Are you D J Shealy?

Claim your profile

Publications (5)17.28 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine with pleiotropic activity that binds to two transmembrane receptors. Its role in mediating the inflammatory response to injury or infection has been well documented and it has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Using synthetic peptide libraries composed exclusively of D-amino acids, two distinct hexapeptide families that block the binding of TNF-alpha to its receptors were identified. In the deconvolution of the library, activity increased from submillimolar to the low micromolar range with the most active compound having an IC50 of 0.33 microM. With the aid of biotinylated constructs of these hexapeptides it was possible to demonstrate that their antagonistic effect is due to specific binding to TNF-alpha and not to its receptor.
    Cytokine 02/1999; 11(1):37-44. · 2.52 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The pleiotropic cytokine tumour necrosis factor-alpha (TNF) is thought to play a central role in infectious, inflammatory and autoimmune diseases. Critical to the understanding and management of TNF-associated pathology is the development of highly specific agents capable of modifying TNF activity. We evaluated the ability of a high affinity mouse/human chimeric anti-TNF monoclonal antibody (cA2) to neutralize the in vitro and in vivo biological effects of TNF. cA2 inhibited TNF-induced mitogenesis and IL-6 secretion by human fibroblasts, TNF-priming of human neutrophils, and the stimulation of human umbilical vein endothelial cells by TNF as measured by the expression of E-selectin, ICAM-1 and procoagulant activity. cA2 also specifically blocked TNF-induced adherence of human neutrophils to an endothelial cell monolayer. Receptor binding studies suggested that neutralization resulted from cA2 blocking of TNF binding to both p55 and p75 TNF receptors on the cells. In vivo, repeated administration of cA2 to transgenic mice that constitutively express human TNF reversed the cachectic phenotype and prevented subsequent mortality. These results demonstrated that cA2 effectively neutralized a broad range of TNF biological activities both in vitro and in vivo.
    Cytokine 02/1995; 7(1):15-25. · 2.52 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of a variety of human diseases including septic shock, cachexia, graft-versus-host disease and several autoimmune diseases. Monoclonal antibodies directed against TNF provide an attractive mode of therapeutic intervention in these diseases. We have generated a murine monoclonal antibody (A2) with high affinity and specificity for recombinant and natural human TNF. To increase its therapeutic usefulness, we used genetic engineering techniques to replace the murine constant regions with human counterparts while retaining the murine antigen binding regions. The resulting mouse-human chimeric antibody should have reduced immunogenicity and improved pharmacokinetics in humans. Molecular analysis of light chain genomic clones derived from the murine hybridoma suggests that two different alleles of the same variable region gene have rearranged independently and coexist in the same hybridoma cell. The chimeric A2 antibody (cA2) exhibits better binding and neutralizing characteristics than the murine A2 which was shown to contain a mixture of two kappa light chains. The properties of cA2 suggest that it will have advantages over existing murine anti-TNF antibodies for clinical use.
    Molecular Immunology 12/1993; 30(16):1443-53. · 2.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: HA-1A, a human IgM mAb, has been shown to significantly reduce mortality in septic patients with Gram-negative bacteremia, especially those with septic shock, in a controlled clinical trial. To confirm the reported specificity of this antibody for the lipid A domain of endotoxin, several assay systems were developed. These assay systems included an ELISA, which measured the binding of HA-1A to lipid A adsorbed to a solid phase; a rate nephelometry assay, which measured the ability of HA-1A to bind and aggregate lipid A in solution; and a dot-blot immunoassay, which measured the ability of HA-1A to interact with lipid A adsorbed to Immobilon-P. In all three assay systems, HA-1A bound in a dose-dependent manner to lipid A prepared from Salmonella minnesota R595 LPS, whereas negative control human IgM mAb or polyclonal antibodies did not. Several experimental approaches were employed to demonstrate the specificity of HA-1A in these assay systems. Both polymyxin B and murine IgG mAb (8A1) with a specificity for lipid A were able to competitively inhibit HA-1A reactivity with lipid A in a dose-dependent manner. Furthermore, a murine IgG anti-Id mAb (9B5.5) developed against HA-1A was also able to block the binding of HA-1A to lipid A in these assay formats. HA-1A reactivity with synthetic lipid A confirmed that HA-1A binding to the natural lipid A was not the result of contaminants in the latter. Finally, the reactivity of HA-1A against a variety of glucosamine-containing and fatty acid-containing compounds was assessed. Some weak interaction was seen with cardiolipin and chitin, but not with serum proteins, lipoteichoic acid, or DNA. Collectively, these results conclusively establish that HA-1A binds to the lipid A region of LPS by an interaction with the V region of the antibody.
    The Journal of Immunology 06/1993; 150(10):4438-49. · 5.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Clinical data suggest that the human immunoglobulin M antiendotoxin antibody HA-1A reduced mortality in patients diagnosed with gram-negative bacteremia and bacteremia with shock. Previous studies have demonstrated that HA-1A binds to the lipid A domain of lipopolysaccharide (LPS). The present study evaluated the ability of HA-1A to interact with LPs isolated from various strains of gram-negative bacteria by using liquid-phase rate nephelometry and solid-phase immunoblotting assays. HA-1A formed immune complexes in solution with LPSs isolated from both rough and smooth gram-negative organisms. Western blot (immunoblot) analysis of these LPS preparations revealed that HA-1A bound to LPS isolated from rough gram-negative organisms and to a rough LPS-like component present in smooth LPS. HA-1A also bound to LPS-protein complexes found in certain commercial rough LPS preparations. Preincubation of HA-1A with lipid A completely blocked subsequent binding of HA-1A to LPS in both liquid- and solid-phase assay formats, suggesting that the interaction of HA-1A with LPS is through the lipid A domain. Evidence that the binding of HA-1A to LPS was mediated through the antigen-combining (Fv) region of the antibody was provided by the finding that a murine anti-idiotypic antibody to HA-1A inhibited binding. These findings suggested that the broad antiendotoxin reactivity exhibited by HA-1A appeared to be due to the ability of HA-1A to bind to the conserved lipid A moiety of LPSs derived from both smooth- and rough-phenotype gram-negative bacterial strains.
    Infection and Immunity 06/1993; 61(5):1756-63. · 4.07 Impact Factor