Healing skin wounds were studied in a series of parabiotic rats . The femurs of one parabiont of each pair were shielded whilst both animals were given 800 r from a Co60 source . The animals were wounded 3 days after irradiation . Each animal with partially shielded marrow was then given tritiated thymidine intraperitoneally daily while the cross-circulation was arrested by clamping . After the thymidine-3 H had cleared the blood, the clamp was released. Animals were sacrificed, and wounds were prepared for radioautography 1, 2, and 6 days after wounding. In the wounds of the shielded animals thymidine-3H was observed in epidermis, endothelium, leukocytes, fibroblasts, and mast cells . Only neutrophilic leukocytes, monocytes, and lymphocytes were labeled, as determined by light and electron microscope radioautography, in the wounds of each nonshielded parabiont . None of the many fibroblasts present were found to contain label in the wounds of the nonshielded parabionts through the 6 day period . These observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells .
The properties of IgG and its subcomponents are being exploited to generate new therapeutics with selected biological activities. In this study, a series of truncated, humanized IgG1 antibodies was expressed in Chinese hamster ovary cells, to evaluate the contribution of structural components to glycosylation and function. The series includes L243 IgG1 (a-MHC Class II) lacking a C H 3 domain pair (DC H 3-IgG1), single-chain Fv fusion proteins with Fc or a hinge-C H 2 domain, Fc with/out a hinge, and a single C H 2 domain. Glycosylation of IgG Fc is important for recognition by effector ligands such as Fcg receptors. HPLC analysis of released and pyridylaminated oligosaccharides indicates that intact IgG1 and scFvFc antibodies are galactosylated and sialylated to levels similar to those observed previously for normal human IgG1. The truncated forms express increased levels of digalactosylated (30±83%) or sialylated (9±21%) oligosaccharide chains with the highest levels observed for the single C H 2 domain. These data show which architectural components influence IgG glycosylation processing and that the (C H 3) 2 pair is particularly influential. When MHC Class II bearing (JY) cells were sensitized with L243 DC H 3-IgG1, scFvFc, or scFvhC H 2 they elicited superoxide production, from U937 cells, at levels of 35±45% relative to that obtained for intact L243 IgG1 (100%). Mild reduction and alkylation of the hinge disulphide bonds of scFvhC H 2 greatly decreased its capacity to trigger superoxide production. Thus, the L243 scFvhC H 2 homo-dimer constitutes the minimal truncated form that binds the MHC Class II antigen and triggers superoxide production through FcgRI.Keywords: antibodies; architecture; Fcg receptors; glycosylation; superoxide.Antibodies are molecules that can bind specifically to antigens, and then activate one or more effector functions [1]. This results in the immobilization, removal and destruction of infecting microoganisms and their toxic products. The IgG class of antibody mediates many effector functions and is the predominant antibody isotype in the blood and interstitial fluids. The IgG molecule is composed of three essentially independent structural and functional regions; two Fab (antigen binding) regions linked by a flexible and exposed hinge to a third Fc region (Fig. 1). The Fc region has multiple binding sites for ligands such as Fcg receptors and C1q through which effector functions are activated, e.g. phagocytosis, oxygen free radical production, and classical complement pathway activation. Structurally, the Fc comprises a portion of the hinge with interheavy-chain disulfide bridges, two unpaired glycosylated C H 2 domains, and paired C H 3 domains. Structure within and proximal to the N-terminus of the C H 2 domain correlates with the expression of functions activated by immune complexes, mediated through each of the three leucocyte Fcg receptors and complement [2±7].Glycosylation of the C H 2 domain at Asn-297 is essential to activation of effector functions triggered by immune com...
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