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Biomedical subjects

D Novick

Publications and source records attributed to D Novick.

At least 73 records · Page 4Linked to original sources
PubMed1990

Two antiviral proteins from tobacco: purification and characterization by monoclonal antibodies to human beta-interferon.

Polyclonal antibodies to human beta-interferon reacted specifically with two plant proteins (gp22 and gp35) by Western blot analysis of crude protein extracts from tobacco leaves infected with tobacco mosaic virus. Immunoaffinity chromatography of these extracts on a column of immobilized monoclonal antibodies to human beta-interferon and then reversed-phase HPLC yielded gp22 and gp35 in a pure state. Both proteins reacted with the Schiff reagent and concanavalin A (indicating their glycoprotein nature) and exhibited antiviral activity (inhibiting tobacco mosaic virus replication in tobacco-leaf discs at concentrations of ng/ml). Each protein was cleaved by cyanogen bromide and the resultant peptides, separated by HPLC, were sequenced as far as the Edman degradation allowed, giving a total of 61 amino acid residues for gp22 and 105 residues for gp35, which represent 30-50% of their expected length. Computer analyses of the sequenced segments revealed no significant homology to human beta-interferon, each other, or any other recorded sequence.

Amino Acid Sequence↗
PubMed1990

Role of interaction of CD2 molecules with lymphocyte function-associated antigen 3 in T-cell recognition of nominal antigen.

The role of the interaction of CD2 molecules with lymphocyte function-associated antigen 3 (LFA-3) in facilitating nominal antigen recognition by T lymphocytes was studied by utilizing an HLA-DR4-restricted CD4+ cytotoxic human T-cell clone specific for human immunodeficiency virus envelope glycoprotein gp120 as a responder and murine fibroblasts transfected with human class II major histocompatibility complex (MHC) and/or human LFA-3 molecules as antigen-presenting cells (APC). Although expression of the DR4 restriction element in fibroblasts is sufficient for T-cell recognition of a gp120 peptide as judged by induction of proliferation coexpression of human LFA-3 on DR4+ APC decreases the molar requirement of nominal antigen by greater than one order of magnitude. Both LFA-3 and the relevant class II MHC molecules are necessary for antigen-independent conjugate formation, but the binding is further enhanced by specific nominal antigen. CD2-LFA-3 interaction is independent of T-cell receptor-MHC interaction and contributes directly to the stabilized conjugate between the T cell and LFA-3-bearing APC; soluble CD2 and monoclonal antibodies to LFA-3 and CD2 reduce T-cell-APC binding to the level mediated by nominal antigen and MHC. During conjugate formation, CD2 but not CD3 molecules are reorganized into the cell-cell interaction site in an antigen-independent manner. Thus, reorganization and/or coassociation of CD2 with CD3 molecules is not essential for T-cell activation.

Animals↗
PubMed1989

Soluble cytokine receptors are present in normal human urine.

Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-gamma, or anti-IFN-gamma-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH2-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH2-terminal sequence of IL-6-R as previously reported. Analysis of the eluted proteins from both IFN-gamma and anti-IFN-gamma-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.

Amino Acid Sequence↗
PubMed1989

Monoclonal antibodies for affinity purification of IL-6/IFN- beta 2 and for neutralization of HGF activity.

Two types of recombinant human IL-6 (rIL-6) were used for the development of specific monoclonal antibodies. The first was produced in E. coli and used for immunization, the second was produced in Chinese Hamster Ovary Cells (CHO) and used for screening. The complete translated sequence of the cDNA coding for human IL-6 was fused, in phase, to protein-A and the hybrid gene was fused to the strong lambda PR promoter. This protein was purified from bacterial extracts by chromatography on rabbit IgG-Sepharose columns. After six injections of the purified protein into mice, sera were tested for their binding titer in a solid phase radioimmunassay (sRIA) and for the specificity of binding by Western blots. In the sRIA, crude supernatants of CHO cells (harboring a plasmid containing the human IL-6 gene and expressing high levels of IL-6 but no protein-A or any bacterial antigen) were bound to a solid support, reacted with supernatants of the hybridomas and finally detected with [125I]-goat anti-mouse antibodies. Spleen cells derived from a mouse showing the highest binding titer were fused to mouse myeloma cells. The hybridomas were screened by the sRIA and several positive clones were isolated and characterized. One of the clones was found to neutralize the hybridoma growth factor activity of the rIL-6 from both sources. The same clone was also used for Western blots and for affinity purification of both natural and recombinant IL-6 (E. coli and CHO).

Animals↗
PubMed1989

Monoclonal antibodies to the human interferon-gamma receptor: blocking of the biological activities of interferon-gamma and purification of the receptor.

Monoclonal antibodies against the human interferon-gamma (IFN-gamma) receptor were developed by injecting mice with a preparation of receptor that was purified from solubilized placental membranes by ligand affinity chromatography. Three antibodies were identified by their ability to block the binding of 125I-labeled IFN-gamma to its receptor on HeLa cells at 4 degrees C. One of these antibodies blocked several biological activities of IFN-gamma, including its antiviral activity, its ability to induce HLA-DR surface antigens, and its ability to protect cells from NK cell-mediated cytotoxicity. This antibody exhibited higher binding capacity to cells at 37 degrees C and was significantly less displaceable by an excess of IFN-gamma as compared with the other two antibodies. Immunoaffinity chromatography of solubilized crude placental membrane preparation yielded a purified receptor that exhibited a molecular weight of 88,000. The purified receptor retained its ability to bind 125I-labeled IFN-gamma in solution.

Animals↗
PubMed1988

Recombinant interferon-beta 2 (interleukin-6) induces myeloid differentiation.

Human IFN-beta 2 cytokine produced in E. coli was purified to homogeneity by immunoaffinity and ion-exchange chromatography. The cytokine inhibits the growth of myeloleukemic M1 cells and induces their morphological and functional differentiation into macrophages. Differentiation was also observed in the histiocytic lymphoma U937 cells. The effect on U937 was synergized by IFN-gamma and under these conditions IFN-beta 2 produced the induction of (2'-5') oligo(A) synthetase typical to IFN action and to differentiation.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Two molecular forms of the human interferon-gamma receptor. Ligand binding, internalization, and down-regulation.

The receptors for human interferon-gamma (IFN-gamma) on peripheral blood monocytes and various cells of nonhematopoietic origin were thoroughly characterized and compared. The receptors of all cell types exhibited a similar affinity for IFN-gamma (Kd approximately 1 x 10(-10) M), and in all cases receptor-mediated endocytosis and ligand degradation were demonstrated. However, the receptors differed in their molecular weights (95,000 in HeLa cells and 140,000 in monocytes, assuming a 1:1 ligand to receptor ratio) as concluded from experiments of cross-linking to 125I-IFN-gamma. Lower molecular weight species were obtained as well, particularly in monocytes. Such species could represent either degradation products or subunit structures. The monocyte and HeLa receptor responded differently to an excess of ligand. A significant receptor down-regulation was observed when monocytes were incubated with an excess of 125I-IFN-gamma, whereas no such down-regulation was observed in HeLa cells or in normal fibroblasts. This differential response was observed both in the presence or in the absence of a protein synthesis inhibitor. The receptor on monocytes was found to be acid-labile whereas that on HeLa cells was resistant to acid treatment. These and additional experiments indicate that the monocyte receptor is inactivated following internalization, whereas the HeLa receptor retains its structure and recycles back to the cell surface. The difference in the properties and fate of these two receptor subtypes is probably related to the differential functions of IFN-gamma in various cell types.

Binding Sites↗
PubMed1987

The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies.

The receptor for human interferon-gamma (IFN-gamma) was purified from foreskin fibroblasts. Triton X-100 extracts obtained from either intact cells or membrane preparations were passed through an immobilized interferon-gamma column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of eluted fractions revealed a major band of Mr = 95,000 and minor bands of Mr = 80,000 and 60,000. Further purification was obtained by steric exclusion and by lectin chromatography. The purified receptor retained the ability to bind 125I-IFN-gamma with a Kd of 2.2 X 10(-10) M, a value close to that obtained with intact fibroblasts (5 X 10(-10) M). A complex of Mr = 105,000-125,000 was visualized by immunoprecipitation of 125I-IFN-gamma cross-linked to the purified receptor followed by SDS-PAGE and autoradiography. A similar complex was obtained when 125I-IFN-gamma was cross-linked to intact cells. Immunization of mice with the excised SDS-PAGE band of Mr = 95,000 elicited antibodies that blocked the antiviral activity of IFN-gamma and immunoprecipitated the cross-linked complex of 125I-IFN-gamma and its receptor.

Antibodies↗
PubMed1987

HLA class I and II, interferon, interleukin 2, and the interleukin 2 receptor expression on labial biopsy specimens from patients with Sjögren's syndrome.

Labial biopsy specimens from eight patients with primary Sjögren's syndrome (SS), 10 patients with secondary SS, and three healthy controls were studied with monoclonal antibodies identifying HLA class I and class II antigens; interferon-alpha, beta, and gamma; interleukin 2 (IL2); and the IL2 receptor (Tac) among others. In the normal biopsy specimens there was evidence of HLA class I and, to a lesser extent, class II antigens in both ducts and acini, though this was much less marked than in the Sjögren's biopsy specimens. Interferon-gamma staining, but not interferon-alpha or beta, was also considerably enhanced in the biopsy specimens from the patients with Sjögren's syndrome. These data support the view that in Sjögren's syndrome the release of interferon-gamma may be involved in the induction of class II determinants. Our observations were broadly similar in both primary and secondary Sjögren's syndrome except that patients with primary Sjögren's syndrome tended to have more diffusely scattered T lymphocytes.

Aged↗
PubMed1987

Natural killer cell activity in hepatocellular carcinoma. In vitro and in vivo responses to interferon.

We have measured natural killer (NK) cell activity in patients with hepatocellular carcinoma (HCC) and have examined the effects of in vitro and in vivo administration of alpha-interferon (IFN) on NK cell activity. The NK cell cytotoxicity of HCC patients was significantly lower than that of patients with cirrhosis or healthy controls. Reduced NK cell cytotoxicity in HCC did not correlate significantly with either the serum alpha-foetoprotein concentration or the patient WHO performance grade. NK cell cytotoxicity in all groups could be increased by prior incubation of effector cells with IFN, but this was significant only in HCC patients, in whom 10 IU/ml of IFN increased NK cell cytotoxicity from 37 +/- 10% to 53 +/- 8% (effector to target ratio, 50:1, mean +/- SEM; p less than 0.05). Further increases in IFN concentration failed to increase NK cell activity further. NK cell cytotoxicity was measured immediately before and 24 h after 2.5 x 10(6) IU/m2 of IFN was given subcutaneously to four HCC patients. NK cell cytotoxicity rose from 27 +/- 9% to 61 +/- 5% (effector to target ratio, 50:1, mean +/- SEM; p = 0.05).

Carcinoma, Hepatocellular↗
PubMed1986

Efficient constitutive production of human IFN-gamma in Chinese hamster ovary cells.

A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-gamma was fused through its 5'-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-gamma gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-gamma cell lines were obtained that produce 1.5-2 million units of IFN-gamma per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-gamma. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-gamma with 1-2 X 10(8) units/mg protein. Like IFN-gamma from human white blood cells, the IFN-gamma from CHO-gamma cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-gamma/ml culture per day.

Animals↗
PubMed1986

Localization of interferons and interleukin 2 in polymyositis and muscular dystrophy.

Muscle biopsies from nine patients with polymyositis, six with muscular dystrophy, six with other muscle diseases and three controls have been studied with a panel of 10 monoclonal antibodies (MoAb) identifying T lymphocytes, HLA-class I antigens, alpha, beta and gamma interferons and interleukin 2 (IL-2). The result confirm that the staining of the sarcolemma with anti-HLA class I antibody is weak or negative, except in areas adjacent to infiltrating leucocytes or where muscle fibre damage is evident. The very similar tissue distribution of alpha, beta and gamma interferons in the polymyositis biopsies supports the hypothesis that interferons are released by the inflammatory infiltrate and induce the class I antigen expression. In contrast, little interferon was demonstrated in the dystrophic muscle implying that class I expression in these disorders must occur by a different mechanism. Little IL-2 was demonstrated in any of the biopsies though some unexplained small dense accumulations were identified by one of the anti IL-2 MoAb.

Adult↗
PubMed1985

Increase of vulnerability to lymphotoxin in cells infected by vesicular stomatitis virus and its further augmentation by interferon.

The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-gamma, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.

Animals↗
PubMed1984

Efficient constitutive production of human fibroblast interferon by hamster cells transformed with the IFN-beta 1 gene fused to an SV40 early promoter.

The coding sequence of the human interferon (IFN)-beta 1 gene, fused 60 bp downstream from the RNA start site of the SV40 early gene, was transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells together with a selectable DHFR gene. Most transformants continuously secreted IFN-beta 1 into the medium. Induction did not stimulate expression of the fused SV40-IFN-beta 1 gene. The role of the SV40 promoter was verified by transforming cells with the unmodified human IFN-beta 1 gene, or by the IFN-beta 1 coding region fused to another poly(rI):(rC)-inducible gene. In these cases, the transformants showed strictly inducible (not constitutive) IFN secretion. By selection for methotrexate resistance, CHO clones with a 10-20-fold amplification of the SV40-IFN-beta 1 DNA were obtained. Such clones constitutively produce up to 350,000 units IFN/ml per 10(6) cells/24 hr, i.e., over 10 times more than fully induced human fibroblasts. In continuous culture with daily changes of medium, accumulation of IFN-beta 1 is constant at a rate of 300,000 molecules per cell/hr. Batches of up to 16 mg of IFN-beta 1 produced by the transformed CHO cells were purified to homogeneity by affinity chromatography on monoclonal antibodies. This IFN appears identical in size, activity, and immunospecificity to the native human IFN-beta 1 glycoprotein.

Animals↗
PubMed1984

Type I and Type II interferon receptors.

Binding site competition studies of 125I-interferon (IFN)-gamma were performed with homogeneous preparations of IFNs alpha 2, beta, and gamma. It was found that only IFN-gamma could compete for the specific binding site of 125I-IFN-gamma in both WISH and FS11 cells. Thus, a clear distinction between the receptor of IFN- alpha and beta on one hand, and the receptor of IFN-gamma on the other hand, was obtained. This result is unambiguous because all IFN preparations which were used were purified to homogeneity by specific monoclonal antibody immunoaffinity chromatography. We propose to assign the names "Type I" for the receptor of IFN-alpha and IFN-beta, and "Type II" for the specific receptor of IFN-gamma.

Binding, Competitive↗
PubMed1983

Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay.

Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h.

Animals↗