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Biomedical subjects

R Eberle

Publications and source records attributed to R Eberle.

At least 73 records · Page 4Linked to original sources
PubMed1986

ELISA for detection of group-common and virus-specific antibodies in human and simian sera induced by herpes simplex and related simian viruses.

A rapid (3.5 h) enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to herpes simplex virus type 1 (HSV-1) and to two antigenically related monkey viruses, simian agent 8 (SA8) and Herpesvirus simiae (B virus). Crude preparations of detergent solubilized infected cells and similarly treated control mock-infected cells served as antigens for coating wells in microplates. Biotinylated protein A and avidin-conjugated alkaline phosphatase were used to detect antibodies in sera from different species (humans, monkeys and rabbits). Three prototype assays are described with three degrees of specificity. Common or specific determinants on the viral antigens could be assayed in simple competition tests using similar antigen preparations to those coating the wells. The specific assays permitted rapid differential serodiagnosis of antibodies to human and simian herpesviruses.

Animals↗
PubMed1986

Rapid identification of herpesvirus simiae (B virus) DNA from clinical isolates in nonhuman primate colonies.

A rapid, simple technique, based on restriction endonuclease analysis of radioactively labeled infected cell DNA, is described for identification of Herpesvirus simiae (B virus) infection in clinical isolates from nonhuman primates. Isolates can be screened within 2-3 days from the time of collection of a specimen from a suspect lesion to final viral identification. Isolates were obtained from eight animals with suspected B virus infections. The results indicated the presence of B virus in each of the eight animals, each isolate unique from the others, but with the dominant prototypic pattern of the laboratory strain of B virus (E2490) and not HSV-1 (KOS), HSV-2 (186), or SA8 (3264).

Animals↗
PubMed1985

The immune response to herpes simplex virus: comparison of the specificity and relative titers of serum antibodies directed against viral polypeptides following primary herpes simplex virus type 1 infections.

Employing an immunoblotting technique, the polypeptide specificity and relative titers of anti-HSV IgG reactive with denaturation-resistant epitopes on HSV proteins were determined in patients experiencing primary HSV-1 infections at various anatomical sites. Early sera from previously seronegative patients with primary HSV-1 infections were found to have comparatively low levels of antibody directed against the major viral glycoprotein antigens (gB, gC, and gD) relative to titers present in sera of individuals with long-standing, latent orofacial HSV-1 infections. Patients with primary infections did however have high titers of antibody directed against a series of low molecular weight HSV polypeptide antigens. These antigens were found to be antigenically related to a structural component of virion nucleocapsids. At later times postinfection, titers of antibodies directed against other viral polypeptides including the major glycoproteins increased to levels more closely approximating those observed in latently infected individuals. These results indicate that the anti-HSV IgG detected by immunoblot analysis which appears earliest following primary infection is not directed against the known major infected cell or virion glycoprotein surface antigens but rather against an internal capsid protein of HSV.

Antibodies, Monoclonal↗
PubMed1985

Discrimination between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide.

Circulating soluble fibrin, observed in the blood of patients with ongoing intravascular coagulation, is generated from the plasma protein fibrinogen by the limited proteolytic action of thrombin. We report the production of a monoclonal antibody that discriminates between fibrin and fibrinogen in blood. The synthetic hexapeptide Gly-Pro-Arg-Val-Val-Glu, representing the amino terminus of the alpha chain of human fibrin, was used as immunogen. This hexapeptide is located within the A alpha chain of fibrinogen but becomes the amino terminus of the fibrin alpha chain, after fibrinopeptide A is removed by the action of thrombin, and thus becomes accessible for antibody binding. The monoclonal antibody we have prepared can discriminate between fibrin and fibrinogen and thus can be used in assay systems to quantitate soluble fibrin or, potentially, to image fibrin-rich thrombi.

Amino Acid Sequence↗
PubMed1984

Modulation of tumour cell colony growth in soft agar by oxygen and its mechanism.

A simple technique for maintaining low oxygen concentrations (0.1-20%) is described. These conditions were then used to study the effect of oxygen on colony growth of neoplastic cells in soft-agar. Physiologically low oxygen concentrations (0.1-10%) compared to 20% O2 were found to enhance plating efficiency and colony size of tumour cells. The optimal oxygen concentration for plating efficiency varied with tumour studied and may be as low as 0.1%. Having established that tumour cell colonies will grow better at 0.1-10% O2 compared to 20% O2, the mechanism by which this enhancement occurs was investigated. Observations on the effect of free radical scavengers and superoxide dismutase on plating efficiency of Ehrlich's ascites tumour cells suggests that this phenomenon occurs through oxygen toxicity mediated by superoxide anion.

Agar↗
PubMed1984

Polypeptide specificity of the early antibody response following primary and recurrent genital herpes simplex virus type 2 infections.

Relative IgG titres to specific viral proteins in acute and convalescent sera from patients with primary and recurrent genital herpes simplex virus type 2 (HSV-2) infections were determined using an immunoblot assay. Patients with recurrent genital infections generally had high titres of antibody reactive with two of the three major viral glycoproteins (gD and gG) as well as with several viral capsid proteins in both acute and convalescent sera. In contrast, patients experiencing primary genital HSV-2 infections exhibited marked differences in the polypeptide reactivity profiles of acute and convalescent sera. Of particular interest was the observation that the earliest and strongest antibody response was directed not against the viral glycoproteins but rather to an internal capsid protein(s) of HSV-2.

Antibody Formation↗
PubMed1984

Replication of simian herpesvirus SA8 and identification of viral polypeptides in infected cells.

The replication of the simian herpesvirus SA8 in Vero cells was examined. The time course of replication of the simian herpesvirus SA8 was found to be similar to that of the herpes simplex viruses. Infectious progeny virions were first detectable by 6 h postinfection and were readily released into the extracellular fluids beginning at 9 h postinfection. All cell lines tested, with the exception of Madin-Darby canine kidney cells, were permissive for SA8. Analysis of SA8-infected cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed over 40 infected cell polypeptides ranging in molecular weight from 158,000 to less than 10,000. Of these proteins, 23 were present in virions. Three classes of infected cell polypeptides could be identified based on the kinetics of their synthesis. Post-translational processing of several SA8-induced proteins was also observed in pulse-chase experiments. Six distinct SA8-specific glycoproteins ranging from 118,000 to 19,500 daltons were also identified in infected cells. Of these glycoproteins, five were present in virions.

Animals↗
PubMed1983

Relative titers of antibodies to individual polypeptide antigens of herpes simplex virus type 1 in human sera.

Human immune sera were analyzed by an immunoblot procedure in order to identify which polypeptides of herpes simplex virus type 1 were recognized and to determine the relative concentrations of antibodies to individual viral antigens. The strongest reactivity in all immune sera examined was directed against the three major herpes simplex virus-specific glycoprotein antigens (gAB, gC, and gD) and a series of uncharacterized low-molecular-weight polypeptides. Titers of antibody to the gC and gD antigens varied among individuals and were often considerably lower than titers of antibody to the gAB antigen. Immune sera also recognized a number of nonglycosylated polypeptides, both structural and nonstructural. No consistent differences were noted between the recognition of particular viral polypeptides by sera from individuals with frequent, recurrent labial lesions and that by sera from individuals with infrequent, recurrent lesions. However, the ratio between the titer of antibody to the gAB antigen and that to the gC antigen did appear to be somewhat lower in sera from persons with a high recurrence rate.

Antibodies, Viral↗
PubMed1982

Multimeric forms of herpes simplex virus type 2 glycoproteins.

Herpes simplex virus type 2-specific glycoproteins present in detergent extracts of infected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under conditions designed to permit detection of multimeric forms of these glycoproteins. Two high-molecular-weight glycosylated species were detected when samples were disrupted at lower temperatures or in the absence of any reducing agents. One multimer having an apparent molecular weight of 275,000 was identified as a multimer of the gA or gB glycoprotein or both. The second glycoprotein, having a molecular weight of approximately 230,000, was identified as a multimeric form of the gC glycoprotein. These data indicate that the gC as well as the gA and gB glycoproteins of herpes simplex virus type 2 may exist in a multimeric form.

Disulfides↗
PubMed1982

Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells.

Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host.

Animals↗
PubMed1981

Assay of type-specific and type-common antibodies to herpes simplex virus types 1 and 2 in human sera.

A reliable and reproducible method for determining specific reactivity to herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in human sera has been developed. Human sera were used to immunoprecipitate HSV-specific glycoprotein antigens from both HSV-1- and HSV-2-infected cell extracts. The viral glycoproteins precipitated from these extracts were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect specific reactivity of the sera with distinct type-specific antigens of HSV-1, HSV-2, or both as well as with type-common glycoprotein antigens. By examining a large number of human sera, this method was found to be more reliable than the standard microneutralization test in discriminating between single-positive (positive for HSV-1 or HSV-2) and double-positive (positive for HSV-1 and HSV-2) sera.

Antibodies, Viral↗
PubMed1981

Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL.

Antigens, Surface↗
PubMed1980

Cell-mediated immunity to herpes simplex virus: specificity of cytotoxic T cells.

This communication deals with the question of which of the viral antigens constitutes the targets for cytotoxic T lymphocytes (CTL) generated against herpes simplex virus type 1 (HSV-1). The approach used was, first, to compare cytotoxicity of CTL against target cells infected with virus in the presence of tunicamycin and 2-deoxy-D-glucose, which are known to inhibit glycoprotein synthesis, and second, to compare cytotoxicity of CTL against target cells infected with wild-type HSV-1 with that against target cells infected with a temperature-sensitive mutant of HSV-1 which, at the nonpermissive temperature, exhibits diminished glycoprotein synthesis. The results show that glycoprotein expression is required for the demonstration of cytotoxic activity of CTL. The level of cytotoxicity against the temperature-sensitive HSV-1 target at the nonpermissive temperature was reduced and correlated with the level of expression of the major envelope glycoprotein region (VP123; molecular weight = 123,000) at the target cell surface as measured serologically by antibody binding studies. The results were interpreted to indicate that HSV-1-induced glycoproteins are the target antigens for anti-HSV CTL and that the principal viral antigens recognized by the CTL may be glycoproteins of the VP123 region.

Animals↗
PubMed1980

Herpes simplex virus glycoproteins: isolation of mutants resistant to immune cytolysis.

Immune cytolysis mediated by antibody and complement is directed against components of the major herpes simplex virus (HSV) glycoprotein complex (molecular weight, 115,000 to 130,000), comprised of gA, gB, and gC, and against glycoprotein gD-all present on the surfaces of infected cells. Tests with a temperature-sensitive (ts) mutant of HSV-1 (tsA1) defective in glycoprotein synthesis at the nonpermissive temperature (39 degrees C) demonstrated that over 90% of mutant-infected cells maintained at 39 degrees C and treated with antibody and complement were not lysed, presumably due to the absence of viral glycoproteins on the surface of infected cells at this temperature. Furthermore, a small number of tsA1-infected cells could be detected among a large excess of wild-type virus-infected cells by virtue of their failure to be lysed at 39 degrees C by antibody and complement. Making use of the involvement of viral glycoproteins in immune cytolysis and the ability of cells infected with glycoprotein-defective mutants to escape cytolysis, we sought mutants defective in the expression of individual viral glycoproteins. For this purpose, antisera directed against the VP123 complex and against the gC and combined gA and gB glycoprotein subcomponents of this complex were first tested for their ability to lyse wild-type virus-infected cells in the presence of complement. Wild-type virus-infected cells were lysed after treatment with each of the three antisera, demonstrating that the gC glycoprotein and the combined gA and gB glycoproteins can act as targets in the immune cytolysis reaction. Next, these antisera were used to select for mutants which were resistant to immune cytolysis. Cells infected with wild-type virus which had been mutagenized with 2-aminopurine and incubated at 39 degrees C were treated with one of the three types of antisera (anti-VP123 complex, anti-gC, or anti-gAgB) and lysed by the addition of complement. Cells which survived immune cytolysis were plated, and virus in the resulting plaques was isolated. Plaque isolates were tested for temperature sensitivity of growth and altered cytopathic effects in cell culture at 34 degrees C (the permissive temperature) and 39 degrees C. A total of 73 mutants was isolated in this manner. Selection with glycoprotein-specific antisera resulted in a 2- to 16-fold enrichment for mutants compared with "mock" -selected mutants using normal rabbit serum. Phenotypically, 24 mutants were temperature sensitive for growth, 27 were partially temperature sensitive, and 22 were not temperature sensitive but exhibited markedly altered cytopathic effects at both permissive and nonpermissive temperatures. Nine mutants of each phenotype (temperature sensitive, partially temperature sensitive, and non-temperature sensitive) were selected at random for confirmatory immune cytolysis tests with the antisera used in their selection. Cells infected with eight of the nine mutants were shown to be significantly more resistant to immune cytolysis at the nonpermissive temperature than were the mock-selected mutants or the wild-type virus from which they were derived.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1980

Preparation and characterization of specific antisera to individual glycoprotein antigens comprising the major glycoprotein region of herpes simplex virus type 1.

The major glycoprotein complex (VP123) of herpes simplex virus type 1 resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was purified and further fractionated into two major and two minor components by chromatography of the isolated VP123 region on SDS-hydroxylapatite columns. The two major components (gC and gA/gB) were purified free of other polypeptides and used to prepare specific antisera to these glycoproteins. Radioimmune precipitation demonstrated that these antisera were specific for the antigens used in their production. These two antisera as well as an anti-VP123 serum were further characterized by immunoprecipitation, neutralization, and membrane immunofluorescence techniques. Results indicate that both of the major glycoprotein antigens are expressed on the surface of virions as well as on the surface of infected cells.

Animals↗