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Biomedical subjects

J Vilcek

Publications and source records attributed to J Vilcek.

At least 145 records · Page 8Linked to original sources
PubMed1983

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon.

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.

Antigens, Surface↗
PubMed1983

Coproduction of interleukin-2 and interferon-gamma in human mononuclear cells.

Interleukin-2 (IL-2) and immune interferon (IFN-gamma) production was studied in Ficoll-hypaque purified human mononuclear cells derived from plateletpheresis residues. As previously reported, costimulation by 5 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 5 microgram/ml of phytohemagglutinin (PHA) caused a marked enhancement of IFN-gamma levels compared to the yields obtained with PHA alone. IL-2 levels were also increased 50-300 times by this induction protocol. Mezerein (MZN), a compound structurally related to TPA, was found to be similar to TPA in enhancing IFN-gamma and IL-2 levels. In addition to TPA and MZN, four other related phorbol esters caused a stimulation of IFN-gamma and IL-2, with the production of IL-2 paralleling the production of IFN-gamma. Kinetic experiments indicate that low levels of IL-2 first became detectable 6 h after induction, whereas IFN-gamma could be demonstrated only later. Furthermore, IL-2 production reached a plateau earlier than IFN-gamma production in the TPA/PHA treated cultures. Dialysis at pH 2 abrogated the IFN-gamma activity but not the IL-2 activity. A three-step purification procedure developed for IFN-gamma isolation effectively separated IFN-gamma from IL-2. Our results show that there is a close correlation between the magnitude of IL-2 and IFN-gamma production under the experimental conditions employed. Physical separation of the two lymphokines is important for future studies on the interactions between IFN-gamma and IL-2 in various cellular immune reactions.

Cells, Cultured↗
PubMed1982

Specific binding of 125I-human interferon-gamma to high affinity receptors on human fibroblasts.

Highly purified human interferon-gamma (IFN-gamma) was iodinated with 125I-Bolton-Hunter reagent to a specific activity of 34 microCi/micrograms of protein. After iodination, molecular sieve chromatography was used to isolate fractions containing IFN-gamma at approximately 80-90% radioactive purity. This preparation of 125I-IFN-gamma bound specifically to high affinity binding sites on human GM-258 fibroblasts. Scatchard analysis of binding data revealed the presence of 2400 binding sites/cell, each binding with an apparent Kd of 1.5 X 10(-10) M. Binding was competitively inhibited in the presence of unlabeled IFN-gamma and, to a lesser degree, by IFN-beta, but not by IFN-alpha. Neutralizing antibodies specific for IFN-gamma efficiently inhibited the specific binding of 125I-IFN-gamma to cells. We conclude that the specific high affinity binding site is the receptor for IFN-gamma.

Cell Line↗
PubMed1982

Systemic lupus erythematosus: presence in human serum of an unusual acid-labile leukocyte interferon.

A previously undescribed species of human leukocyte, or alpha, interferon is present in the serum of many patients with systemic lupus erythematosus. It was shown to be alpha-interferon by neutralization with specific antiserums, affinity column chromatography, and antiviral activity on bovine cells. However, 23 of 30 interferon samples tested were inactivated by incubation at pH 2, a characteristic of human "immune," or gamma, interferon. Multiple samples of interferon from the same patient had similar biological properties, but samples from different patients were not all identical, suggesting that several variants of this species of human alpha-interferon may exist.

Humans↗
PubMed1982

Molecular weight of human gamma interferon is similar to that of other human interferons.

The molecular weight (as determined by molecular sieve chromatography) of human gamma interferon, formerly referred to as immune or type II interferon, is between 40,000 and 70,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gamma interferon activity was recovered mainly from two regions of the gels corresponding to molecular weights of 20,000 and 25,000. The results suggest that in native form human gamma interferon may be aggregated.

Glycoproteins↗
PubMed1982

Human T cell hybridomas secreting immune interferon.

Human T-cell hybridomas were established by hybridization of concanavalin A-stimulated human peripheral blood lymphocytes with a 6-thioguanine-resistant mutant cell line, designated SH9, derived by irradiation from a cloned human cutaneous T lymphoma line, Hut102-B2. High levels of interferon (IFN) were demonstrated in the supernatants of hybridoma L265 and its subclones. Whereas no IFN was detected in SH9 cell cultures, up to 1,330 units of IFN per ml were produced spontaneously by the hybrids. On induction with 12-omicron-tetradecanoylphorbol 13-acetate, IFN synthesis in hybridoma cultures was enhanced 8- to 16-fold. Neutralization with specific antisera and determination of antiviral activities in human and bovine cells showed that the IFN secreted by the hybridomas was immune IFN (IFN-gamma). Analysis of DNA content, karyotype, and cell surface phenotype, including T cell specific antigens and receptors, confirmed the T cell hybrid nature of L265 clones. No correlation was found in the hybridomas between IFN production and the expression of HTLV, a retrovirus released by Hut102-B2 and SH9 cells.

Gene Expression Regulation↗
PubMed1982

Purification of two subspecies of human gamma (immune) interferon.

Interferon (IFN)-gamma was produced in cultures of human leukocytes by combined stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA). IFN-gamma was purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAE-Sephacel at pH 8.0. Treatment of such partially purified IFN-gamma preparations with the anionic detergent NaDodSO4 (0.1% at 20-25 degrees C) decreased biological activity to approximately 5-20%. When analyzed by NaDodSO4/polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-gamma. Native IFN-gamma was found to have a lower affinity for alkyl agarose columns than human IFN-alpha or IFN-beta did, suggesting that IFN-gamma is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-gamma to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.

Humans↗
PubMed1982

Production of human alpha- and beta- interferons by human-rodent hybrids.

Attempts were made to demonstrate human alpha interferon production in virally induced human-rodent cell hybrids. In all hybrids investigated human beta-interferon production was detected when the relevant human chromosomes were retained, but only low levels of human alpha interferon could ever be detected even when human chromosome 9, which contains at least eight alpha-interferon genes, was present in the hybrid. The detection of human alpha interferon was made possible only by the use of specific antisera to human alpha and beta interferon and the sensitive virus yield-inhibition assay. Evidence is presented for independent regulatory mechanisms governing the expression of human alpha and beta interferons in human-rodent hybrids.

Animals↗
PubMed1982

Acid-labile human leukocyte interferon in homosexual men with Kaposi's sarcoma and lymphadenopathy.

Some immunologic parameters in homosexual patients with Kaposi's sarcoma (KS) or unexplained lymphadenopathy resemble findings in patients with autoimmune diseases such as systemic lupus erythematosus (SLE). Many patients with SLE have an unusual acid-labile form of human leukocyte interferon (HuIFN-alpha) in their serum. Sera from 91 homosexual men were tested for the presence of HuIFN. Of 27 patients with KS, 17 had significant titers of HuIFN in their serum. Ten of 35 patients with lymphadenopathy and three of four patients with other clinical symptoms also had circulating HuIFN. In contrast, only two of 25 apparently healthy subjects had serum HuIFN. All 32 samples of HuIFN had antiviral activity on bovine cells, a characteristic of HuIFN-alpha, and all of 14 representative samples tested were neutralized by antibody to HuIFN-alpha. In addition, the HuIFN-alpha in six of eight representative patients was inactivated at pH 2 and therefore appears to be similar to the HuIFN-alpha found in patients with SLE. These findings suggest that an autoimmune disorder may underly lymphadenopathy and KS in homosexual men.

Homosexuality↗
PubMed1982

Gamma interferon induction in human thymocytes activated by lectins and B cell lines.

Human thymocytes in culture synthesized small quantities of interferon (IFN) when stimulated by the lectins concanavalin A or phytohemagglutinin. IFN production by lectin-activated thymocytes was enhanced in the presence of live B lymphoblastoid cells, irradiated B lymphoblastoid cells, or the conditioned medium from B lymphoblastoid cell cultures. The IFN synthesized in mixed cultures had characteristics of IFN-gamma, whereas the IFN synthesized by B lymphoblastoid cells alone could be identified as IFN-alpha on the basis of its neutralization with specific antisera and stability at pH 2. These findings indicate that human thymocytes in culture synthesize IFN-gamma and that B lymphoblastoid cells and their products considerably stimulate IFN-gamma synthesis by lectin-activated human thymocytes in culture. This stimulation was not diminished in the presence of antibodies to IFN-alpha, indicating that IFN-alpha production by B lymphoblastoid cells was not responsible for the stimulatory effect. Removal of adherent cells from thymocyte suspensions did not abrogate IFN-gamma production.

B-Lymphocytes↗
PubMed1982

Immunological profile of amyotrophic lateral sclerosis patients and their cell-mediated immune responses to viral and CNS antigens.

The 'immunological profile' of amyotrophic lateral sclerosis (ALS) patients was established from standard tests for B- and T-cell function. This showed no significant difference from age and sex-matched other neurological (CNS) disease controls and normal subjects. Immune complex (IC) levels in ALS serum differed significantly from normal controls but not from CNS controls. There was no relation between the various indices of immune activity of IC levels and the clinical disability of the ALS patient or progression of the disease. Distribution of complement-fixing antibodies to poliovirus was similar to sera of ALS and control groups. The in vitro cell-mediated immune responses to poliovirus, however, were significantly greater in ALS patients than in CNS controls and were inversely related to the ALS disability score. Poliovirus has not been demonstrated in the CNS or extra-CNS tissues of ALS patients by conventional means but, if latent or defective poliovirus or related virus were present, this could account for sensitization and a possible autoimmune mechanism. ALS patients exhibited in vitro cellular immunity to ALS and normal CNS subfractions. These responses were not related to the ALS disability score or progression of the disease and probably represent epiphenomena.

Adenoviridae↗