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Biomedical subjects

B R Bloom

Publications and source records attributed to B R Bloom.

At least 181 records · Page 10Linked to original sources
PubMed1985

Persistent viral infection affects tumorigenicity of a neuroblastoma cell line.

A mouse neuroblastoma cell line (clone NS20Y) is highly tumorigenic in syngeneic A/J mice. When this clone was persistently infected with measles virus (NS20Y/MS) it failed to grow or form tumors in conventional A/J or nude mice, even when large numbers of cells were inoculated. As doubling time, serum dependence, and anchorage-independent growth on agar did not differ significantly between NS20Y and NS20Y/MS, lack of tumorigenicity of the persistently infected cells is unlikely to be due to an intrinsic property of the cells. NS20Y/MS cells were found to be effectively rejected in athymic nude as well as conventional syngeneic mice. However, injection of mice with either anti-interferon or anti-asialo GM1 serum, both of which have been shown to deplete natural killer (NK) cells in vivo, enabled NS20Y/MS cells to form large tumors. Unexpectedly, treatment of mice with silica also allowed the NS20Y/MS cells to form tumors. Under these conditions, it was shown that silica caused a significant decrease in NK activity as late as 7 days after a single injection. Although NS20Y/MS were not susceptible to NK cell lysis in vitro, the in vivo data suggest that NK cells are in fact the prime mechanism in the rejection of this persistently virus-infected neuroblastoma cell line by athymic and conventional syngeneic mice. The results indicate that NK activity may be greater or more sensitively detected in vivo than in vitro.

Animals↗
PubMed1985

Prazosin, an alpha 1-adrenergic receptor antagonist, suppresses experimental autoimmune encephalomyelitis in the Lewis rat.

Prazosin, an antagonist of alpha 1-adrenergic receptors, has been found to suppress the clinical and histological expression of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. Suppression was more significant in females than in males and was a dose-dependent phenomenon. Analysis of the effect of other adrenergic receptor antagonists supports the conclusion that the suppressive effect of prazosin is a consequence of blockade of the alpha 1-receptor since treatment with either the alpha 2-antagonist yohimbine or the beta-antagonist propranolol exacerbated the disease, whereas treatment with the long-acting mixed alpha 1/alpha 2-antagonist phenoxybenzamine had some suppressive activity. Treatment with prazosin was also able to suppress clinical and histological signs of EAE in animals sensitized by adoptive transfer with activated spleen or lymph node cells. Whether prazosin acts through altering vascular permeability or the immune response, or both, remains to be determined.

Animals↗
PubMed1985

Natural killer cell recognition of target cells expressing different antigens of vesicular stomatitis virus.

Natural killer (NK) cells have the capability of lysing virus-infected, transformed, and embryonal cells, yet the nature of the target structure(s) recognized remains unclear. The availability of well-characterized temperature-sensitive (ts) mutants of vesicular stomatitis virus, defective in expression of individual viral-encoded polypeptides at the nonpermissive temperature (39 degrees C), offered an approach to elucidating NK-cell recognition of virus-infected cells. Target cells were infected with ts mutants in three functions: the viral surface glycoprotein (G protein; ts 045); the matrix (M) protein (ts G31, ts G33), and the polymerase (ts G11). Cells infected with wild-type virus and all ts mutants at the permissive temperature (31 degrees C) were killed by murine spleen cells. Similar to results on cytotoxic T lymphocytes, target cells infected by ts 045 defective in expression of G protein at 39 degrees C were not killed by NK cells. Unexpectedly, cells infected at 39 degrees C with the M-protein mutants also were not killed, although G protein was expressed at the cell surface. Target binding studies indicated that conjugates were not formed by cells infected with the ts mutants at the nonpermissive temperature. That expression of G protein was not sufficient for NK cell-mediated cytotoxicity was established in experiments in which a plasmid (pSVGL) containing the gene for vesicular stomatitis virus G protein was transfected into COS cells. Although G antigen was expressed on the plasma membrane, the cells were not lysed. These results suggest either that recognition of virus-infected cells depends on an appropriate conformation imparted to the viral G protein by association with the M protein or that NK cells can recognize alterations in the structure of the cell membrane induced by insertion of viral M and G molecules.

Animals↗
PubMed1985

Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines.

Antibodies, Monoclonal↗
PubMed1985

Immunogenic potentials of copolymer I in normal human lymphocytes.

Copolymer I (COP I), a nonencephalitogenic polypeptide analogous to myelin basic protein, is currently being tested for possible effectiveness in treating MS. Peripheral blood mononuclear cells from normal human donors respond blastogenically to the L-form of COP I. This response was greater than that obtained with either bovine or guinea pig myelin basic protein (GPMBP), with no specificity for a particular T-cell subset. Analyses of culture supernatants demonstrated the presence of interleukin-2 and gamma interferon. Mononuclear cells from human fetal cord blood also responded blastogenically to COP I, but the magnitude of the response did not differ from that induced by GPMBP.

Animals↗
PubMed1985

In situ characterization of the cellular immune response in American cutaneous leishmaniasis.

American cutaneous leishmaniasis is a spectrum of granulomatous disease caused by related species of an intracellular parasite. The host response in localized cutaneous leishmaniasis (LCL) is effective in that few organisms can be found in tissue lesions. In contrast, diffuse cutaneous leishmaniasis (DCL) patients mount a poor response with numerous parasites present in multiple skin lesions. Immunopathological correlates were sought in LCL and DCL with immunoperoxidase techniques using monoclonal antibodies directed against T lymphocyte subpopulations and interleukin-2 in tissue lesions. Both LCL and DCL granulomas showed a mixture of T lymphocyte subpopulations with the ratio of helper:suppressor phenotypes less than one. This ratio and localization of cells is more similar to the ineffective lepromatous leprosy granuloma than the effective tuberculoid leprosy granuloma. In contrast, interleukin-2 was identified in equivalent numbers of cells in LCL and tuberculoid leprosy, an order of magnitude greater than DCL and lepromatous leprosy lesions. Cells expressing Tac, the receptor for interleukin-2, were present in approximately equal numbers in all disorders. The immunological effectiveness of granulomas appear to related less to the numbers and location of T cell phenotypes than to the functional aspects of these cells, particularly the ability to generate lymphokines.

Granuloma↗
PubMed1984

Biochemical analysis of mutants of a macrophage cell line resistant to the growth-inhibitory activity of interferon.

While a multiplicity of cellular and biochemical effects are mediated by interferons on cultured cells, the mechanisms involved in the direct growth-inhibitory activity of interferons remain problematic. We have previously found that variants in cAMP metabolism in a macrophage cell line, J774.2, were at least 50-fold less sensitive to the growth inhibitory activity of interferons (IFN) than the parental clone. To test the hypothesis that cAMP mediates the growth inhibition produced by IFN in these cells, interferon-resistant variants were selected and characterized with respect to cAMP synthesis and function. Approximately one-third of the IFN-resistant clones were found to be resistant to growth inhibition produced by cholera toxin, but not 8Br-cAMP. IFN was fully able to protect all of the interferon-resistant/choleratoxin-resistant (IFNr/CTr) clones against infection by vesicular stomatitis virus and markedly stimulated 2', 5'-oligodenylate synthetase activity. These IFNr/CTr variants were shown to have a defect in adenylate cyclase. The remaining IFN-resistant clones were fully susceptible to the growth-inhibitory effects of cholera toxin because their basal and stimulated adenylate cyclase activity is similar to that of the parental clone. IFN failed to protect these IFNr/choleratoxin sensitive clones against infection by vesicular stomatitis virus and failed to stimulate 2', 5-oligodenylate synthetase, suggesting that they have defective or deficient IFN receptors. In addition, IFN failed to increase intracellular cAMP levels in both IFNr/CTr and IFNr/choleratoxin sensitive clones. These results provide firm genetic and biochemical evidence that the growth inhibitory effects of IFN on this cell line are mediated by cAMP.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Immunological unresponsiveness in leprosy.

The various forms of leprosy form a clinical and immunological spectrum which offers extraordinary possibilities for insight into immunoregulatory mechanisms in man. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which ultimately results in killing of bacilli in the tissues, albeit often with damage to nerves. At the lepromatous pole, patients exhibit selective immunological unresponsiveness to antigens of M. leprae. Even though all currently known protein species of M. leprae and BCG are cross-reactive, lepromatous patients unreactive to M. leprae antigens frequently respond strongly to tuberculin. In vitro experiments suggest the existence of lepromin-induced suppressor activity, mediated by both monocytes and T cells. The T suppressor cells have the T8 phenotype of which 50% express the activation markers, Ia and FcR. The one unique species of antigen of the leprosy bacillus is a phenolic glycolipid, and it appears that the TS cells largely recognize the terminal trisaccharide of this unique antigen. Depletion of the TS cells restores in vitro reactivity of lymphocytes to lepromin in a portion of lepromatous patients, and addition of IL-2-containing supernatants partially restores responsiveness to M. leprae antigens. Vaccination of lepromatous patients with a mixture of M. leprae and live BCG restores cell-mediated immunity in the majority of lepromatous patients, and concomitantly reduces the in vitro suppressor activity and number of activated T8 cells. These experiments suggest the existence of stage-of-disease related suppressor cells in leprosy which appear to block the responsiveness of TH capable of responding to either specific or cross-reactive mycobacterial antigens. The mode of action of these TS appears to be the inhibition of production of IL-2 and other lymphokines. Successful immunotherapeutic vaccination appears to overcome this block in the majority of patients.

Antigens, Bacterial↗
PubMed1984

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.

Animals↗
PubMed1984

Oxygen metabolism in cloned macrophage cell lines: glucose dependence of superoxide production, metabolic and spectral analysis.

The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the nature of the defect in oxidative metabolism in a variant clone derived from it, J774.C3C, were studied. Upon stimulation with phorbol myristate acetate (PMA), the parental clone produced approximately 1 nmol O2-/min/10(6) cells, whereas the variant clone produced no detectable O2- under the same conditions. Sustained O2- production by J774.16 was totally dependent on extracellular glucose; in glucose-free medium, the cells initiated O2- production but could not sustain it. When cells were stimulated with PMA, glucose-C-1 oxidation of J774.16 cells increased 20-fold while that of J774.C3C remained at resting levels. O2- production in J774.16 cells was inhibited by some agents known to block mitochondrial electron transport before coenzyme Q, such as rotenone and tetrathiafulvalene, whereas antimycin A enhanced O2- production. A dissociation between O2- production and glucose-C-1 oxidation was observed when J774.16 was treated with certain metabolic inhibitors. Quinacrine, 2,4-dinitrophenol, chlorpromazine, and trifluoperazine inhibited O2- production completely under conditions in which glucose-C-1 oxidation was reduced only by 30%. Rotenone inhibited O2- production with no effect on glucose-C-1 oxidation whereas antimycin A augmented O2- production 50% but inhibited glucose oxidation by 20%. Glucose transport studies, with 2-deoxy-D-glucose, showed that the Km for glucose transport of both clones was about 1 mM, indicating that cells could effectively transport glucose even at low concentrations. The Vmax for glucose transport in both J774.16 and variant J774.C3C cells doubled after PMA stimulation, indicating that the variant was effectively stimulated by PMA, even though O2- was not produced. Similarly, PMA induced protein phosphorylation in both clones. No differences between clones J774.16 and J774.C3C in hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, or glutathione peroxidase activities could be found. When dithionite-reduced and -oxidized difference spectra of plasma membranes of these clones were compared, comparable levels of b-type cytochrome were found in both clones. However, CO difference spectra indicated that CO was bound to a b-type cytochrome (presumed to be b-245) in clone J774.16 but not in J774.C3C.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Activation of murine macrophage cell lines. Possible involvement of protein kinases in stimulation of superoxide production.

The mechanism by which phorbol myristate acetate (PMA) activates mononuclear phagocytes to produce O2- was investigated in two cloned murine macrophage-like cell lines. J774.16 cells produce O2- upon stimulation by PMA and aggregated IgG, but a variant clone derived from it, J774.C3C cells, do not, probably because of a functionally defective cytochrome b. 4-O-methyl 12-O-tetradecanoyl-phorbol-13-acetate (4-O-methyl TPA) did not stimulate O2- production in either clone. Studies on the ionic requirements for production of O2- indicated that the extracellular Ca2+ and Mg2+ concentrations did not affect O2- production of J774.16 cells. Furthermore, calcium flux studies with the use of 45Ca2+ indicated that PMA caused no detectable influx of Ca2+, produced a modest release of presumed loosely cell-associated 45Ca2+, but did not detectably change the rate of Ca2+ efflux. By using quin 2 acetoxymethyl ester, no evidence could be adduced that PMA caused intracellular Ca2+ redistribution. Because membrane potential changes are seen in other systems of activation, membrane potential changes in the macrophages stimulated by PMA were measured both by means of the fluorescence ionophore, dis-S-C3-(5), and by direct measurement by using a K+-electrode. Under conditions that stimulated O2- production, no membrane depolarization was observed. Under the conditions for stimulating O2- production, PMA also did not induce increased turnover of membrane phospholipids or arachidonic acid. Although J774.16 and J774.C3C produce prostaglandin D2 at resting state, PMA neither released arachidonic acid from membrane lipids, nor induced release of prostaglandins and thromboxane at least for 10 min after stimulation. Similarly, PMA failed to increase transmethylation of phospholipids or break down the methylated phospholipids. PMA and aggregated IgG, however, induced protein phosphorylation in both J774.16 and J774.C3C cells. Among these, mainly two sets of phosphorylated proteins with m.w. of 42,000 and pI 6.85 to 7.3 and m.w. of 58,000 and pI 8.15 to 9.3 are common in both stimuli. Biologically inactive 4-0-methyl TPA did not induce phosphorylation of these proteins. From the above results, we conclude that PMA induces O2- production in a macrophage line without detectable changes in membrane potential; Ca2+ flux, intracellular Ca2+ increase or redistribution, or increase of membrane lipid turnover. Because two stimuli that induce O2- production in J774.16 cells induced phosphorylation of common proteins, whereas a biologically inactive analog did not, protein kinases, possibly protein kinase C, may be involved in the activation for O2- production.

Animals↗
PubMed1983

Growth of Trypanosoma cruzi in a cloned macrophage cell line and in a variant defective in oxygen metabolism.

A continuous cloned murine macrophage-like cell line, clone 16 derived from J774, has been found upon appropriate stimulation to be capable of oxidizing glucose by the hexose monophosphate shunt and producing O2- and H2O2. A variant in oxidative metabolism, clone C3C, was selected from this cell line which under similar conditions is unable to produce significant amounts of O2- and H2O2. When cells of the parental clone 16 were infected with epimastigotes of Trypanosoma cruzi, there was significant killing or growth inhibition of the parasites at 3 to 4 days after infection. In contrast, the parasites grew in the oxidative variant, clone C3C. Trypomastigote forms of T. cruzi were found to be only partially killed in the parental clone 16 but grew abundantly in the oxidative variant. Infection of the parental clone, but not the variant, was sufficient to stimulate oxygen metabolism as demonstrated by the increased reduction of nitro blue tetrazolium. Studies on the killing of T. cruzi epimastigotes in cell-free suspension by xanthine-xanthine oxidase indicated that 90% of the killing was catalase sensitive and due to H2O2, with at most 7 to 8% killing which could be inhibited by scavengers of . OH and singlet oxygen (1O2). In the in vitro experiment with H2O2 produced by glucose and glucose oxidase, the 50% lethal doses of epimastigotes and trypomastigotes were 6.0 and 8.7 nmol of H2O2 per min per ml, respectively, indicating that trypomastigotes were more resistant to killing by H2O2 than epimastigotes were. A reconstitution experiment of trypanocidal activity in clone C3C by ingestion of zymosan particles coupled with glucose oxidase showed that H2O2 was essential for this cytocidal process in the macrophage cell line. These results provide clear evidence for killing of an intracellular parasite by a continuous macrophage-like cell line and suggest the importance of the oxidative cytocidal mechanism in this process.

Animals↗
PubMed1983

Role in nude mice of interferon and natural killer cells in inhibiting the tumorigenicity of human hepatocellular carcinoma cells infected with hepatitis B virus.

The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 10(6) cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 10(6) cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 10(6) PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response. The nature of the response was delineated by treating nude mice challenged with tumor cells with sheep anti-mouse interferon globulin (anti-IFN). When 2 X 10(6) cells were injected, tumor growth occurred in 75% of anti-IFN-treated mice, whereas controls injected with the same number of cells, but not receiving anti-IFN, failed to develop tumors. The tumors in the anti-IFN-treated mice were highly invasive and the latency period until tumor appearance was reduced to 3-5 d. An inverse correlation was found between susceptibility of the hepatoma cells to natural killer (NK) activity in vitro and resistance to tumor growth in vivo. In vitro cytotoxicity for PLC/PRF/5 cells was eliminated by anti-NK 1.1 and complement, establishing the effector cell as an NK cell. NK cell activity 14 d after inoculation of mice with PLC/PRF/5 cells was augmented against PLC/PRF/5 target cells but not against SK-HEP-1 cells. Treatment of mice with ALS, irradiation, or anti-IFN abolished NK activity against PLC/PRF/5 cells. Co-cultivation of nude mouse spleen cells with PLC/PRF/5 but not with HBsAg or SK-HEP-1 cells induced secretion of murine IFNalpha. These results suggest that the IFN/NK cell system may play a role in limiting tumorigenicity and invasiveness of HBV-infected human hepatocellular carcinoma cells by a mechanism similar to that found for other cells persistently infected with viruses.

Animals↗
PubMed1983

IgE-binding factors from mouse T lymphocytes. I. Formation of IgE-binding factors by stimulation with homologous IgE and interferon.

Incubation of normal mouse spleen cells with homologous IgE resulted in the formation of soluble factors that inhibited rosette formation of mouse Fc epsilon R+ cells with IgE-coated ox erythrocytes. The soluble factors could be absorbed with mouse or rat IgE coupled to Sepharose and recovered from the beads by acid elution. However, the factors had no affinity for either human IgE or mouse IgG. The IgE-binding factors were derived from T cells. Production of the factors required Lyt1+ T cells and Fc gamma R+ cells, which suggests that the factors are derived from Fc gamma R+ Lyt 1+ T cells. The molecular size of IgE-binding factors was approximately 15,000 daltons. When IgE-binding factors were formed by BALB/c spleen cells, nearly one-half of the factors had affinity for lentil lectin, and the remaining half of the factors failed to bind to the lectin. The proportion of the two species of IgE-binding factors differed depending on mouse strains. The majority of the factors formed by B6D2F1 spleen cells had affinity for lentil lectin, but those formed by SJL spleen cells failed to bind to the lectin. The IgE-binding factors were also induced by incubation of normal spleen cells with polyinosinic-polycytidylic acid (pI:pC). The nucleotide stimulated splenic adherent cells to form "inducers" of IgE-binding factors, which in turn induced normal lymphocytes to form IgE-binding factors. The inducers of IgE-binding factors were inactivated (or neutralized) by antibodies specific for mouse Type I interferon. It was also found that purified mouse beta interferon could induce the formation of IgE-binding factors. IgE-binding factors induced by pI:pC consisted of two different molecules: one had a m.w. of 15,000 daltons, and another had a m.w. of between 40,000 and 60,000 daltons.

Animals↗