Electrochemically reduced graphene oxide on CoCr biomedical alloy: Characterization, macrophage biocompatibility and hemocompatibility in rats with graphene and graphene oxide.

Electrochemically reduced graphene oxide (ErGO) films on a biomedical grade CoCr alloy have been generated and characterized in order to study their possible application for use on joint prostheses. The electrodeposition process was performed by cyclic voltammetry. The characterization of the ErGO films on CoCr alloys by XPS revealed sp2 bonding and the presence of CO and CO residual groups in the graphene network. Biocompatibility studies were performed with mouse macrophages J774A.1 cell cultures measured by the ratio between lactate dehydrogenase and mitochondrial activities. An enhancement in the biocompatibility of the CoCr with the ErGO films was obtained, a result that became more evident as exposure time increased. Macrophages on the CoCr with the ErGO were well-distributed and conserved the characteristic cell shape. In addition, vimentin expression was unaltered in comparison with the control, results that indicated an improvement in the CoCr biocompatibility with the ErGO on the material surface. The in vivo response of graphene and graphene oxide was assessed by intraperitoneal injection in wistar rats. Red blood cells are one of the primary interaction sites so hemocompatibility tests were carried out. Rats inoculated with graphene and graphene oxide showed red blood cells of smaller size with a high content in hemoglobin.

[125I]diiodoinsulins. Binding affinities, biologic potencies, and properties of their decay products.

UNLABELLED: Insulin was iodinated with 0.3-0.4 mol 125I/mol insulin using the lactoperoxidase method. About one-third of the radioactivity incorporated into insulin was in diiodoinsulins and about 40% of these molecules contained diiodotyrosine in residue 14 of the A chain. Most of the remaining molecules contained one A14-monoiodotyrosine and one monoiodotyrosine in either position A19, B16, or B26. The binding affinity and biologic potency of this heterogeneous diiodoinsulin preparation was not significantly different from that of A14-[125I]monoiodoinsulin in rat adipocytes, whereas it was slightly reduced in hepatocytes and IM-9 lymphocytes. From the iodine distribution and previous data on the binding affinity of each of the four monoiodoinsulin isomers it was calculated that A14-diiodotyrosine-insulin possesses full binding affinity and biologic potency in adipocytes. Diiodoinsulins isolated from another iodoinsulin preparation (iodate method) contained 58% A19-diiodotyrosine-insulin, and most remaining molecules contained one A19-monoiodotyrosine. The binding affinity of this mixed diiodoinsulin preparation was approximately one-fourth of that of A14-monoiodoinsulin in adipocytes, IM-9 lymphocytes, and hepatocytes. It was calculated that A19-diiodotyrosine-insulin is nearly devoid of binding affinity. The diiodoinsulins (lactoperoxidase method) decayed to iodide (probably from diiodotyrosine-insulin) or to polymers with little specific but a markedly increased nonspecific binding. In addition, the polymers had a marked tendency to adsorb to cellulose acetate filters. CONCLUSIONS: 1. The binding affinities of diiodoinsulins range from very low values to values at least as high as that of insulin depending on the positions of the iodine moieties. 2. The relative binding affinities vary among tissues. 3. Polymeric decay products give high nonspecific binding.

Antibodies to dietary antigens in rheumatoid arthritis--possible molecular mimicry mechanism.

Antibodies in serum from some patients with rheumatoid arthritis, recognize bovine albumin present in the milk, as determined by immunoprecipitation analysis from 125I-milk extracts. This antigen was also immunoprecipitated from bovine sera. These and ELISA studies showed that BSA is preferentially recognized over other proteins present in the milk. Panel studies demonstrated that although the average reactivity for BSA was high, only one third of the sera tested displayed a reactivity above the mean. The possibility of a molecular mimicry mechanism in RA between this food antigen and other human antigens was investigated. A sequence alignment analysis showed that the residues 141-157 of bovine albumin significantly differed from the corresponding fragment of human albumin, but were highly homologous with human collagen type I, C1q and vitamin D binding protein. In support of the immunogenicity of this fragment, we found that representative RA sera displayed a specific reactivity for a synthetic peptide containing the BSA residues responsible for the homology. Furthermore, most of the epitopes recognized on BSA by the RA sera seem to be conformationally dependent as heat denaturation or reduction followed by alkylation lead to a diminished recognition.

Autoantibodies from rheumatoid arthritis patients recognize antigens on the synoviocyte surface.

We have found autoantibodies in the sera from rheumatoid arthritis (RA) patients which recognize two cell surface antigens of approximately 70 kDa and 28 kDa from synoviocyte extracts as detected by immunoprecipitation analysis. These polypeptides were immunoprecipitated from extracts containing mainly macrophage-like synoviocytes (type A) but not from extracts of homogeneous fibroblast-like synoviocytes (type B). These autoantigens are not selectively expressed by RA synoviocytes, since both RA and non-rheumatoid synovia were reactive for RA sera. From the panel of different RA sera tested, 64% immunoprecipitated the 70 kDa band, and 27% recognized the 28 kDa polypeptide. These differences in the specificity of the sera seemed to be related to the clinical state of the donor. The sera from patients suffering from other autoimmune diseases such as autoimmune thyroiditis and systemic lupus erythematosus (SLE) do not appear to be reactive for these specificities, but sera from patients with Sjögren's syndrome, psoriatic arthritis, and Crohn's disease showed a weak cross-reactivity with the 70 kDa polypeptide. This autoreactivity against synovial cells in RA supports the idea that these cells participate in the initial immune response of the disease.

Biochemical analysis of synoviocytes from normal and arthritic rats. Evidence for an activated state associated with adjuvant polyarthritis.

Adjuvant-induced polyarthritis in rats is a common model system used for the study of the synovitis that occurs in rheumatoid arthritis. Synoviocytes A, the major cell type covering the internal surface of the joint, could be involved in the pathogenesis of rheumatoid arthritis because of their increased proliferation and the intraarticular manifestations of the disease. So far only a few molecular studies have been reported on synoviocytes upon arthritis induction. We report here changes in polypeptides, between control and arthritic synoviocytes, by using two different radiolabeling methods and two-dimensional gel electrophoresis analysis. Major differences were found using metabolic labeling on regions of tropomyosins, cyclin, tubulins and vimentin. In addition, external surface labeling of the cells with lactoperoxidase showed clear differences between control and arthritic synoviocytes in the region of 77-100-kDa proteins. Some of these differences can be reproduced by certain macrophage activators such as phorbol myristate acetate and lipopolysaccharide acting on synoviocytes in vitro and in vivo respectively. These results exclude the possibility that the changes observed were due to a possible infiltration of other cell types in the arthritic synovia and strongly support the existence of an activated state of synoviocytes associated with arthritis induction.

Evidence for common antigens on human non-adherent synoviocytes (type A) and peripheral monocytes.

The characterization of a homogeneous non-adherent synoviocyte (Type A) cell population (greater than or equal to 95%) from non-rheumatoid patients by culturing the cells in the presence of forty percent foetal calf serum is reported. These cells were able to phagocyte latex beads, iron particles, fluoresceinated zymosan and yeast. Furthermore, non-adherent synoviocytes were capable of being infected by the obligate intracellular parasite of peripheral monocytes Leishmania donovani. Indirect immunofluorescence experiments with specific anti-human monocyte (OKM1) antibody and specific antisynoviocyte serum, showed the presence of common surface structures between synoviocytes A cells and peripheral monocytes. Fifty five percent of the synoviocytes were also positive for HLA Dr antiserum. Analysis by two dimensional gel electrophoresis showed that peripheral monocytes and synoviocytes secreted identical polypeptides in vitro. These results strongly suggest a relationship between synoviocytes A and mononuclear phagocyte system.

Synoviocytes type A bind exogenous antigens recognized by antibodies present in rheumatoid arthritis.

The presence of antibodies in rheumatoid arthritis (RA) patients to antigens on the synoviocyte surface has recently been reported (Scand. J. Immunol. 27, 295, 1988). Here we have further characterized these antigens and found that they are exogenous proteins acquired from the bovine serum used in the culture medium. By immunoprecipitation and ELISA studies, we have identified bovine albumin and transferrin as the antigens recognized by the RA antibodies. These specificities were found not only in the sera but also in the synovial fluid from RA patients. A comparative study with a large panel of RA sera did not show a correlation in the antibody specificities for bovine albumin, bovine transferrin, or the 65-kDa heat shock protein from Mycobacterium bovis. Similar experiments using rabbit and monkey sera as well as human synovial fluid and serum as a source of antigen did not reveal any reactivity with a highly positive RA serum. By sequence alignment, a high degree of homology between residues 142-156 from bovine albumin and residues 65-78 from human pro-collagen alpha 1 (I) was found. The capacity of the synoviocytes to bind exogenous antigens and the presence of antibodies to bovine proteins, normally present in the diet, suggest a role for these type A synoviocytes as well as a possible involvement of food antigens in the pathogenesis of RA.