James R. Dasch

Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives.

Whereas xenogeneic collagen has provided a safe and effective biomaterial for numerous medical applications, there are few instances in which data permit the correlation of the immunologic profile of well-defined devices with their clinical sequelae. A major exception is the use of injectable bovine dermal collagen for soft-tissue contour correction. The low incidence of hypersensitivity has been studied in the context of clinical efficacy and safety with several devices. The findings indicate that such immunity usually results in the manifestation of local symptoms of dermal inflammation at sites of treatment that resolve as the implant is resorbed by the host. In contrast, more immunogenic hemostatic agents may elicit a more frequent or vigorous immune response that is not clinically visible or relevant in that application. Recent experiences with collagen-based devices for the repair and regeneration of bone have also demonstrated that the presence of immunity to their collagenous or non-collagenous components does not necessarily predict adverse clinical sequelae. Indeed, numerous specific data indicate that this immunity can exist as an epiphenomenon with no effect on osteogenesis. To get a true composite picture of biocompatibility, significant steps must be taken to characterize biomaterials properly and to ensure that immunologic, clinical, histologic, and other pertinent laboratory data are viewed in relation to one another and not in isolation.

Transforming growth factor-βs are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.

Effects of trifluoperazine, a calmodulin antagonist, on rabbit T- and B-cell responses to mitogens and antigen☆

Trifluoperazine (TFP), an inhibitor of the calcium-binding protein, calmodulin (CaM), was used to assess the role of calmodulin in the responses of rabbit lymphoid cells to stimulation with mitogen and antigen. After binding goat anti-rabbit Fab antibody, rabbit B cells lose their surface immunoglobulin (Ig) through endocytosis and then reexpress this protein during the next 24 hr. This reexpression was markedly inhibited by TFP. The brief and early addition of TFP markedly inhibited the increased [3H]thymidine (Tdr) uptake by rabbit T cells treated with concanavalin A and B cells exposed to anti-Fab. TFP greatly inhibited the induction by keyhole limpet hemocyanin (KLH) of the in vitro syntheses of antibody, Ig, and protein by KLH-primed lymph node cells (LNC). The earlier the TFP the greater was the inhibition of induction of these syntheses. However, once induced, synthesis and secretion of antibody were not inhibited by TFP. In striking contrast to the inhibition by TFP of the mitogenic and antigenic responses of lymphoid cells was the lack of effect of this drug on resting lymphocytes. Since TFP was not cytotoxic for either resting or mitogen- or antigen-stimulated LNC, it is highly unlikely that the observed inhibitory effects of this drug were due to its cytotoxicity. We postulate that an early signal for the activation of LNC proliferation, differentiation, and the syntheses of antibody, Ig, and protein involves a calcium-CaM-mediated reaction. Based on this work and that of others, the calcium-CaM complex may mediate an interaction between the ligand-occupied surface receptor and the cytoskeleton.

Characterization of monoclonal antibodies recognizing bovine bone osteoglycin

Six monoclonal antibodies (mAb) are described that bound to the bovine bone glycoprotein, osteoglycin. The protein osteoglycin was originally called Osteoinductive Factor (OIF). The antibodies were characterized with respect to their reaction patterns in Western blots, indirect immunoprecipitation, and binding epitope. The antibodies bound to one of two sequential determinants, either residues 62-76 or 95-105, in the C-terminal region of mature osteoglycin. One mAb, 2C11, was found to be useful for affinity purification of osteoglycin. Another mAb, 3B2, was able to immunohistochemically stain osteoblasts, osteocytes, chondrocytes, occasional osteoclasts and nail bed epithelial cells in rat neonatal forelimb. The mAbs will provide an essential tool for the further characterization of this unique glycoprotein.