected in bile following hydrocodone ingestion.

Two similar cases are reported here in which Tussionex, a preparation containing hydrocodone and phenyltoloxamine, caused or contributed to death. Toxicological analyses revealed a high concentration ratio of hydromorphone to hydrocodone in the bile in both cases. It is postulated that the finding of hydromorphone is due to the metabolism of hydrocodone.

Toxicologic assessments in acute heroin fatalities.

The recent improvements in analytic methods enable routine morphine detection in blood in microgram or nanogram quantities. It is now possible to assess acute death from heroin use by toxicologic analyses. A review of available data indicates a rapid distribution of morphine even in sudden fatalities, to the various organs of the body. Blood morphine levels in most acute heroin-involved deaths range from 0.1 to 1.0 microgram/ml, while morphine concentration in liver ranges from 0.1 to 10.0 microgram/gm. In rapid death, the blood to liver ratio is approximately 1:5. Blood and liver appear to be the specimens of choice in determining fatality due to heroin; however a distribution study that included other tissues such as brain, bile, and urine would afford a more meaningful evaluation in forensic investigation. The correlation of the survival periods of decedents to concentrations of morphine in tissues is discussed. Since morphine concentration decreases precipitously in antemortem blood immediately after administration of heroin, the assurance of detecting and determining morphine is greater in blood specimens from decedents who died within 1 hr after drug taking than from those who survived for a longer period. Blood levels of morphine also appear to be regulated by dosage. The role of ethanol and other drugs, including excipients in illicit heroin preparations, in acute narcotism is still poorly understood. Morphine is produced in the antemortem metabolism of codeine. A close evaluation of toxicologic data is necessary to determine whether the morphine detected, if a metabolite, is a conversion product of codeine, heroin, or both. In any event, the cause of death involving heroin is determined only after information from history and pathology, as well as toxicology, are carefully correlated.

The carboxy terminus of human immunodeficiency virus type 1 gp160 limits its proteolytic processing and transport in transfected cell lines.

Mutagenesis of the transmembrane domain and cytoplasmic tail of human immunodeficiency virus type 1 envelope glycoprotein gp160 revealed that its intracellular transport and processing in transfected cell lines were modulated by a functional domain included in the carboxy-terminal sequence consisting of residues 751 to 856.

Kinetics of heroin deacetylation in aqueous alkaline solution and in human serum and whole blood.

A kinetic study of heroin hydrolysis in alkaline aqueous solution at room temperature was conducted by a gas chromatographic method to measure the consecutive reactions of diacetylmorphine to monoacetylmorphine and of monoacetylmorphine to morphine. A first-order reaction was observed in both instances, and the rate for the deacetylation of heroin was greater than that of monoacetylmorphine. The rates of in vitro hydrolysis of diacetylmorphine in human whole blood and in serum were compared by the same method. Diacetylmorphine was hydrolyzed twice as rapidly in blood as in serum. While morphine was an end product of hydrolysis in the blood, it was absent in the serum.

Intracellular transport and secretion of hepatitis B surface antigen in mammalian cells.

The oligosaccharide processing and secretion of hepatitis B surface antigen (HBsAg) was studied in Chinese hamster ovary cells stably transfected with the gene coding HBsAg. HBsAg was secreted from cells with a relatively long half time (ca. 5 h). This appeared to be a characteristic of HBsAg itself, since HBsAg-producing cells infected with vesicular stomatitis virus transported the viral envelope glycoprotein to the cell surface with normal kinetics (half time of ca. 30 min). The secreted HBsAg was comprised of both the unglycosylated (P20) and the glycosylated (G25) polypeptides, characteristic of HBsAg isolated from human serum or secreted from other cell lines (C. W. Crowley, C.-C. Liu, and A. D. Levinson, Mol. Cell. Biol. 3:44-55, 1983; M. F. Dubois, C. Pourcel, S. Rousset, C. Chang, and P. Tiollais, Proc. Natl. Acad. Sci. U.S.A. 77:4549-4553, 1980; C.-C. Liu, D. Yansura, and A. D. Levinson, DNA, 1:213-221, 1982; G. M. Macnab, J. J. Alexander, G. Lecatsas, E. M. Bey, and J. M. Urbanocvicz, Br. J. Cancer, 24:509-515, 1976; A. M. Moriarity, B. H. Hoyer, J. W.-K. Shih, J. L. Gerin, and D. H. Hamer, Proc. Natl. Acad. Sci. U.S.A. 78:2606-2610, 1981; D. L. Peterson, J. Biol. Chem., 256:6975-6983, 1981). The glycosylated polypeptide (GP25) contained complex oligosaccharide chains. Cell-associated HBsAg also was comprised of both an unglycosylated and a glycosylated polypeptide; however, the glycosylated form (GP23) contained only high-mannose oligosaccharide chains. No oligosaccharide processing of the high-mannose chains could be detected within the cells. Thus, most of the time before secretion of HBsAg from cells must have been spent in a pre-Golgi or early Golgi compartment. Glycosylation was inhibited completely by tunicamycin, although unglycosylated particles were still secreted from cells and were antigenic. The secretion and oligosaccharide processing of HBsAg were inhibited with high concentrations of monensin, but at lower concentrations of monensin HBsAg was still secreted, although only half of the oligosaccharide chains were processed to the complex form.

Intracellular assembly and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum.

Hepatitis B surface antigen (HBsAg) particles are secreted by Chinese hamster ovary cells that are stably transfected with the S gene of hepatitis B virus. The assembly of HBsAg into cylindrical and spherical particles occurred intracellularly within the endoplasmic reticulum. HBsAg particles accumulated within large dilated areas of the endoplasmic reticulum and remained within these structures for most of the time prior to secretion from the cells. Once the particles were formed, the HBsAg polypeptides did not appear to become associated with subsequent intracellular organelle membranes or the plasma membrane. HBsAg within the dilated structures did not bind wheat germ agglutinin, indicating that its oligosaccharide chains had not yet been processed to the complex form (containing terminal sialic acid-N-acetylglucosamine residues). The oligosaccharide chains of HBsAg are processed to the complex form and can be detected on the HBsAg after secretion, but this event was not detected within cells. In addition, HBsAg was not observed on the cell surface by indirect immunofluorescence or immunoprecipitation, although immunoelectron microscopy revealed some staining at or near the cell surface. These results suggested that HBsAg was either secreted from cells without being incorporated into the plasma membrane, or that the levels of HBsAg in the plasma membrane were below the limits of detection.