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B B Aggarwal

Publications and source records attributed to B B Aggarwal.

At least 199 records · Page 11Linked to original sources
PubMed1985

Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts.

Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with [3H]propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. [3H]hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of [3H]hTNF-beta to the cells. We conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.

Animals↗
PubMed1985

Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.

Cells, Cultured↗
PubMed1985

Human tumor necrosis factor. Production, purification, and characterization.

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.

Amino Acid Sequence↗
PubMed1985

Primary structure of human lymphotoxin derived from 1788 lymphoblastoid cell line.

The amino acid sequence of human lymphotoxin derived from a 1788 lymphoblastoid cell line was determined. Peptide fragments obtained by trypsin, lysine-C peptidase, cyanogen bromide, and acetic acid cleavage of the intact protein were purified by reverse-phase high performance liquid chromatography and analyzed by amino acid composition and by automated Edman degradation. The protein is 171 amino acids long with a molecular weight of 18,664. It contains one asparagine-linked glycosylation site and lacks cysteine. The salient features of the amino acid sequence of lymphotoxin are described.

Acetates↗
PubMed1985

Human lymphotoxin.

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Amino Acid Sequence↗
PubMed1985

Human interleukin 1 is a cytocidal factor for several tumor cell lines.

Highly purified interleukin 1 (IL 1) obtained from stimulated human monocytes appeared to be growth inhibitory and cytocidal for a human melanoma cell line, A375. Although IL 1 did not have an immediate cytolytic effect, with time in culture the growth of the target cells was irreversibly inhibited. The cells eventually lysed and decreased markedly in number; the IL 1 effect can therefore be said to be cytocidal. IL 1 activity could not be separated from the cytocidal activity by a variety of chromatography procedures by using conventional and high-performance liquid chromatography (HPLC). The A375 melanoma cell line was also sensitive to another human cytokine alpha-lymphotoxin (alpha-LT) derived from a human B cell line. IL 1 also appeared to be partially growth inhibitory and cytocidal for a LT-sensitive mouse fibroblast cell line, L929; but not for LT-resistant cells, including a subline of L929; a human epithelial carcinoma cell line, HeLa; a human osteosarcoma cell line, HOS; and a mouse SV40-transformed kidney cell line, TU5. However, the LT-sensitive mouse fibroblast cell line, L-M, was resistant to IL 1. Therefore, the cytocidal activity of IL 1 only partially overlapped the target cell selectivity of alpha-LT. Although natural IFN-alpha and recombinant IFN-beta were appreciably growth inhibitory for the A375 cell line, natural and recombinant IFN-alpha and recombinant IFN-beta and IFN-gamma exhibited little cytocidal activity. Purified IL 1 did not have any antiviral activity, and conversely, IFN and alpha-LT were not co-mitogenic for thymocytes. Furthermore, by ELISA and radioimmunoassays, antibodies against human alpha-LT, tumor necrosis factor, and IFN-gamma did not react with IL 1, indicating that IL 1 is antigenically distinct from these other cytokines. These in vitro results suggest that IL 1 may play a role in host defense against some tumors as a cytocidal factor.

Animals↗
PubMed1985

Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors.

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

Antibodies, Monoclonal↗
PubMed1984

Human lymphotoxin. Production by a lymphoblastoid cell line, purification, and initial characterization.

Human lymphotoxin was purified to homogeneity from a serum-free tissue culture supernatant of a lymphoblastoid 1788 cell line. The purification scheme consisted of DEAE-cellulose chromatography, preparative isoelectric focusing, lentil lectin-Sepharose chromatography, and preparative polyacrylamide gel electrophoresis. The purified glycoprotein was homogeneous by the criteria of high pressure liquid chromatography and polyacrylamide gel electrophoresis run under both nondenaturing and denaturing conditions. The specific activity of the purified lymphotoxin is approximately 40 X 10(6) units/mg. The protein has an apparent molecular weight of approximately 20,000, and RF of 0.33 on 7.5% polyacrylamide gels at pH 8.8 and an isoelectric point of 5.8. A tryptic digest of the purified native material produced two major fragments of approximately 15,000 and 5,000 Da. The amino acid compositions of the intact molecule and of the tryptic fragments are presented.

Amino Acids↗
PubMed1984

The synergistic anti-proliferative effect of gamma-interferon and human lymphotoxin.

Highly purified preparations of rHuIFN-gamma and HLT synergistically inhibit the in vitro proliferation of WI38VA13 cells, a SV40 virus-transformed derivative of the human cell line W138. The growth of nontumorigenic WI38 cells, established from normal diploid embryonic lung tissue, was not inhibited under identical incubation conditions; rMuIFN-gamma and HLT have the same in vitro effects on the murine melanoma B16. Thus, the combination of rMuIFN-gamma and HLT synergistically inhibits the growth of B16 cells, although incubation with either lymphokine alone has no significant anti-proliferative effect. Flow cytometric analysis of the cell cycle of asynchronous cells indicates that when B16 melanoma cells are treated with rMuIFN-gamma and HLT in combination, more cells accumulate in the G0/G1 phase than when the cells are treated with rMuIFN-gamma alone. It appears that populations of rapidly proliferating tumor cells, which normally contain a large percentage in the S+G2+M phases of the cell cycle, become arrested in the quiescent G0/G1 phase by the combination of IFN-gamma and HLT, resulting in the inhibition of cell growth.

Animals↗
PubMed1983

Subunits of an avian (ostrich) follicle-stimulating hormone and their hybridization with subunits of mammalian gonadotropins.

Follicle-stimulating hormone (FSH) from the ostrich, Struthio camelus, was dissociated by methods previously used to prepare subunits from mammalian gonadotropins. Two chemically dissimilar subunits were obtained from the ostrich FSH and these resembled the alpha- and beta-subunits of mammalian FSH in amino acid composition. The subunits were relatively inactive in radioimmunoassay, radioreceptorassay, and bioassay when tested alone, but significant activity was regenerated upon their recombination. Incubations of mixtures of one of the ostrich subunits with the opposing subunit of ovine FSH also resulted in a significant regeneration of binding and biological activity in FSH assays. These hybrid recombinants demonstrated that the species specificity of the FSH molecule is conferred entirely by the source of the beta-subunit; in fact, hybrids formed between ovine LH-alpha and either ovine or ostrich FSH-beta were more potent in FSH assays than were those containing even two homologous FSH subunits. In contrast, there was little regeneration of LH bioactivity when FSH subunits (from ostrich or sheep) were combined with the subunits of ovine LH, although some LH binding activity was obtained when the mixture contained either one of the LH subunits; i.e., even LH-alpha + FSH-beta showed significant enhancement of binding activity for LH receptors. Thus, the subunits of avian FSH show biochemical and functional homologies to those of mammalian FSH, but there is only limited interchangeability with ovine LH subunits.

Amino Acids↗
PubMed1981

Effect of the alpha subunit of equine LH on the FSH induced cAMP production in rat seminiferous tubule cells.

Previous observations from our laboratory have shown that equine LH can suppress the FSH induced cyclic AMP production in rat seminiferous tubule cells in vitro. The present investigation was carried out to determine the effect of various subunits in this system. The ratios (w/w) of subunits to equine FSH tested was 1:3, 3:1 and 30:1 with a standard dose of 0.3 microgram of the FSH. It was noted that equine LH-beta was not effective up to a 10 microgram concentration in inhibiting the cyclic AMP production induced by equine FSH. Under these conditions, equine LH-alpha suppressed the FSH activity in a dose dependent manner. However, alpha subunits derived from several other species of LH were without any effect on FSH action. Histidine modified derivative of equine LH and its alpha subunit, both of which lack biological activity in the rat Leydig cell assay for LH, were found to be inactive as inhibitors of the equine FSH response. Thus, these results show that the suppressive effect of equine LH on FSH action in the rat seminiferous tubule is a function of the equine LH alpha subunit.

Animals↗
PubMed1981

Studies on the disappearance of equine chorionic gonadotropin from the circulation in the rat: tissue uptake and degradation.

Equine CG (eCG) both radioiodinated and unlabeled, was injected iv into male rats, and the disappearance of the hormone from the circulation was studied. The uptake and inactivation of the hormone by various tissues was also examined. The disappearance curve of eCG consisted of two exponential components with apparent t1/2 of 0.2 and 6 h. Gel filtration analysis of a blood sample 60 min after injection of eCG indicated that residual plasma eCG is intact and is active in both RIA and bioassay. Along with the disappearance of radiolabeled eCG from plasma, its uptake and release was recorded in liver, kidneys, and testis. The label also appeared rapidly in the urine and the thyroid gland. However, gel filtration profiles of urine samples did not show the presence of any intact eCG. Furthermore, overnight in vitro incubation of eCG with plasma, liver, and kidney tissues destroyed 58%, 64%, and 96% of the biological activity of the hormone, respectively. Thus, these results suggest that eCG disappears from the circulation in a biphasic manner, and the plasma, liver, and kidneys may play an important role in the inactivation and metabolism of the hormone.

Animals↗