Biomedical subjects

P Stumbles

Publications and source records attributed to P Stumbles.

3 recordsLinked to original sources

PubMed1998

Rat interleukin-4 assays.

The present article describes procedures to measure rat IL-4 protein. The RT-PCR technique has been successfully and widely used to measure IL-4 mRNA, but it does not determine IL-4 protein synthesis. Assays to measure rat IL-4 protein based on its biological activity were developed using the mAb OX-81, which inhibits rat IL-4 activity. Two bioassays were attempted based on the ability of IL-4 to induce the proliferation of T cell blasts and to increase MHC class II expression on resting B cells. A second mAb against rat IL-4 was used in a sandwich ELISA to detect rat IL-4. This ELISA is satisfactory although its sensitivity is not as high as that of the bioassay. According to our experience, the bioassay based on the induction of class II MHC molecules on B cells is the technique of choice for rat IL-4 determination because it proved specific, sensitive and reproducible.

Animals↗

PubMed1997

Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.

Amino Acid Sequence↗

PubMed1995

Activation of CD4+ T cells in the presence of a nondepleting monoclonal antibody to CD4 induces a Th2-type response in vitro.

In vitro experiments using purified rat CD4+ T cells in primary and secondary mixed leukocyte cultures (MLC) have been carried out to explore the mechanism of inhibition of cell-mediated autoimmune disease in the rat by a nondepleting monoclonal antibody (mAb) to CD4. Previous work has shown that W3/25, a mouse anti-rat CD4 mAb of immunoglobulin G1 isotype, completely prevents the development of the paralysis associated with experimental allergic encephalomyelitis (EAE) in Lewis rats, but does so without eliminating the encephalitogenic T cells. The in vitro experiments described in this study have shown that when CD4+ T cells were activated in the presence of the anti-CD4 mAb in a primary MLC, the synthesis of interferon (IFN) gamma, but not interleukin (IL) 2, was completely inhibited. After secondary stimulation, now in the absence of the mAb, the synthesis of IL-4 and IL-13 mRNA was greatly enhanced compared with that observed from CD4+ T cells derived from primary cultures in which the mAb was omitted. As IL-4 and IL-13 are known to antagonize cell-mediated immune reactions, and as EAE is cell-mediated disease, the data suggest that the W3/25 mAb controls EAE by modifying the cytokine repertoire of T cells that respond to the encephalitogen. The capacity for the mAb to suppress IFN-gamma synthesis provides, in part, an explanation for this change in cytokine production. These findings are discussed in terms of what is known of the factors that determine which cytokine genes are expressed on T cell activation. Possible implications for the evolution of T cell responses in human immunodeficiency virus infection are also discussed.

Animals↗