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R H Neubauer

Publications and source records attributed to R H Neubauer.

At least 19 recordsLinked to original sources
PubMed1991

A sensitive method for the detection of beta-galactosidase in transfected mammalian cells.

A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.

Chlorophenols↗
PubMed1989

Stable indicator cell lines exhibiting HIV-1 tat function.

Since HIV tat function is essential for the HIV infectious cycle, it represents an important possible target of therapeutic intervention for HIV infection. Stable human cell lines were derived that express high levels of beta-galactosidase under the combined control of the transacting HIV-1 tat gene product and the cis-acting HIV-1 LTR. The tat gene product induces LTR-linked gene expression approximately 1000-fold in this system. The high level of expression of beta-galactosidase under HIV tat and LTR control in stable cell lines allows rapid spectrophotometric quantitation of beta-galactosidase enzymatic activity from fewer than 5000 cells seeded in a microtiter plate well. Such cell lines provide a virus-free system for the high-capacity screening of compounds for the ability to interfere with HIV tat-mediated transactivation of gene expression.

Cell Line↗
PubMed1988

Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity.

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

Animals↗
PubMed1987

Semiautomated colorimetric assay for in vitro screening of anticancer compounds.

An in vitro tetrazolium dye (MTT) reduction technique was modified and evaluated for use in the large-scale screening of anticancer compounds by examining the activity of ten clinically used drugs against 16 different human and murine cell populations. Cell populations included colon and mammary adenocarcinomas, melanomas, leukemias, and freshly isolated normal cells. Cell lines were grown in microtiter plates for 18-20 hours prior to a 72-hour continuous exposure to the drugs. Cultures were initiated at cell densities which maximized both the difference in dye reduction and the number of cell doublings between the beginning and end of the drug exposure period. Drug potency, expressed as the 50% inhibitory concentration (IC50), was comparable whether the effect on cell doublings or dye reduction was determined. There was good agreement between this method and the more labor-intensive, conventional method of counting trypan blue dye-excluding cells in a hemacytometer. Implemented as a large-scale, high-capacity system, our adaptation of the MTT technique is a rapid, sensitive, reproducible first-line screening device for detecting anticancer compounds with cytostatic or cytocidal activity.

Antineoplastic Agents↗
PubMed1985

Cytotoxic activity of normal and Herpesvirus saimiri-transformed nonhuman primate cells.

Owl monkey mononuclear cells were separated from peripheral blood by centrifugation on Ficoll gradients, removal of adherent cells, and subsequent separation on discontinuous Percoll gradients. Lymphocytes recovered from the various fractions were tested for cytotoxic reactivity immediately after isolation. Low-density cells, enriched in large granular lymphocytes (LGL), demonstrated cytotoxic activity against the human natural killer-susceptible cell lines MOLT 4 and K562. In addition, IL-2-independent T-cell lines which had been obtained by immortalization with the primate herpesvirus Herpesvirus saimiri showed cytotoxicity, even after prolonged culture in vitro, similar to that demonstrated by fresh LGL. Cytotoxic activity of these lines was regulated by IL-2 in a fashion which appeared to be independent of the growth-promoting effects of this lymphokine. These results indicate a function for IL-2 beyond its role in supporting cellular proliferation. Cytotoxic activity could also be demonstrated in culture fluids from one of these cell lines (70N2). In addition, these results indicate the usefulness of immortalized cell lines (like 70N2) as a potential source for studies of the biochemical characterization and purification of supernatants containing cytotoxic factors.

Animals↗
PubMed1985

Stimulation of in vitro immunoglobulin production by interferon-alpha.

The effect of various natural and recombinant DNA-derived human interferon-alpha (IFN-alpha) on immunoglobulin (Ig) production by human B cells was investigated. The cell populations examined included peripheral blood mononuclear cells (PBMC) and highly purified B cell and helper T cell populations obtained by negative selection by using monoclonal antibodies and a fluorescence-activated cell sorter. In the presence of all forms of IFN-alpha tested, IgG and IgM production by PBMC increased twofold to fourfold. This increase was noted in the absence of pokeweed mitogen (PWM), was not affected by depletion of monocytes, required that IFN-alpha was present early in the culture period, and reached maximal levels around 500 U/ml IFN-alpha. Both IgG and IgM production were affected, but the magnitude of the IgM response was greater. The augmentation of Ig production was noted with the recombinant DNA-derived subtype, IFN-alpha F, two analogs, IFN-alpha Con1 and IFN-alpha Con2, as well as with buffy-coat-derived (leukocyte) IFN-alpha. The recombinant DNA-derived forms of IFN-alpha appeared to differ in their ability to augment Ig production. In the presence of PWM, IFN-alpha Con1 failed to increase Ig production by PBMC. In contrast to these results with PBMC, IFN-alpha Con1 increased the Ig production of purified B cells 10- to 20-fold in the presence of PWM. This increase reached maximal levels around 500 U/ml IFN-alpha Con1. Although purified B cells responded to IFN-alpha and PWM, maximal responses occurred in the presence of low numbers of helper T cells. Cell dilution experiments suggested that the effect observed with purified B cells was the result of the interaction of B cells with residual cells, e.g., helper T cells, remaining in the preparations.

Antibody Formation↗
PubMed1979

Biochemical identification of primate lymphoid cell-surface glycoproteins.

We have employed the galactose oxidase-tritiated sodium borohydride labelling method to examine the surface glycoproteins of cotton-topped marmoset and other primate cell lines either established from tumors or transformed in vitro by different lymphotropic herpesviruses. The labelled surface glycoproteins were separated on acrylamide gels in the presence of sodium dodecyl sulfate (SDS) and analyzed by fluorography. Our results indicate that (1) lymphocytes of the same class from different primate species are similar but can be distinguished; (2) T and B lymphocytes of the same species can be differentiated; (3) cotton-topped marmoset lymphocytes of the same class show marked similarities regardless of tumor or in vitro origin or virus used for transformation; (4) three cell lines established from different EBV-induced tumors of the same marmoset show essentially the same labelling pattern, supporting the hypothesis that they originated from a single clone.

Animals↗
PubMed1979

Antibody responses to Herpesvirus papio antigens in baboons with lymphoma.

An Epstein-Barr virus-related herpesvirus, termed Herpesvirus papio (HVP), was isolated from baboons (Papio hamadryas) at the Institute of Experimental Pathology and Therapy, Sukhumi, USSR, where there is a continuing outbreak of lymphoma. In the present study sera from diseased baboons and from age- and sex-matched control animals were examined for antibodies to HVP antigens. Results showed that animals with lymphoid disease had antibodies to HVP virus capsid, early, soluble, and nuclear antigens at higher frequencies and at higher titers than did control animals. Antibody titers were not age- or sex-related. No concordancy was detected for antibodies to soluble and nuclear antigens. The sera were also examined for antibodies to two other widely distributed viruses of hamadryas baboons, cytomegalovirus and foamy virus. The results of these studies did not indicate a disease-related role for either of these viruses.

Animals↗
PubMed1979

Establishment of a lymphoblastoid cell line and isolation of an Epstein-Barr-related virus of gorilla origin.

A B-lymphoid cell line was established from a normal gorilla. The cells contained Epstein-Barr virus-related antigens, and herpesvirus particles were demonstrated by electron microscopy. DNA-DNA reassociation kinétics revealed 30 to 40% hybridization to Epstein-Barr virus with 50 genomes per cell. Examination of the viral nuclear antigen with gorilla sera showed this to be a unique isolate termed Herpesvirus gorilla. H. gorilla transformed gibbon B-lymphocytes in vitro.

Animals↗
PubMed1979

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

We prepared mouse hybrid cell lines which produced antibodies against herpes simplex virus type 1 and 2 nucleocapsids. Cell lines 1D4 and 3E1, respectively, secreted immunoglobulin G1 herpes simplex virus type 1 and immunoglobulin G1 herpes simplex virus type 2 antibodies which immunoprecipitated proteins designated p40 and p45 from homologous nucleocapsid preparations but precipitated no proteins from heterologous preparations. In contrast, guinea pig antisera prepared against either herpes simplex virus type 1 or 2 p40 precipitated p40 and p45 from both homologous and heterologous preparations. These findings suggest that p40 and p45 possess similar antigenic determinants and that the monoclonal antibodies that were tested reacted preferentially with the homologous determinants.

Antibodies, Viral↗
PubMed1979

Correlation between Epstein-Barr virus membrane antigen and three large cell surface glycoproteins.

A correlation between Epstein-Barr virus membrane antigen (MA) and three surface glycoproteins has been established on the basis of radio-immunoprecipitation and immunoabsorption experiments. For radio-immunoprecipitation, Epstein-Barr virus-infected cells were radiolabeled either with neuraminidase-galactose oxidase tritiated borohydride, a procedure highly specific for surface glycoproteins, or with a general tritiated amino acid mixture. Intact cells were incubated with MA(-) or MA(+) human sera, washed free of unbound immunoglobulins, and then lysed with Nonidet P-40. The antigen-antibody complexes were bound to protein A-Sepharose and after elution with sodium dodecyl sulfate were analyzed by acrylamide gel electrophoresis in sodium dodecyl sulfate. MA(+) sera specifically precipitated three glycoproteins with molecular weights of 236,000, 212,000, and 141,000 from B95-8 cells induced with 12-O-tetradecanoylphorbal-13-acetate (TPA) and from Raji cells superinfected with P3HR-1 virus. These glycoproteins were not detected on Epstein-Barr virus-negative Ramos cells treated with TPA or on B95-8 cells treated simultaneously with TPA and phosphonoacetic acid. Soybean lectin-Sepharose bound all three glycoproteins, and lectin-Sepharose-bound glycoproteins from TPA-induced P95-8 cells absorbed MA-specific antibody from MA(+) human sera. The data strongly suggest that either all three glycoproteins have MA determinants or they are part of a complex in which one or more of the components constitute the reactive antigen.

Animals↗
PubMed1978

Further characterization of a herpesvirus-positive orang-utan cell line and comparative aspects of in vitro transformation with lymphotropic old world primate herpesviruses.

An orang-utan (Pongo pygmaeus) suspension line, CP81, was shown to lack myeloid markers of lysozyme activity an d phagocytosis but to be positive for lymphocytic N-alkaline phosphatase activity, and to release a B-cell-tropic herpesvirus. This herpesvirus, termed Herpesvirus pongo, had 30--40% DNA homology with EBV and was present at 2-3 genome copies per CP-81 cell. Gibbon lymphocytes transformed by H. pongo, Epstein-Barr virus (EBV), and H. papio (of baboon, Papio hamadryas, origin) were found to be virus antigen-positive B cells. Gibbon lymphocytes transformed by H. pongo and EBV and transplanted to nude mice by the intracranial (IC) route (had a 75% and a 45% success rate, respectively), while transplants of similar cells transformed by H. papio were only 10% successful. None of these lines transplanted subcutaneously (SC) nor manifested a high degree of colony formation in 0.33% agarose (less than or equal to 0.5%), Gibbon lymphocytes transformed by H. pongo were hypodiploid while those transformed by EBV or H. papio were diploid. CP-81 cells themselves could be transplanted both IC (100%) and SC (70%) and showed a relatively high degree of colony formation in agarose (6.4-7.6%). B95-8 cells (marmoset, Saguinus oedipus-EBV) could be transplanted IC (66%) but not SC and had a low but significant ability to grow in agarose (1.6%). 594S (baboon, P. hamadryas-H. papio) cells could be transplanted IC (25%) but not SC, and grew to very low levels in agarose (0.1%).

Alkaline Phosphatase↗
PubMed1978

Selective stimulation and differentiation of early antigens in lymphoblastoid cell lines producing Epstein-Barr-like viruses.

Comparisons of early antigens (EA) of EBV-related lymphotropic herpes virus of Old World primates have proved difficult to accomplish because antigen levels are low in infected cells and the ratio of virus capsid antigen to EA is generally high. To overcome these difficulties, we have developed a procedure which combines antigen stimulation with inhibition of late virus functions and results in the selective stimulation of EA. Using this procedure, we have examined the EA of EBV, herpesvirus papio, h. pongo, and h. pan. The results show that the EA of these viruses are composed of both R (restricted) and D (diffuse) components and that the EA, while related, are distinguishable.

Animals↗
PubMed1978

Characteristics of cell lines established from Epstein-Barr virus induced marmoset tumors.

The inoculation of a cotton-topped marmoset with B95-8 strain of EBV resulted in the induction of a multifocal lymphoma and lymphoblastoid cell lines were established from liver, spleen and mesenteric lymph node tumors. The cell lines were remarkably similar to each other with respect to the presence of EBV and its expression, the surface properties of the cells and their stability, and the functional products of the cells. Karyotypic examination of the cell lines revealed the common loss of a single chromosome. The slight differences noted in karyotypes suggest some divergence from a tumor stem cell and imply a clonal origin. Thus, EBV-induced lymphomas in cotton-topped marmosets resemble Burkitt's lymphoma in man.

Animals↗
PubMed1978

Spontaneous esophageal carcinoma and epithelial cell line of an adult rhesus monkey.

A continuous epithelial cell line, 816A, was established from a lymph node of an adult rhesus monkey with metastatic esophageal carcinoma. These cells are characterized by the presence of desmosomes and a markedly heteroploid karyotype. At a relatively early culture age, electron microscopy showed both budding and extracellular type C virus. Antigen reactive with antisera to Mason-Pfizer monkey virus was observed by complement-fixation. The level of this antigen decreased with increased culture age. To our knowledge, the 816A cells represent the only established simian or human cell line of esophageal carcinoma origin.

Animals↗
PubMed1978

Augmentation of the in vitro mitogenic response of owl monkey peripheral blood lymphocytes by levamisole and loss of this effect with the development of herpesvirus saimiri-induced lymphoma.

Levamisole (LMS) has been shown to be capable of enhancing the proliferative response of normal owl monkey peripheral blood lymphocytes (PBL) to PHA, and, to a lesser degree, of increasing the level of spontaneous DNA synthesis. With the development of herpesvirus saimiri-induced lymphoma, these stimulatory responses were lost. LMS was not capable of stimulating tumour cells to normal functions, or of reversing the disease-induced suppressed functions on normal cells.

Animals↗
PubMed1978

In vitro lymphocyte transformation by Epstein-Barr virus (EBV)-like viruses isolated from Old-World non-human primates.

EBV-like viruses and lymphoid cell lines have been isolated from baboons and an orangutan. The cell lines have properties of B- or undifferentiated lymphocytes and have antigens and DNA related to those of EBV. The baboon virus has a broad in vitro transformation host range among lymphocytes of Old-World simian species whereas the orangutan isolate has a narrower host range. Baboon and orangutan viruses as well as EBV have shown transforming activity for gibbon lymphocytes. Baboon virus is infectious for rhesus monkeys and baboons but has not induced neoplastic disease in these species.

Animals↗