1. A method is described for the trace iodination of immunoglobulins and other serum proteins by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. gammaG immunoglobulin that had been labelled to a specific radioactivity of 5muc/mug. by use of carrier-free [(125)I]iodide gave no evidence of denaturation when analysed by electrophoresis and density-gradient ultracentrifugation. 3. Tryptic hydrolysis and peptide ;mapping' of a completely characterized peptide radioiodinated by this method showed that the [(125)I]iodide was bound to tyrosyl residues. 4. Proteins differ in their susceptibility to iodination by this method. Human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions. 5. Lactoperoxidase catalyses the iodination of proteins much more readily than does horseradish peroxidase.

1. Radioactive iodide was covalently bound to living cells from normal mouse spleen and a variety of lymphoid tumours by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. About 3x10(5)-6x10(5) molecules of [(125)I]iodide/cell could be incorporated without affecting cell viability. 3. Electron-micrographic radioautography showed that the radioactive label was associated with the outer surfaces of the cells. 4. Radioiodinated proteins were solubilized in 9m-urea-0.2m-mercaptoethanol and analysed by gel-filtration and disc electrophoresis. 5. Comparison of distinct tumour lines by disc electrophoresis showed qualitative and quantitative differences in protein distribution patterns.

We used mapping with synthetic overlapping peptides in combination with molecular modeling to analyze the IgG antibodies that humans naturally produce against human T-cell receptor .8 chains and to localize the recognized peptide autoantigens in the three-dimensional structure of the molecule. Healthy individuals produce low levels of antibodies against T-cell receptor peptides, and these can be increased in autoimmune diseases. We characterized the reactivities in detail because IgG molecules reactive with self peptides occur in preparations of intravenous immunoglobulin and can be isolated by immunoaffinity chromatography. Natural IgG antibodies were directed against three major peptides. One corresponds to the first complementarity-determining region of the variable region. A second corresponds to the third framework of the variable region. The third is located in the constant region and is predicted to be a loop that extends out of the 18-barrel structure. This peptide is one that would give a characteristic structural distinction between the fl-chain constant region and the constant regions of immunoglobulin light chains to which 13 chains are homologous. The capacity to bind these peptides is found in small fractions of normal polyclonal IgG, which contains both Kc chains and A chains. The activity is antibody-like in being confined to the Fab fragment and in its capacity to discriminate among homologous synthetic peptides corresponding to distinct f-chain variable-region genes.We propose that a recognition and regulatory process naturally occurs that parallels the immune network for the regulation of the production of antibodies.Humans produce antibodies to a variety of self antigens without noticeable ill effects (1-4). The production of antibodies themselves is regulated in part by autoimmune responses to defined portions of the antibodies, most notably combining site-related markers or idiotypes (5, 6) and constant-region markers detected by IgM antibodies termed rheumatoid factors (7). Because of the crucial importance of immunoglobulin-like T-cell receptors (TCRs) in the initiation of specific immunity, we chose to focus on natural human antibodies to synthetic peptides based on human TCR /3-chain gene sequence. We assessed by enzyme-linked immunosorbent assay (ELISA) the capacity ofsera to react with synthetic TCR peptide autoantigens. These sera were obtained from ostensibly healthy individuals 20-90 years of age and from patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Hexadecapeptides representing the TCR YT35 ,3 chain, which contains V,8, D1.1, J01.2, and Cp1 segments (8), were overlapped by 5 residues to model the antigenic structures (9, 10) of the TCR P fragment homologous to immunoglobulin A light chain (11).We found that reaction of human IgG with particular synthetic TCR autoantigens occurs in low levels in healthy individuals and can rise in autoimmune diseases such as RA. We present immunochemical data characterizing the nature and locations of the T...

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

No abstract

Knowledge of the nature of the receptor for antigen and its role in the specific activation of lymphocytes is essential to the elucidation of the mechanisms of immune recognition and differentiation. The fact that antibody formation (1-5) and cellmediated immunity (6-9) can be inhibited by antiglobulin reagents suggests that this receptor shares antigenic determinants with immunoglobulin. Complete analysis of the lymphocyte receptor for antigen requires isolation of the molecule and direct measurement of its binding properties for antigen. Furthermore, the manner in which the receptor is attached to the cell surface and the mechanism by which combination with antigen activates immune differentiation must be ascertained.As an initial step in this chain of biochemical proof, we have developed a method for the covalent attachment of radioactive iodine to accessible surface proteins of living lymphocytes (10, l l). A sufficient amount of radioisotope was incorporated to facilitate fractionation of these surface proteins by a variety of biochemical and immunological means. We previously reported the isolation of low molecular weight "yM immunoglobulin from the surfaces of murine and neonatal human thymic lymphocytes (12). Uhr and his colleagues (13, 14) have independently employed a similar approach to isolate "7S" "yM from splenic lymphocytes of BALB/c mice.In the present communication we report the radioiodination, isolation, and partial characterization of surface immunoglobin from neonatal human thymic lymphocytes and a variety of murine lymphocyte populations. These lymphocyte populations were chosen to provide information on the nature of surface immunoglobulin associated with bone marrow-derived lymphocytes (B ceUs) t and thymus-influenced lymphocytes (T cells). Materials and MethodsCell Sources and Methods of Preparatlon.--Male and female CBA/H/Wehi and (CBA ;K C57BL)F1 mice, weighing 20-25 g each, were used as a source of normal spleens and thymuses.

The induction of T helper (TH) lymphocytes by distinct TH ligands results in a differentiation to TH1/TH2 subsets based on their unique pattern of cytokine secretion and effector functions. We hypothesized that the relative proportion of TH1/TH2 directly relates to cardiac fibroblast (CF) function and thereby cardiac extracellular matrix (ECM) composition and cardiac diastolic function in the absence of injury or altered wall stress. We compared the effect of selective TH1 with TH2 inducers on cardiac gene expression, ECM composition, and diastolic function in C57BL/J mice. Twelve weeks after immune modulation, the left ventricular stiffness (beta) was significantly increased in the TH1 group and decreased in the TH2 group (P < 0.01). The TH2 group also demonstrated significantly increased end-diastolic and end-systolic volumes (P < 0.01). Cardiac gene expression patterns for pro-matrix metalloproteinase (MMP)-9 and -13 were increased by greater than fivefold in the TH2 group and significantly decreased in the TH1 group (P < 0.05). The total cardiac collagen and cross-linked collagen were significantly increased in the TH1 group and decreased in the TH2 group (P < 0.01). Coculturing lymphocytes harvested from the treated mice with naive primary CF demonstrated a direct control of the lymphocytes on CF pro-collagen, pro-MMP gene expression, and MMP activity. These results suggest that the TH phenotype differentially affects diastolic function through modulating CF pro-collagen and pro-MMP gene expression, MMP activity, and cardiac collagen cross-linking, resulting in altered ECM composition. Thus modulation of TH lymphocyte function could promote adaptive remodeling in heart failure and postmyocardial infarction.

No abstract