Osteoblast response to thermally oxidized Ti6Al4V alloy.

We have recently reported that thermal oxidation treatments of Ti6Al4V at 500 degrees and 700 degrees C for 1 h result in the formation of an outer "ceramic" layer of rutile that do not decrease the high in vitro corrosion resistance of the alloy. In the present work, surface roughness was measured and found marginally increased as a consequence of oxidation of the alloy at 700 degrees C, but not at 500 degrees C. We have evaluated the biocompatibility of the oxidized surfaces, by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared with polished alloy, both thermal treatments increased osteoblast adhesion measured as cell attachment, beta1 integrin and FAK-Y397 expression, as well as cytoskeletal reorganization. Compared with treatment at 500 degrees C, thermal oxidation at 700 degrees C enhanced cell adhesion. Treatment at 700 degrees C transiently impaired cell proliferation and viability, which were not altered in alloys oxidized at 500 degrees C. Several markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, and mineralized nodule formation were found either unaffected or differentially increased by alloys treated either at 500 degrees or 700 degrees C. In addition, thermal oxidation at 700 degrees C also increased osteoprotegerin secretion. Taken together, our results indicate that thermal oxidation treatments at 500 degrees or 700 degrees C for 1 h improve the in vitro biocompatibility of Ti6Al4V.

In vitro corrosion behaviour and osteoblast response of thermally oxidised Ti6Al4V alloy.

In this work, the influence of thermal oxidation treatments of Ti6Al4V at 500 degrees C and 700 degrees C for 1 h on the in vitro corrosion behaviour and osteoblast response is studied. The potential of these treatments, aimed to improve the wear surface performance as biomaterial, relies in the formation of an outer "ceramic" layer of rutile. The corrosion behaviour was evaluated in simulated human fluids by electrochemical impedance spectroscopy and anodic polarisation tests. The effect of these thermal oxidation treatments on osteoblastic behaviour was studied in primary cultures of human osteoblastic cells. Results show that thermal oxidation treatments do not decrease the high in vitro corrosion resistance of the Ti6Al4V alloy. Osteoblast adhesion studies indicate that thermal oxidation treatments do not impair the material biocompatibility. Moreover, the thermal oxidation at 700 degrees C enhances the in vitro osteoblastic cell attachment compared to the thermal oxidation at 500 degrees C.

Molecular cloning and characterization of an IgE-reactive protein from Anisakis simplex: Ani s 1.

Ingestion of the parasitic nematode Anisakis simplex in undercooked fish can cause severe allergic reactions in some individuals. Using pooled human sera from sensitized patients we have probed an expression library for A. simplex antigens. One positive clone was found to encode a full length 21 kDa protein with strong homology to nematode troponins. The recombinant protein was expressed as a GST-fusion protein and found by immunoblot analysis to react with sera from 20% of allergic patients. The presence of functional EF-hand Ca(2+) binding motifs was demonstrated by gel-shift analysis.

CD48-deficient mice have a pronounced defect in CD4(+) T cell activation.

We have generated mice deficient in the expression of the lymphocyte cell surface antigen CD48 (Blast-1, BCM1, sgp-60) by gene targeting in embryonic stem cells. Mice homozygous for the CD48 mutation (CD48(-/-) mice) are severely impaired in CD4(+) T cell activation. Proliferative responses to mitogens, anti-CD3 mAb, and alloantigen are all reduced. Experiments in which T cells and antigen-presenting cells from either wild-type or CD48(-/-) mice were cocultured reveal that CD48 is important on both T cells and antigen-presenting cells. The most dramatic impairment was observed in experiments in which highly purified T cells were stimulated through the T cell receptor in the presence of the phorbol ester, phorbol 12-myristate 13-acetate. The results of these experiments raise the possibility that CD48 plays a role in signaling through the T cell receptor.

Presence of circulating antibodies against brush border antigens (Fx1A) in a patient with membranous nephropathy and bilateral pyeloureteral stenosis. Comparison with idiopathic membranous nephropathy.

In a patient with membranous nephropathy and bilateral pyeloureteral stenosis with hydronephrosis, we examined the possibility that an increase in the intratubular pressure could facilitate the passage of the Fx1A antigens to the circulation. Elevated serum anti-Fx1A antibodies were detected in this particular patient by ELISA on three occasions during the disease follow-up, even though he was in clinical remission. These antibodies reacted in vitro with the tubular brush border of a normal human kidney. The anti-Fx1A antibodies isolated from the patient's sera by affinity chromatography competed with the rabbit anti-Fx1A antisera binding to plates coated with human Fx1A antigen. In immunoblotting studies the isolated specific IgG antibodies from that patient reacted with a 180 kDa antigen of the human Fx1A and with less intensity with 75 kDa and 50-55 kDa polypeptides. In none of 12 patients with idiopathic membranous nephropathy could the circulating anti-Fx1A antibodies be demonstrated. On the whole, this particular case suggests that on some occasions increased intratubular pressure could cause the release of Fx1A antigens, facilitating an autologous immunocomplex nephritis. These antigens, by contrast, do not seem to play any role in most cases of membranous nephropathy in man.

Expression and synthesis of cross-reactive idiotypic antibodies by peripheral blood lymphocytes and their suppression by anti-idiotypes in patients with IgA nephropathy.

We have recently described that patients with IgA nephropathy present high serum levels of anti-BSA idiotypic antibodies that were well correlated with the existence of hematuria. Furthermore, these Id were found in circulating and renal deposited immune complexes. In the present work, we examined the expression of surface idiotypic determinants on PBL by flow cytometry and their in vitro production, using as reagent anti-idiotypic antibodies previously well characterized. The presence of cross-reactive Id-bearing cells was observed in 5 out of 6 patients studied, with frequencies ranging from 3 to 12% of lymphocytes. After 7 days of culture, the spontaneous synthesis of idiotypic antibodies by PBL was found elevated in 6 out of 13 (46%) patients. A major Id cell expression and production was noted in patients with active disease as defined by hematuria. The preincubation of PBL with 20 and 50 micrograms of anti-idiotypic antibodies/2 x 10(6) cells for 3 days induced a significant inhibition of cross-reactive Id production in a dose-dependent fashion, with a degree of suppression between 12 and 50% in five out of six patients studied. In the above assays, as negative controls, we used the anti-Id antibodies previously adsorbed on an Id-Sepharose column. On the whole, these results suggest that patients with IgA nephropathy present dysfunctions in the Id-Anti-Id network that could play an important role in the pathogenesis of this disease.

The sgp-60 molecule is linked to the plasma membrane via a glycosylphosphatidylinositol anchor.

We have recently identified a novel murine glycoprotein termed sgp-60, which is expressed on the cell surface of T and B lymphocytes. Because of the profound modulatory effects of sgp-60 on activation through the T cell receptor/CD3 complex, we have examined the membrane attachment domain of the molecule. sgp-60 is not expressed on the surface of variants of a T-T hybridoma cell line that are defective in glycosylphosphatidylinositol (GPI) anchor biosynthesis. In wild-type but not in mutant cells, sgp-60 can be labeled with palmitic acid. Furthermore, the molecule can be removed from the cell surface of both T and B lymphocytes by enzymatic digestion with a phosphatidylinositol-specific phospholipase C. We conclude that the sgp-60 molecule is linked to the plasma membrane via a GPI anchor.

Detection and characterization of circulating and glomerular immune complexes in experimental IgA nephropathy.

The different models of experimental IgA nephropathy described so far have provided insight into pathogenesis; however, the evidence for a role of IgA immune complexes (IC) has only been gained in passive systems. In an active model of IgA nephropathy, induced in mice by repeated injections of dextran, some of the mechanisms that could explain the formation of glomerular IgA deposits are studied in this report. Serum total IgA and anti-dextran IgA antibody levels increased significantly over the period of immunization. Only 13-30% of mice had total and/or specific IgA IC, determined by Raji cell and PEG assay in ELISA. Analytical ultracentrifugation showed that IgA IC were of small (7-13 S) or intermediate (13-17 S) size. There was a close correlation between total serum IgA levels and the presence of IC-containing IgA anti-dextran antibodies, with the existence of IgA in the mesangium. The percentage of animals (n = 76) with IgA mesangial deposits increased over the immunization period (88% at 10 weeks). Forty-three per cent of mice had polymeric IgA in the mesangium; by contrast, only 12% had dextran deposits. On the whole, these data suggest that in the dextran-induced IgA nephropathy, the glomerular IgA could be the result of circulating IgA complexes and/or IgA polymers deposition.

[New immunopathogenic aspects of IgA nephropathy]. / Nouveaux aspects immunopathogéniques de la néphropathie à IgA.

IgA nephropathy (Berger's disease) has become recognized worldwide as one of the most common of the primary glomerulonephritis. The mesangial granular deposits suggested an immune complex disease. The available data evidence that the IgA circulating immune complexes in these patients are heterogeneous. Recent analysis, performed after dissociating the complexes, found both IgA1 and IgG. In fact, high serum levels of IgA rheumatoid factor and shared antibody idiotypes were found in a large proportion of those patients. A close relationship was noted between the presence of cross-reactive idiotypes on mesangial immunoglobulins and the existence of increase levels of serum idiotypes and many patients have increased rates of IgA synthesis either spontaneously or after stimulation of the peripheral blood mononuclear cells by various mitogens. A lot of abnormalities on B and T lymphocytes, related with the IgA immune regulation, have been described. Most of deposited IgA in the mesangium is polymeric and belongs to the IgA1 class. Patients with IgA nephropathy have very often antibodies against exogenous and endogenous antigens. Among the most frequently found are the antibodies against dietary, viral and bacterial antigens as well as against the Fc and Fab portions of immunoglobulins, nuclear and glomerular antigens. The mechanisms of mesangial damage in IgA nephropathy are not well known. Mesangial cells are capable of producing and releasing various lipidic and proteic mediators. The stimulation of mesangial cells, cultured in vitro by IgA or IgG immune complexes induced the release of PAF, PGE2 and superoxide anion. A better knowledge of the mechanism implicated in the abnormality of IgA immune regulation, as well as of the glomerular inflammation response could afford a new therapeutic approach to this nephropathy.

Beneficial effect of fibronectin administration on chronic nephritis in rats.

We studied the effect of fibronectin (FN) on the course of chronic nephritis (induced by daily injections of ovalbumin) and on the clearance and catabolism of immune complexes in Wistar rats. Rats with chronic nephritis were treated with FN (2.5 mg/kg/48 hours) for 15 days after proteinuria was first detected. In rats with untreated nephritis, urinary protein levels increased from 40 +/- 22 mg/day (mean +/- SD) to 339 +/- 68 mg/day during the 15 days of the study (P less than 0.0005). This statistically significant increase was not observed in rats treated with FN (mean +/- SD 58 +/- 46 mg/day to 124 +/- 112 mg/day). Rats treated with FN showed a higher total serum protein level than did the untreated animals (mean +/- SD 6.4 +/- 0.3 gm/dl versus 5.1 +/- 0.5 gm/dl; P less than 0.0125), as well as a significant reduction in mesangial and glomerular basement membrane deposits. Untreated nephritic rats demonstrated delayed plasma clearance of 125I-labeled aggregated IgG (plasma half-life [T1/2] 3.03 +/- 0.6 minutes) and less catabolism of these aggregates at 30 minutes (mean +/- SD 15 +/- 1.7%) than did the normal rats (T1/2 1.5 +/- 0.2 minutes, 22 +/- 2.8%, respectively; P less than 0.0005). Both parameters were within normal limits in the FN-treated rats (T1/2 1.6 +/- 0.4 minutes, 22 +/- 6%, respectively). In vitro, FN induced a significant increase in aggregated IgG catabolism by Kupffer cells and peritoneal macrophages from normal rats. These results show that FN reduces the proteinuria and histologic lesions of chronic nephritis in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated plasma fibronectin levels in rats with immune and toxic glomerular diseases.

We measured plasma fibronectin levels by a rocket immunoelectrophoresis in rats with chronic serum sickness induced by repeated injections of ovalbumin and in rats with epithelial nephropathy induced by a single injection of adriamycin. In the early phases of the immune model, rats presented granular deposits of IgG in the mesangial area with no or descrete proteinuria (less than 40 mg/24 h). Fibronectin levels in that group were significantly higher (450 +/- 90 micrograms/mL) than in normal rats of the same age (350 +/- 46; p less than 0.01). When animals presented IgG deposits in the capillary wall, an important nephrotic syndrome developed in most of them. Fibronectin levels then increased very significantly (863 +/- 153 micrograms/mL; p less than 0.0005). In the model of adriamycin nephropathy, fibronectin significantly increased (580 +/- 110 micrograms/mL; p less than 0.0005) from the first week, when proteinuria was in a range 40-60 mg/24 h. However, the levels were higher (860 +/- 175 micrograms/mL; p less than 0.0005) when a complete nephrotic syndrome developed. At this time, plasma fibronectin levels correlated directly in both models with the degree of proteinuria and inversely with the total serum protein concentration. Our results show that plasma fibronectin levels increased very early in animals with immune and toxic damage of the kidney. The highest elevated values found thereafter, when a full nephrotic syndrome was present, suggest an increased synthetic rate of that glycoprotein linked to that situation.

Defective hepatic handling of IgA immune aggregates by mice with experimental IgA nephropathy.

In an experimental model of IgA nephropathy induced in mice by chronic immunization with dextran, we tested the hypothesis that a defect in the hepatic handling of IgA could be an important determinant in the deposition of IgA in the mesangium. In mice injected with 1-16 doses of 1 mg of dextran (after a preimmunization period of 21 days) the blood clearance of IgA immune aggregates was significantly delayed in relation to control animals, becoming normal at 24 injections. This alteration seems specific since the clearance of IgG aggregates was normal. The percentage of isolated hepatocytes with Fc receptors for IgA decreased significantly over the whole period of dextran immunization. The binding rate of 125I-IgA aggregates to hepatocytes of mice with 24 dextran injections was twice lower than that of control animals. By contrast, the percentage of Kupffer cells with IgA receptors increased over ensuing dextran injections. A progressive increase in the IgA blood levels and in the percentage of mice with mesangial IgA deposits was seen along the period of study. At 24 injections most animals presented moderate to intense mesangial proliferation and abundant electron-dense deposits. On the whole, these data suggest that the early impairment in the liver IgA clearance capacity observed in these animals could facilitate the presence of circulating immune complexes (IC) and their deposition in the mesangium. The increase in serum IgA, seen thereafter, together with the normalization of the IgA clearance capacity, suggest that other pathophysiological mechanism(s) (e.g. in situ IC formation or IgA polymers deposition) must also be involved in this model of experimental IgA nephropathy.

Characterization of circulating idiotypes containing immune complexes and their presence in the glomerular mesangium in patients with IgA nephropathy.

The possible pathogenic role for idiotype-anti-idiotype interactions in kidney diseases has recently been suggested. Since patients with IgA nephropathy often present antibodies against alimentary antigens, like bovine serum albumin (BSA), we isolated an idiotypic antibody with BSA specificity from one of these patients. By means of a specific anti-idiotypic antibody raised in rabbits, we have studied the participation of these idiotypes in circulating and renal deposited immune complexes (IC) in patients with IgA nephropathy. On indirect immunofluorescence, the presence of cross-reactive idiotypes was detected in the glomeruli of 12 out of 42 (28%) patients with IgA nephropathy, but in none of 15 membranous or mesangiocapillary nephritis examined. The staining was located within mesangial and paramesangial areas, with a similar, but less intensive, pattern distribution than IgA. Previous adsorption of rabbit anti-idiotype antibodies on an idiotype-Sepharose column completely abolished that staining. A close relationship was found between the presence of cross-reactive idiotypes on mesangial immunoglobulins and the existence of increased levels of serum idiotypes and idiotype-containing IC. Serum analytical ultracentrifugation showed that circulating IC containing idiotypes have chiefly a large (greater than 19 S) and intermediate (13 S-19 S) size, while those containing anti-BSA antibodies were only between 7 S-13 S fractions, or absent. Our results suggest that in patients with IgA nephropathy, shared idiotypes participate in the formation of circulating and renal deposited IC. It is possible that the apposition of free anti-idiotype to idiotype already bound to glomeruli, and vice versa, could contribute to increasing the amount and size of mesangial immune deposits, and, therefore, facilitate or perpetuate tissue injury.

Presence of shared idiotypes in serum and immune complexes in patients with IgA nephropathy.

Patients with IgA nephropathy often present a large array of antibodies against diet antigens and this disease can be experimentally induced by alimentary antigens. In this report, we have described the isolation from a patient with IgA nephropathy of antibovine serum albumin (BSA)-antibody idiotypes that are specifically recognized by auto-and heteroantiidiotypic antibodies. The fact that antigen (BSA) but not monomeric or aggregated human IgG inhibited the binding of antiidiotypic antibodies to the idiotypes, suggested that the idiotypic determinants are in or near the antigen binding site and that it is not a rheumatoid factor. By means of the heteroantiidiotypic antibodies raised in rabbits we observed the presence of increased levels of shared idiotypes in serum and/or immune complexes (IC) of 48 out of 70 (68.5%) genetically unrelated patients with IgA nephropathy. The close correlation (P less than 0.005) between the presence of IgA-IC, measured by Raji cell assay, and the existence of high levels of serum idiotypes, suggest that a portion of circulating IC could consist of idiotype-antiidiotype. A strong concordance between the presence and levels of idiotypes and the clinical activity, as defined by the existence of haematuria, was also noted. The discrepancies and absence of correlation observed in our study among the levels of anti-BSA antibodies of different classes and serum levels of idiotypes, circulating IC and haematuria could suggest that the antibodies reacting with the heterologous antiidiotypic antibodies could be directed to other more pathogenic antigens than dietary antigens. All together, our results suggest that IgA nephropathy might belong to the group of diseases that occur in susceptible individuals with a limited potential in the immunological response repertoire.