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Biomedical subjects

H Rabin

Publications and source records attributed to H Rabin.

At least 19 recordsLinked to original sources
PubMed1991

Structure, secretion, and bacterial specificity of an endogenous lectin from cystic fibrosis lung.

Endogenous heparin-binding lectin purified from postmortem lung samples of two cystic fibrosis (CF) patients was compared to lectin derived from normal tissue with respect to structure, carbohydrate specificity, interaction with alginate derived from CF isolates of Pseudomonas aeruginosa, and secretion within the lung. Lectin was purified from extracts of lung tissue by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. Lectin purified from either CF lung or control tissue ran as two peptides of approximately 16,000 and 13,000 molecular weight on electrophoresis in sodium dodecyl sulfate. The lectins displayed similar carbohydrate specificity and interacted in much the same way with bacterial alginate. An increase in lectin secretion was seen in CF lungs affecting the bronchial epithelial cells and the mucosal glands. The data suggest that the major changes seen in endogenous heparin-binding lectin in CF are related to the quantity and distribution of lectin secretion.

Adhesins, Bacterial↗
PubMed1988

Distribution and immunochemical characterization of a keratin-like antigen in epithelial tumors using mouse and human monoclonal antibodies.

Using conventional murine hybridoma technology, we have produced a monoclonal antibody (MAb), 89E5, which recognizes two keratin-like polypeptides (Mr 53,000 and 45,000), which are preferentially expressed by epithelial tumors. In addition to detection of tumor cells by immunohistochemistry, MAb 89E5 was able to localize to tumor xenografts in nude mice after iodination of its F(ab')2 fragments. To develop potentially less immunogenic antibodies to antigens defined by MAb 89E5, studies were performed to produce a human counterpart to the mouse MAb. The mouse 89E5 MAb was used to purify the 89E5 polypeptides from tumor cell lines. The partially purified 89E5 antigen was then used to sensitize human splenic lymphocytes in vitro. Immortalization of the sensitized cells by cell fusion resulted in a human IgM MAb, PA1, which showed the same reactivity pattern on a panel of cell lines as did the mouse MAb 89E5. Immunofluorescent studies showed that both 89E5 and PA1 had staining patterns on epithelial cells indicative of antibodies to cytokeratin. Furthermore, PA1 immunoprecipitated two polypeptides (Mr 53,000 and 45,000) which comigrated with the 89E5 polypeptides. Competitive binding assays showed that the PA1 MAb and 89E5 MAb recognized closely associated epitopes. As with the 89E5 MAb, PA1 was reactive with tumor tissues in immunohistochemical studies. These studies indicate that the PA1 MAb is a human counterpart of the mouse 89E5 MAb. Direct comparison of human MAb and mouse MAb against the same antigen could yield valuable information on the efficacy of using human MAb in vivo.

Animals↗
PubMed1988

Characterization of murine monoclonal antibodies to HIV-1 induced by synthetic peptides.

We have used short synthetic peptides, 12 and 13 amino acids in length, conjugated to carrier proteins to develop monoclonal antibodies (MAb) to the envelope glycoprotein of 120 (kD) (gp120) and the 3' open reading frame protein (3-orf) of the human immunodeficiency virus type 1 (HIV-1). The peptides employed were chosen because of their strong hydrophilicity and in the case of the gp120 peptide because it represents a highly conserved hydrophilic region in the envelope protein. The MAb developed displayed appropriate specificities with their respective peptides and reacted with appropriate HIV-1 components (i.e., a 120 kD glycoprotein and a 27 kD protein, respectively) as determined by Western blot analysis. In indirect immunofluorescence assays the MAb strongly stained syncytia present in cultures of HTLV-3B-infected H9 cells. The MAb to the envelope component reacted with the RF isolate of HIV-1, as well as with the 3B isolate in immunofluorescence.

Amino Acid Sequence↗
PubMed1987

Tissue distribution, immunochemical characterization, and biosynthesis of 47D10, a tumor-associated surface glycoprotein.

This report describes a new monoclonal antibody (MAb) designated 47D10 which was produced by immunizing mice against a human lung adenocarcinoma line, A549. The MAb 47D10 reacts with a surface antigen found in 95% of adenocarcinomas of the pancreas as well as on high percentages of adenocarcinomas from colon, breast, lung, and bile duct. The antigen was not detected in normal pancreas, in pancreatitis, or in a variety of normal tissues with the exception of colon and mature granulocytes. Lymphocytes and erythrocytes were also negative. The binding of 47D10 to tumor cells was unaffected by treatment of cells with neuraminidase. Immunoprecipitation followed by polyacrylamide gel electrophoresis showed that 47D10 MAb recognized a group of glycoproteins ranging in molecular weight from 67,000-98,000 on A549 lung carcinoma cells. Pulse-chase labeling showed two precursor proteins with molecular weights of 69,000 and 67,000 which were processed to the larger polypeptides in 1.5 h. At least part of the carbohydrates associated with the 47D10 antigen was asparagine linked because the antigen was sensitive to endoglycosidases, and tunicamycin inhibited the biosynthesis of 47D10 antigen. The 47D10 antigen was expressed on the cell surface because it could be detected on live A549 cells by enzyme-linked immunosorbant assays as well as by immunofluorescent staining. Furthermore, 47D10 antigens on tumor cell lines and granulocytes were vectorially labeled with 125I. The antigen found on granulocytes showed a higher molecular weight of 150,000-180,000, which was digested by endoglycosidase F to polypeptides with molecular weights ranging from 23,000-27,000. In contrast, the degradation product of the A549 antigen was a Mr 39,000 polypeptide after treatment with endoglycosidase F. The immunochemical characteristics of 47D10 antigen suggest that it is distinct from other antigens associated with pancreatic tumors, such as carcinoembryonic antigen, 19-9, and Du-PAN-2. By virtue of its broad range of tumor cell reactivity and low activity on normal cells, the 47D10 MAb may represent an important immunological reagent for differential diagnosis, especially of pancreatic carcinoma.

Animals↗
PubMed1987

Nuclear DNA-binding proteins determined by the Epstein-Barr virus-related simian lymphotropic herpesviruses H. gorilla, H. pan, H. pongo and H. papio.

Nuclear DNA-binding proteins were extracted from lymphoblastoid cell lines transformed with Epstein-Barr virus (EBV) or with the related lymphotropic herpesviruses of gorilla (Herpesvirus gorilla), chimpanzee (H. pan), baboon (H. papio) or orang-utan (H. pongo). They were immunoblotted with the sera of all four simian species in comparison with EBV antibody-positive human sera. Eight nuclear proteins were identified, and were designated GONA-1 and GONA-2 for H. gorilla-determined nuclear antigens, PANA-1A, PANA-1B and PANA-2 for H. pan, PONA-2 for H. pongo and HUPNA-1 and HUPNA-2 for H. papio-determined nuclear antigens. One of two tested HUPNA-2-positive baboon sera and one PONA-2-positive orang-utan serum also reacted with EBNA-2 in EBV-transformed cells. A human serum that contained antibodies to all five EBNA proteins cross-reacted only with PANA-2 and PONA-2. Monospecific anti-peptide antibodies against EBNA-2, type A, also reacted with PONA-2, but not with the other simian nuclear antigens. The data provide evidence that EBV-like simian lymphotropic herpesviruses induce EBNA-like nuclear antigens and that EBNA-2 and some simian EBNA-related proteins contain an epitope that has been conserved during the evolution of the EBV family of viruses.

Animals↗
PubMed1986

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody.

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.

Antibodies, Neoplasm↗
PubMed1986

Cloned helper T lymphocytes exposed to interleukin 2 become unresponsive to antigen and concanavalin A but not to calcium ionophore and phorbol ester.

Two cloned murine helper T lymphocyte (HTL) lines were used to investigate the immunoregulatory properties of highly purified interleukin 2 (IL2). The clone, designated J6.19, secretes lymphokines, including IL2, and proliferates when stimulated with ovalbumin in the presence of I-Ak-bearing spleen cells, while the alloreactive clone, L2, secretes lymphokines, including IL2, and proliferates when stimulated with Mlsa,d-bearing spleen cells. When either clone was exposed to a high concentration of pure IL2 for 1 to 2 days in the absence of either antigen or spleen cells, the HTL became unresponsive to rechallenge with antigen. Unresponsiveness to antigen was indicated by an inability of HTL to proliferate or secrete usual amounts of IL2 or colony-stimulating factor. Within 5-7 days after exposure to IL2, the cloned HTL again responded to antigen. Thus, in addition to being a growth factor for HTL, IL2 can limit the magnitude of the HTL response to antigen. L2 or J6.19 cells could also be induced to secrete lymphokines by the lectin, concanavalin A (Con A) or by a combination of the calcium ionophore, A23187, and phorbol myristate acetate (PMA). After exposure of L2 or J6.19 cells to sufficient IL2 to induce unresponsiveness to antigen, cells were also unresponsive to Con A, as indicated by a reduction in the level of lymphokines secreted. In contrast, lymphokine levels stimulated by A23187/PMA were comparable to those produced by cells not exposed to IL2. The failure of antigen to stimulate lymphokine release and proliferation by HTL previously exposed to IL2 therefore may result from an inability of HTL to recognize antigen or to transduce effectively the antigen recognition signal. Several T cell surface molecules are known to be involved in antigen activation of HTL; these include the antigen receptor and the "associative recognition" structures L3T4 and LFA-1. We observed that L2 cells, rendered unresponsive to antigen by exposure to IL2, expressed normal levels of antigen receptor, as identified by the monoclonal antibody, KJ16-133.18. Furthermore, expression of L3T4 and LFA-1 was not decreased. Unresponsiveness to antigen induced by IL2 thus could not be correlated with decreases in the expression of antigen receptors, L3T4, or LFA-1. Unresponsive HTL may therefore be capable of recognizing antigen but the signal generated by antigen binding may be attenuated during its transduction, resulting in the failure of cloned HTL to proliferate or secrete lymphokines at the usual levels.

Animals↗
PubMed1986

A monoclonal antibody reactive with an activated ras protein expressing valine at position 12.

Activated ras transforming genes have been described in a variety of neoplasms and encode 21,000-Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown to contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.

Amino Acid Sequence↗
PubMed1986

Leukocyte migration inhibition detects cross-reacting antigens between cells transformed by Epstein-Barr virus (EBV) and EBV-like simian viruses.

The leukocyte migration inhibition (LMI) technique was used to measure the T cell-mediated immune response of Epstein-Barr-virus (EBV)-seropositive human donors to antigens associated with B cell lines of simian origin, transformed by simian EBV-like viruses, Herpesvirus papio (HVP), H. pan, H. gorilla and H. pongo. Extracts of cell lines carrying three of the four simian viruses (from gorilla, chimpanzee and orangutan) induced a positive LMI response, whereas lines carrying baboon-derived HVP were ineffective. None of the simian virus-transformed lines elicited an LMI reaction in human EBV-seronegative individuals. Leukocytes from patients with acute infectious mononucleosis (IM) failed to respond to any of the lines transformed by EBV or the simian EBV-like viruses. Such lines express the virally encoded nuclear antigen, but have only a low level of viral cycle-associated antigens. Extracts of the EBV-carrying human cell line P3HR-1 induced with 12-O-tetradecanoylphorbol-13-acetate to express high levels of early and virus capsid antigens (EA, VCA, respectively) however, elicited a strong response with leukocytes from patients with acute IM. During convalescence, IM patients became responsive to EBV, H. gorilla-, H. pan- and H. pongo-transformed lines, indicating that the LMI reaction induced by these simian virus-transformed lines was directed against the antigens expressed in immortalized cells rather than against antigens of the lytic cycle. It is highly probable that this reaction reflects a cross-recognition of the nuclear antigens associated with these four transforming viruses (excluding H. papio) at the level of human T cells.

Antibodies, Viral↗
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

The proliferation and function of human mononuclear leukocytes and natural killer cells in serum-free medium.

We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF.

Cell Division↗
PubMed1985

Modulation of interleukin 2 release from a primate lymphoid cell line in serum-free and serum-containing media.

The ability to grow a clone of the cell line, MLA144, which is a constitutive producer of interleukin 2 (IL-2), in serum-free medium permitted the study of the direct effect of various agents on cell growth and IL-2 production in a homogeneous population. Bovine serum albumin (BSA) at 4 mg/ml was optimal for cell growth and IL-2 production. Selenium at 10 ng/ml enhanced IL-2 production nearly twofold and lithium at 42 ng/ml also enhanced IL-2 production by nearly twofold. Neither compound at these levels altered cellular proliferation. Two other compounds, iron and zinc, known to be associated with cellular proliferation and/or immunoregulation did not alter IL-2 production. Catalase or horseradish peroxidase was able to substitute for BSA and maintain the long-term growth of the MLA144 clone with only a 30% decrease in the rate of cellular proliferation and a 50% decrease in IL-2 production compared to cells maintained in the serum-free formulation with BSA. Addition of 0.5 mg of BSA to the catalase serum-free formulation increased the production of IL-2 to 70% of that of cells cultured in the BSA-containing serum-free formulation. The catalase-containing serum-free formulation has the advantage of consisting of only three proteins, catalase, insulin, and transferrin, at a very low protein content. The catalase-containing serum-free medium also supported the long-term growth of a human T-cell line, HSB-2.

Animals↗
PubMed1985

Herpesvirus saimiri-induced lymphoblastoid rabbit cell line: growth characteristics, virus persistence, and oncogenic properties.

A nonproducer lymphoblastoid cell line (7710) containing the herpesvirus saimiri (HVS) genome was established from the HVS-positive spleen of a male, inbred New Zealand White rabbit (III/J strain) which had developed a well-differentiated lymphoma after inoculation of HVS and 12-O-tetradecanoylphorbol-13-acetate (TPA). Antibodies to HVS early and late antigens were detected in the serum of rabbit 7710 by indirect immunofluorescence and immunoprecipitation. The cell line was of T-cell origin, did not produce HVS, and could not be superinfected with HVS. However, HVS early antigens could be induced in the cells with n-butyric acid and TPA or TPA alone. On the other hand, late antigens were never observed, and infectious virus could not be rescued by cocultivation of 7710 cell with OMK cells. The 7710 cells were T-cell growth factor dependent, even after many in vitro passages. The 7710 cell line contained multiple copies of a nonintegrated, covalently closed circular HVS genome. As is characteristic of some other HVS-transformed nonproducer lymphoid cell lines, a large segment of unique light (L) DNA was missing in the persistent circular viral DNA present in 7710 cells. This deletion spanned at least 42.5 kilobases, corresponding to the segment between 12.3 and 50.7 map units of full-length, infectious virion L-DNA. The 7710 cells failed to induce tumors in athymic nude mice, but inbred rabbits inoculated with as few as 100 of these cells developed fatal lymphomas. Chromosomal analysis showed that tumors were due to the growth of donor cells. Cells recovered from one of the rabbits inoculated with 7710 cells also contained HVS DNA and, after in vitro culture, induced the same type of lymphoma when inoculated into two other III/J-strain rabbits. None of the previously described HVS-transformed cell lines have been able to induce tumors in either their host species or nude mice. Thus, our demonstration that the 7710 cell line is readily transplantable in syngeneic rabbits represents the first available model which allows analysis of many biological and molecular aspects of the in vivo oncogenicity of HVS.

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↗
PubMed1985

In vitro immunization of human lymphocytes. I. Production of human monoclonal antibodies against bombesin and tetanus toxoid.

A procedure for in vitro sensitization of human lymphocytes against bombesin conjugated to tetanus toxoid (BTT) is described. Bombesin is a tetradecapeptide associated with small cell lung carcinoma. We found that antibody responses against bombesin as well as tetanus toxoid could be generated in vitro by culturing nylon-separated human splenic lymphocytes for 6 days with lipopolysaccharide, phytohemagglutinin-activated lymphocyte supernatants, human AB serum, and bombesin conjugated to tetanus toxoid. Cells sensitized by this procedure were fused to murine myeloma cells, NS-1. The specificities of resulting hybrids were analyzed by enzyme-linked immunoassays and competitive inhibition experiments. Hybrids secreting anti-bombesin (IgM) or anti-tetanus toxoid (IgM or IgG) were obtained. The ratio of IgG to IgM antibodies against tetanus toxoid could be increased by using antigen coupled to Sepharose beads. The sensitization procedure described here offers a system for the study of antigenic stimulation of human B lymphocytes in vitro and for the production of human monoclonal antibodies with the desired specificities.

Animals↗
PubMed1984

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A.

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.

B-Lymphocytes↗