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Biomedical subjects

D A Weigent

Publications and source records attributed to D A Weigent.

54 records · Page 3Linked to original sources
PubMed1983

Potentiation of lymphocyte natural killing by mixtures of alpha or beta interferon with recombinant gamma interferon.

Human lymphocytes were treated with human alpha (IFN-alpha), beta (IFN-beta), or recombinant gamma (IFN-gamma) interferons separately or in combination to determine their ability to enhance natural killing against mouse L cell targets. Our results showed that recombinant IFN-gamma was approximately 50 times more active per unit of antiviral activity than either IFN-alpha or IFN-beta. Moreover, the levels of natural killing by lymphocytes treated with combinations of IFN-alpha and IFN-beta were additive, whereas combinations of recombinant IFN-gamma and IFN-alpha or recombinant IFN-gamma and IFN-beta were synergistic. The development of natural killing in lymphocytes treated with recombinant IFN-gamma did not occur more rapidly but reached higher levels (62%) than that observed with lymphocytes treated with IFN-alpha or IFN-beta (15%). The results suggest the importance of IFN-gamma and mixtures of IFN-gamma with IFN-alpha or IFN-beta in the enhancement of natural killing activity against virus infections and neoplasia.

Adjuvants, Immunologic↗
PubMed1983

Interleukin 2 enhances natural killer cell activity through induction of gamma interferon.

Highly purified interleukin 2 (IL 2), free of interferon activity, enhanced natural killer (NK) cell activity against tumor cells in mouse spleen cell cultures and in human peripheral lymphocyte cultures in a manner similar to that of interferon (IFN). We determined that IL 2 enhanced NK activity indirectly in a cascade manner by the induction of gamma IFN (IFN-gamma) in the cultures, which actually mediated the enhanced killing. Accordingly, lymphocyte cultures treated with IL 2 alone produced 10 to 100 U of IFN per ml in 6 to 24 h of culture. The IFN was typed as IFN-gamma by specific antibodies. Specific antibodies either to natural IFN-gamma or to a synthetic peptide corresponding to the human IFN-gamma N-terminal amino acids, when added to cultures treated with IL 2, completely blocked IL 2 enhancement of NK cell activity for both the mouse and human systems. IL 2-induced proliferation was not affected by the antibodies. Thus, the enhancement of NK cell activity by IL 2 is completely mediated by IL 2-induced IFN-gamma. The findings clearly indicate a cascade effect whereby one lymphokine (IL 2) induces the production of another. The latter lymphokine (IFN-gamma) then mediates an important biological effect (natural killing).

Animals↗
PubMed1983

Microassay for Sindbis virus and interferon activity.

A simple and rapid microplaque assay for Sindbis virus was developed which uses microtiter plates and overlay medium consisting of methylcellulose and specific antibody to Sindbis virus. Discrete plaque formation was consistently observed on baby hamster kidney (BHK-15) cells within 24 h. The assay was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique on chicken embryo cells in 35-mm petri dishes. The microplaque assay could be accurately applied to measuring interferon activity, particularly at low interferon levels. Overall, the microassay methods described here for assay of Sindbis virus yields and interferon activity retain the accuracy of chicken cell macroplaque assays while offering greater simplicity, rapidity, and economy of materials. This assay is also potentially applicable for use with other togaviruses.

Antibodies, Viral↗
PubMed1981

Human B lymphocytes produce leukocyte interferon after interaction with foreign cells.

Enriched human B-cell populations cocultivated with xenogeneic or allogeneic tumor cells produced 1,000 to 10,000 U of leukocyte interferon per ml. In contrast, cocultivation of enriched plastic-adherent or T-cell populations with xenogeneic or allogeneic cells produced only 10 to 30 U of interferon. The population of cells producing the interferon absorbed to nylon wool and not sheep erythrocytes. They showed a strong mitogenic response to the B-cell mitogen Escherichia coli lipopolysaccharide but not the T-cell mitogen staphylococcal enterotoxin A. In addition, treatment of this cell population with goat anti-human immunoglobulin M and complement depleted the cell population synthesizing the interferon. Together, these in vitro findings strongly suggest that the cells producing most of the interferon after interacting with foreign cells belong to the B-cell population. These results also suggest that the cells that produce most of the leukocyte interferon after interacting in vivo with tumors or other cells made foreign to the body by certain viruses most likely belong to the B-lymphocyte population.

Animals↗
PubMed1981

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma).

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.

Animals↗
PubMed1980

Transfer of interferon-induced viral resistance from human leukocytes to other cell types.

Nonsensitized human leukocytes cocultured with various xenogeneic epithelioid and fibroblastic cells produced human leukocyte interferon and shortly thereafter transferred antiviral activity to the xenogeneic cells. Antiviral activity in the cocultured xenogeneic cells was not due to cell-mediated cytotoxicity as measured by specific 51Cr release and staining with vital dyes. The transfer of antiviral activity from leukocytes to xenogeneic cells was blocked by rabbit antiserum to human leukocyte interferon. Transferred viral resistance failed to develop in actinomycin D-treated xenogeneic cells, even though these cells induced human leukocyte interferon. Based on these findings, it appears that interferon made in the cocultures acts on the leukocytes to effect the transfer of interferon-induced viral resistance to the xenogenic cells, possibly by transmission of an inducer for the antiviral state. These studies strongly suggest a new and efficient host defense against virus infection which does not require killing of noninfected or recently infected cells.

Animals↗
PubMed1980

Reduction of DNA transforming activity in culture by 6-mercaptopurine.

The transforming activity of DNA isolated from 6-mercaptopurine-treated cultures of Bacillus subtilis strain UTH-8505 is markedly reduced when compared to that of DNA obtained from control cultures. The lower transforming activity appears to be a property of the isolated DNA; i.e., various treatments either to restore the activity or to indicate the presence of inhibitory substances that isolate with the DNA suggest a defect in the nucleic acid per se. The reduced transforming activity is not gene specific since the ability of DNA from 6-mercaptopurine-treated cultures to transform several mutants of different genetic loci is lowered. The dose-dependent effect is correlated with the extent of trichloroacetic acid-insoluble radioactivity associated with the DNA from 6-[35S]mercaptopurine-treated cultures. However, the level of apparent drug incorporation is low, being only 1 molecule equivalent to 6-mercaptopurine in 17,500 base residues of DNA having 40% of control transforming activity. The amount of 6-thioguanine incorporation possibly associated with the reduced transforming activity is even less, about one 6-thioguanine moiety per 100,000 base units. If base analog substitution accounts for the reduced transforming activity, exceedingly low levels of incorporation are sufficient to alter this biological property of B. subtilis DNA.

Bacillus subtilis↗
PubMed1978

Contrast of Glycogenesis and protein synthesis in monkey kidney cells and HeLa cells infected with Chlamydia trachomatis lymphogranuloma venereum.

Glycogen metabolism of monkey kidney (LLC-MK-2) cells and HeLa 229 cells infected with a Chlamydia trachomatis lymphogranuloma venereum 440 L (LGV) was studied. The growth cycle of LGV in both host cells was similar; however, a greater number of infectious organism developed intracellularly and were released into the medium during LGV infection of HeLa 229 cells than MK-2 cells. A rapid infection accompanied by a high rate of glycogen synthesis and a short period of accumulation was found in GeLa 229 cells infected with LGV. LGV infected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells; however, the rate of glycogen synthesis was lower and the period of accumulation was longer. The LGV agent grew in cycloheximide-treated cells in the absence of host cell protein synthesis. Protein synthesis associated with LGV throughout the developmental cycle was similar in both cell types and could be abolished by chloramphenicol. The continued synthesis of glycogen in the presence of cycloheximide suggested that the synthesis of glycogen was directed by the organism in both MK-2 cells and HeLa 229 cells.

Cell Line↗
PubMed1976

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.

Bacillus subtilis↗
PubMed1976

Regulation of histidinol phosphate aminotransferase synthesis by tryptophan in Bacillus subtilis.

The effect of tryptophan on the synthesis of histidinol phosphate aminotransferase and prephenate dehydrogenase has been examined. The genes specifying two enzymes for tryptophan biosynthesis (anthranilate synthase and tryptophan synthase-B) were found to be derepressed in a temporal sequence according to their chromosomal location. The genes for histidinol phosphate aminotransferase and prephenate dehydrogenase were derepressed simultaneously approximately 8 min after tryptophan synthase-B. When excess tryptophan was added to a derepressed culture, the pattern of repression of trpE (anthranilate synthase), trpB (tryptophan synthase-B), hisH (histidinol phosphate aminotransferase), and tyrA (prephenate dehydrogenase) was found to be simultaneous. Methyl tryptophan-resistant mutants, which synthesize elevated levels of the tryptophan enzymes, also synthesized elevated levels of histidinol phosphate aminotransferase. Qualitatively similar data were obtained in a temperature-sensitive tryptophanyl-transferase ribonucleic acid synthetase mutant grown at elevated temperatures. The time at which messenger ribonucleic acid was synthesized for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in the presence of actinomycin D indicated that ordered enzyme synthesis was a result of ordered transcription of the corresponding portion of the genome. The effect of the drug rifampin on enzyme synthesis was also examined. The addition of this drug halted the transcription of anthranilate synthase very rapidly, but later regions of the tryptophan region continued to be transcribed. The transcription of the hisH and tyrA genes was also shut off rapidly after rifampin was added. The significance of these observations to the control of transcription of the hisH gene by tryptophan is discussed.

Anthranilate Synthase↗
PubMedDate not supplied

Immunoreactive growth hormone-releasing hormone in rat leukocytes.

In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone-releasing hormone (GHRH) in vitro. By using RNA slot-blot analysis, we detected maximum basal levels of specific GHRH mRNA in the cytoplasm of rat leukocytes after an 8 h in vitro incubation. Northern gel analysis demonstrated that the specific GHRH RNA was polyadenylated and had a molecular mass of approximately 0.8 kDa. Further studies using antibody affinity chromatography followed by size separation on high-performance liquid chromatography (HPLC) columns showed two peaks of immunoreactive (ir) material, a large molecular weight species, and a smaller molecular weight species at approximately 5 kDa. The smaller molecular weight irGHRH appeared to be de novo synthesized since it could be radiolabeled with tritiated amino acids. Both molecular species were detectable in enzyme-linked immunosorbent assay (ELISA) with specific antibodies made to the first 23 amino acids as well as specific antibody obtained commercially made to the entire molecule (1-43). Although the larger molecular weight form appeared to be the more predominant, only the lower molecular weight form could block the binding of 125I-hGHRH to pituitary cells. Most importantly, the lower molecular weight leukocyte-derived GHRH stimulated an increase in the level of GH RNA in the pituitary. We conclude that lymphocytes produce an irGHRH that is similar to hypothalamic GHRH in terms of bioactivity, antigenicity, and molecular weight. The findings demonstrate a potential regulatory loop between the immune and neuroendocrine tissues.

Animals↗
PubMedDate not supplied

Cloning and nucleotide sequencing of rat lymphocyte growth hormone cDNA.

The messenger RNA for rat growth hormone (GH) was isolated from rat spleen lymphocytes and cloned as a cDNA after PCR. Four clones, including part of the prehormone sequence and the full-length mature GH sequence, were obtained and sequenced. The sequence data revealed that rat lymphocyte GH was identical to that reported for pituitary GH. The results do not support the expression by alternate splicing of the 20-kD variant described in the pituitary. We also cloned the promoter of lymphocyte GH and analyzed its nucleotide sequence including 300 base pairs of the 5'-flanking region. The promoter sequence we obtained did not exactly match that reported in the literature because of reading compressions on the gel. However, parellel sequencing of thymus, spleen and pituitary GH promoter sequences gave identical patterns of compressions in the gel. The results suggest that the sequence in all the tissues was the same. Collectively, these studies demonstrate that lymphocytes express authentic GH mRNA and suggest that GH gene expression may be regulated in immune tissue by similar transcriptional factors to those described in the pituitary.

Animals↗
PubMedDate not supplied

Growth hormone induces interferon gamma production and may play a role in the presentation of alloantigens in vitro.

Several reports support the view that growth hormone (GH) promotes proliferation and cytotoxicity by T cells in a mixed leukocyte culture (MLC). The present study was undertaken to begin to determine the mechanism of action of GH on the MLC in vitro. First, we determined that peripheral blood mononuclear cells (PBMC) cultured with mitomycin-treated allogeneic PBMC in an MLC in the presence of exogenously added rhGH develop an augmented proliferative (25-100%) and cytotoxic response (50-600%). We next examined the possibility that GH may promote alloresponses by inducing gamma-interferon (IGN gamma) production. In these experiments, in situ hybridization was used to determine the frequency of cells expressing mRNA for IFN gamma. It was observed that GH increased significantly the frequency of cells expressing mRNA for IFN gamma (100-800%). To determine the site of action of rhGH, we evaluated the response of purified T cells to alloantigens in the presence of rhGH. The addition of rhGH to an MLC had no demonstrable effect when purified T cells were used as the responding population. However, when T cells were reconstituted with autologous mitomycin-treated PBMC and used as the responding population, rhGH augmented proliferation and cytotoxicity. Taken together, these data show that rhGH augments proliferation, cytotoxicity and IFN gamma production during an MLC, and at lease part of the action of rhGH appears to be on the autologous antigen-presenting cell.

Adult↗
PubMedDate not supplied

Induction of interferon by bacteria, protozoa, and viruses: defensive role.

Interferon is established as one of the natural defenses against virus infection. The evidence that interferon may serve a defensive role against certain protozoa is less complete and consists mainly of induction of interferon during protozoal infection, as well as interferon protection of mice against malarial infection. The evidence that interferon may function as a defense against certain bacterial infections is extended with data indicating that a wide variety of bacteria can induce interferon in the mouse and in cultured human peripheral lymphoid cells. This induction of interferon by bacteria and the ability of interferon under many conditions to activate neutrophils and macrophages raises the possibility that interferon may serve a defensive role against some bacteria.

Animals↗