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Biomedical subjects

R Thorpe

Publications and source records attributed to R Thorpe.

At least 109 records · Page 6Linked to original sources
PubMed1992

Viral infection induces cytokine release by beta islet cells.

Viral infection has been suggested to play a triggering role in the pancreatic beta cell destruction which occurs in insulin-dependent diabetes (IDDM). However, the underlying mechanism of this phenomenon is unknown. In this study a human insulinoma cell line has been infected with measles, mumps and rubella viruses since a temporal association is reported between the clinical onset of IDDM and diseases caused by these viruses. The infection with measles and mumps viruses induced the release of interleukin-1 (IL-1) and interleukin-6 (IL-6) by the cell line as assessed by a bioassay and up-regulated the expression of human leucocyte antigen (HLA) class I and class II antigens as evaluated by cytofluorimetric analysis. Stimulation with rubella virus induced the release of IL-6 only and had no effect on HLA antigen expression. These data show for the first time that IL-1 and IL-6 secretion by an insulinoma cell line may occur after viral infection and suggest that cytokine release and increased expression of HLA molecules by beta cells may act to induce the immune response towards beta cells in IDDM.

Cell Line↗
PubMed1991

Interleukin-4 proliferative signal transduction involves the activation of a tyrosine-specific phosphatase and the dephosphorylation of an 80-kDa protein.

Interleukin-4 (IL-4) is a cytokine that expresses its biological effects by binding to specific membrane receptors. Although the diverse biological properties of this molecule have been characterized extensively the biochemical mechanisms by which extracellular binding events lead to biological responses remain unclear. IL-4 can stimulate the proliferation of several hemopoietic cell types, and we have taken advantage of its ability to induce the growth of leukemic cell lines to investigate the role that protein phosphorylation events might play in IL-4 mitogenic signal transduction. We show that the addition of IL-4 to several leukemic cell lines of different origin causes the rapid dephosphorylation of an 80-kDa phosphoprotein (p80) from tyrosine residues. This event occurs in a dose-responsive manner closely correlating to that of biological activity, and both are blocked by an anti-IL-4-specific antiserum. The ability of sodium orthovanadate to prevent IL-4-induced dephosphorylation of p80 suggests that this event is mediated by a protein-tyrosine-phosphatase (EC 3.1.3.48). The importance of the role that tyrosine-specific dephosphorylation plays in mediating IL-4 mitogenic signal transduction is substantiated by the ability of sodium orthovanadate in cell culture to block effectively IL-4-induced proliferation at doses that enhance the proliferation stimulated by either granulocyte-macrophage colony-stimulating factor or interleukin-3.

Adenosine Triphosphate↗
PubMed1991

Studies on the immunogenicity of Chinese hamster ovary cell-derived recombinant gp120 (HIV-1IIIB).

Recombinant DNA-derived gp120 (HIV-1IIIB) expressed in chinese hamster ovary cells elicited specific humoral and cell-mediated immune responses in a variety of mammals. Antisera from immunized rabbits, sheep and goats recognized virus-derived gp120 and its precursor (gp160). Neutralizing antibodies were also elicited, but only in a few animals, and this may be related to the protein's susceptibility to cleavage through the neutralizing domain. However, in rabbits the degree of cleavage of gp120 had little or no effect on its antigenicity or immunogenicity. All antisera had limited cross-reactivity to envelope glycoproteins from a panel of HIV-1 isolates suggesting that immunodominant antibody epitopes are in variable regions of the recombinant gp120. Antigen-specific T-cell responses were detected in immunized macaques and were found to be stronger and more prolonged when gp120 was administered in Freund's adjuvant rather than alum.

Animals↗
PubMed1991

Development of immunoassays for human interleukin 3 and interleukin 4, some of which discriminate between different recombinant DNA-derived molecules.

Two panels of hybridomas were produced that secreted monoclonal antibodies (MAbs) against recombinant DNA-derived human interleukin 3 and interleukin 4 (rhIL-3 and rhIL-4). From each panel, sensitive immunoradiometric assays (IRMAs) were developed which were capable of detecting the recombinant molecule used as the immunogen but were unable to recognize natural or other recombinant forms of the same cytokine. Subsequent studies using the MAbs from each panel showed that a number of the MAbs appeared only to recognize that particular recombinant molecule used as immunogen, with little or no binding to other recombinant forms of the molecule. By using MAbs that were found to be unrestricted in their recognition for different recombinant forms of the cytokines, it was possible to develop an IRMA for IL-4 that was capable of detecting natural IL-4 as well as all the recombinant forms equally. An IRMA was also developed for IL-3 but was not of equivalent sensitivity in detecting the different recombinant forms of IL-3 used in the study. The recombinant DNA-derived cytokine molecules used to raise the two panels of MAbs contained amino acid substitutions relative to the natural sequences, and these findings indicate that caution should be exercised when using immunoassays to estimate natural sequence molecules if antibodies raised to modified rDNA-derived molecules are used.

Animals↗
PubMed1991

Antisera raised against recombinant soluble CD4s can have reactivity to recombinant HIV-1 envelope glycoproteins.

Antisera from sheep immunized with two different recombinant soluble CD4 preparations both exhibited cross-reactive antibodies specific for recombinant gp120s and gp160s prepared in different expression systems in immunoblots and radiobinding assays. The cross-reactivity was broadly mapped to the amino-terminal 70K cleavage fragment of gp120 and more closely identified to residues 204-274 using a series of truncated recombinant gp120s. However, this reactivity could not be detected against virally encoded gp160/120.

Animals↗
PubMed1991

Nature of the endogenous pyrogen (EP) induced by influenza viruses: lack of correlation between EP levels and content of the known pyrogenic cytokines, interleukin 1, interleukin 6 and tumour necrosis factor.

Fever in influenza results from the release of endogenous pyrogen (EP) following virus-phagocyte interaction and its level correlates with the differing virulence of virus strains. However, the different levels of fever produced in ferrets by intracardial inoculation of EP obtained from the interaction of different virus strains with ferret of human phagocytes did not correlate with the levels of interleukin 1 (IL-1), IL-6 or tumour necrosis factor in the same samples as assayed by conventional in vitro methods. Hence, the EP produced by influenza virus appears to be different to these cytokines.

Animals↗
PubMed1991

Cytokines in sera from insulin-dependent diabetic patients at diagnosis.

Cytokines are known to play an important role in autoimmunity and have been suggested to be involved in the pathogenesis of insulin-dependent diabetes (IDDM). In the present study we have measured IL-1, IL-2, IL-4, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) (using both immunoassays and bioassays) in sera from 50 patients affected by IDDM at the time of clinical diagnosis and 51 age and sex matched controls. Detectable levels of IL-1, IL-2, IL-6 and IFN-gamma were found in the serum of a small percentage of subjects and were not significantly different between patients and controls. IL-4 was detectable in a higher number of both patients and controls and circulating TNF-alpha (greater than 1 U/ml) was found in a percentage of patients (24%) significantly higher than controls (P less than 0.01). Raised levels of TNF-alpha were detectable using an immunoenzymatic assay whereas TNF bioactivity in these samples was negligible. We conclude that the presence of immunoreactive TNF-alpha in the patient's sera may reflect an increased localized production of this cytokine at pancreatic level. However, the measurement in serum of other cytokines does not add information on the role that they may play in the pathogenesis of IDDM.

Cytokines↗
PubMed1990

Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays.

Murine monoclonal antibodies and a sheep polyclonal antiserum against recombinant human granulocyte colony-stimulating factor (GCSF) have been produced. These have been used to develop an immunoassay which can detect 250 pg/ml (25 U) of both natural and recombinant human GCSF. The assay involves forming a complex between GCSF and a monoclonal anti-GCSF, binding of the complex to microtitre wells coated with sheep anti-GCSF and detection of the bound complex with 125I-labelled sheep anti-mouse IgG. Unlike the classical bone marrow assay and other cell line based bioassays for GCSF, the immunoassay was specific for the cytokine, showing no cross-reactivity with GM-CSF, IL-6, IL-3 or IL-1 alpha and -beta. The assay does not exhibit interfering matrix effects when used for the estimation of human GCSF in serum.

Animals↗
PubMed1990

The production and purification of PCR-derived recombinant simian immunodeficiency virus p27 gag protein; its use in detecting serological and T-cell responses in macaques.

The polymerase chain reaction (PCR) was used to amplify a region of the gag gene, encompassing the core protein p27, from genomic DNA of cells infected with SIVmac251 (32H isolate). The 767 base pair PCR product was cloned into the bacteriophage M13 and fully sequenced before sub-cloning into the expression vector pUC19. The 30 kilodalton (kDa) fusion protein of lacZ-p27 was expressed as a soluble protein in E. coli JM101 cells and purified to greater than 90% purity by affinity chromatography. The affinity purified product was used in serological and T-cell assays to assess immune function in cynomolgus macaques immunised or challenged with immunodeficiency virus derived material. This reagent and accompanying methods provide valuable assays for monitoring the efficacy of vaccines for SIV as a model for human AIDS.

Animals↗
PubMed1990

Iodine and selenium deficiency associated with cretinism in northern Zaire.

Selenium status was determined in an endemic-goiter area and in a control area of Zaire. Compared with the reference values of a noniodine-deficient area, serum selenium in subjects living in the core of the northern Zaire endemic-goiter belt (Karawa villages) was seven times lower in 52 school-children and similarly low in 23 cretins; erythrocyte glutathione peroxidase (RBC-GPX) was five times lower in schoolchildren and still two times lower in cretins (P = 0.004). In a less severely iodine-deficient city of the same endemia (Businga), selenium status was moderately altered. RBC-GPX activity was linearly associated with serum selenium concentration up to a value of 1140 nmol/L and leveled off at approximately 15 U/g Hb at greater selenium concentration. At Karawa villages, selenium supplementation normalized both the serum selenium and the RBC-GPX. This combined iodine and selenium deficiency could be associated with the elevated frequency of endemic myxedematous cretinism in Central Africa.

Adolescent↗
PubMed1990

Modulation of the biological activity of thyrotrophin by anti-human thyrotrophin monoclonal antibodies.

The mechanism by which monoclonal antibodies enhance the biological activity of a number of hormones is poorly understood. One such antibody (GC73), which binds to human but not bovine TSH, enhances the bioactivity of human TSH in vivo. We have investigated whether GC73 enhancement of TSH bioactivity involves potentiation of hormone-receptor activation assessed by the cyclic AMP (cAMP) responses of both primary human thyrocyte cultures and a TSH-responsive human thyrocyte cell line (SGHTL-45). GC73 had no effect on basal cAMP production. In contrast to its enhancement of the bioactivity of human TSH in vivo, it markedly inhibited the cAMP response to 1 and 10 mU human TSH/ml in primary thyrocytes. This effect was dose-dependent with neutralization of the bioactivity of TSH occurring at 2 mg GC73/ml. GC73 had no effect on the bioactivity of bovine TSH. In contrast, a second anti-TSH monoclonal antibody (TC12), which binds to both human and bovine TSH, inhibited the bioactivity of both species of TSH. Similar results were obtained using SGHTL-45 cells, although the peak concentrations of cAMP were lower. We conclude that binding of GC73 to human TSH resulted in inhibition rather than enhancement of the in-vitro biological activity of human TSH. We suggest that GC73 enhancement of human TSH bioactivity seen in vivo does not result from a mechanism involving potentiation of receptor activation by human TSH.

Antibodies, Monoclonal↗
PubMed1990

Cell surface phenotype, cytokines, and antibody gene expression in immortalized human B cell lines.

In an attempt to gain some insight into the many factors influencing antibody gene expression in human B cell lines, we have examined in detail the relationship between cell surface phenotype, cytokines, and the growth and antibody-producing capacity of a panel of immortalized human B cell lines. The cell panel comprised lines secreting either high or low titers of antibodies against Rhesus D, hepatitis B surface, and tetanus toxoid antigens. All the transformed cell lines exhibited a cell surface phenotype characteristic of well-differentiated peripheral blood cells strongly expressing CD23 and CD38 while weakly expressing CD10 and CD21. There was no obvious relationship between the antibody-body-secreting and proliferative capacity of the cell lines and their cell surface phenotype. Antibody secretion by the cells was rarely improved by the addition of a wide range of doses of recombinant IL-2, IL-4, or IL-6. In addition, such treatment frequently inhibited proliferation. Supernatants from some of the cell lines promoted the growth of unrelated cell lines but failed to influence antibody production. Such supernatants contained the highest concentration of IL-1, TNF beta, TGF beta, and soluble CD23. In contrast, the heterohybrid supernatant which inhibited cell growth secreted low levels of these cytokines. None of the cell lines secreted detectable amounts of IL-2, IL-4, INF gamma, or GCSF. There was no obvious relationship between cytokine production and antibody secretion. Finally, LPS had a slight but variable effect on antibody secretion but failed to influence cell growth.

Antibody Formation↗
PubMed1989

Procoagulant activity of T lymphoblastoid cells in extrinsic and intrinsic coagulation systems.

We have measured the procoagulant activity (PCA) of four T lymphoblastoid cell lines (Jurkat, CEM, HSB-2 and Molt 4) as well as normal peripheral blood T lymphocytes, before and after stimulation with phytohaemagglutinin (PHA), using clotting and amidolytic methods. Of the four cell lines only one, Jurkat, gave enhanced PCA after stimulation with PHA. This activity was shown to be tissue factor-like by its dependence on factor VII in plasma and in an amidolytic assay with purified factors VII and X. Jurkat was also the only one of the four cell lines to secrete interleukin-2. All four cell lines promoted the generation of large amounts of thrombin in platelet-free plasma in glass tubes. This activity was dependent on the presence of plasma factor VIII, and was probably due to phospholipids in the cell membranes. Normal T lymphocytes gave intrinsic PCA in the thrombin generation test which was only 15% of that of the lymphoma cells. These results show that some T lymphocytes can develop PCA in both intrinsic and extrinsic systems and this should be taken into account in studies of the PCA of mixed leukocyte populations.

Blood Coagulation↗
PubMed1989

Demonstration of cytokines in biological medicines produced in mammalian cell lines.

Commercially produced biological medicines may contain cytokines secreted by mammalian cell lines. Several such cell lines were found to produce interleukin 6 and, after stimulation, to secrete interleukin 1, tumour necrosis factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. High levels of interleukin 6 were detected in several vaccines and rDNA-derived proteins, and certain vaccines contained interleukin 1, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Some preparations of human monoclonal antibodies were also found to contain interleukin 1 and tumour necrosis factor. Cytokines may contribute to certain types of adverse reactions to these products.

Animals↗