P. Mahieu

Tumour necrosis factor (TNF) gene polymorphism influences TNF-α production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans

TNF‐α is involved in infectious and immuno‐inflammatory diseases. Different individuals may have different capacities for TNF‐α production. This might determine a predisposition to develop some complications or phenotypes of these diseases. The aims of our study were to assess the inter‐individual variability of TNF‐α production and to correlate this variability to a single base pair polymorphism located at position −308 in TNF gene. We studied 62 healthy individuals. TNF‐α production after LPS stimulation was evaluated using a whole blood cell culture model. The TNF gene polymorphism was studied by an allele‐specific polymerase chain reaction. Other cytokines produced in the culture, soluble CD14 concentrations and expression of CD14 on blood cells were also measured. Among the 62 individuals, 57 were successfully genotyped. There were 41 TNF1 homozygotes and 16 TNF1/TNF2 heterozygotes. TNF‐α production after LPS stimulation of whole blood cell culture was higher among TNF2 carriers than among TNF1 homozygotes (929 pg/ml (480–1473 pg/ml) versus 521 pg/ml (178–1307 pg/ml); P < 0.05). This difference was even more significant after correction of TNF‐α production for CD14 expression on blood cells. In conclusion, the single base pair polymorphism at position −308 in the TNF gene may influence TNF‐α production in healthy individuals.

Synthesis of collagen and fibronectin by glomerular cells in culture.

The biosynthesis of collagen and fibronectin molecules by cultivated glomerular epithelial or mesangial cells was studied at confluency using radioactive proline or lysine as precursors. Collagen represented 0.5% of the total protein synthesized by the glomerular epithelial cells. About 60% of this collagenous protein were associated to the cell layer, whereas about 40% were secreted into the culture medium. Two major collagenous polypeptides were observed with apparent molecular weights of 185K and 170K, and were identified as two gene products of type IV procollagen. They exhibited ratios of 3- to 4-hydroxyproline, of total hydroxyproline to proline, and of hydroxylysine to lysine characteristic of type IV procollagen. They were degraded by bacterial collagenase. The patterns of peptides obtained after digestion of the 185K and 170K chains of this type IV procollagen with pepsin and V8 protease were identical to those obtained after digestion of type IV procollagen chains purified from a murine tumor (EHS sarcoma). Finally. a purified antibody to type IV collagen specifically immunoprecipitated the collagenous protein produced by the glomerular epithelial cells. By contrast, the mesangial cells synthesized about 5% of collagenous protein. 90% of this collagen were secreted into the cultured medium, whereas about 10% remained associated to the cell layer. Type I, III and IV procollagens were synthesized by the mesangial cells. Fibronectin was found in the medium and cell layer of both epithelial and mesangial cells. Fibronectin molecules were identified by their resistance to bacterial collagenase, their susceptibility to pepsin digestion, and their specific adherence to collagen. It was composed of disulfide-linked peptides of 220K daltons. The data therefore demonstrate that: (a) the glomerular epithelial and mesangial cells synthesize fibronectin molecules and type IV procollagen in vitro; (b) the cultivated mesangial cells also synthesize type I and III collagens. The implications of these findings in certain pathological circumstances, such as diabetes mellitus, are now being investigated.

Proliferative glomerulonephritis in rats: evidence that mononuclear phagocytes infiltrating the glomeruli stimulate the proliferation of endothelial and mesangial cells

Abstract. A proliferative, non‐crescentic, glomerulonephritis (GN) was induced in rats preimmunized with rabbit IgG by injecting a sub‐nephrotoxic dose of rabbit anti‐GBM IgG. Control rats either received anti‐GBM IgG only, or were totally irradiated (800 rads, kidneys protected) 2 days before the second injection. All the GN rats developed a severe proteinuria within 2–4 days after the injection of anti‐GBM IgG, contrarily to the control rats. At the same time, many mononuclear cells, of predominantly extra‐renal origin, infiltrated the glomeruli. Glomeruli were isolated from GN, normal and control rats and were cultivated in RPMI medium. In normal and control rat cultures, epithelial and mesangial cells were observed. In GN rat cultures, not only epithelial and mesangial cells, but also endothelial and macrophagic cells were identified; the outgrowth capacity of the mesangial cells was enhanced. These data were particularly evident in cultures of GN glomeruli isolated within 2–4 days after the induction of the renal disease, exactly when the glomeruli were infiltrated by a large number of mononuclear phagocytes. It is suggested that the mononuclear phagocytes infiltrating the glomeruli of rats with this model of GN stimulate the proliferation of endothelial and mesangial cells in vitro.

Increased Intestinal Permeability to (51 Cr) EDTA Is Correlated with IgA Immune Complex-Plasma Levels in Children with IgA-Associated Nephropathies

ABSTRACT. Intestinal permeability was investigated in 10 normal young adults, in 11 control children and in 9 children presenting with either Berger disease (4 cases) or Henoch‐Schönlein nephritis (5 cases), making use of (51 Cr) EDTA as a probe molecule. All subjects exhibited a normal creatinine clearance at the time of testing. After oral administration of (51 Cr) EDTA, 24‐hour urine radioactivity was measured and results were expressed in percentage of the oral dose administered. Means and SD were 2.35%±0.77, 2.51%±0.70, and 5.10%±2.35 for normal adults, control children and patients with IgA‐associated nephropathies, respectively. The differences of permeability between controls and patients were statistically significant (p < 0.01). In addition, a significant, direct, linear correlation has been established between the percentage of (51 Cr) EDTA excreted in 24‐hour urine and IgA immune complex‐plasma levels. Our results therefore support the hypothesis that increased gut permeability could play a role in the pathogenesis of IgA‐associated nephropathies.

Soluble Interleukin-2 Receptor in Crohn's Disease Assessment of Disease Activity and Prediction of Relapse *

In Crohns disease, the activity of the disease is sometimes difficult to evaluate and the evolution of the disease is difficult to predict. The soluble interleukin-2 receptor serum level has been reported to correlate with clinical activity of the disease and with mucosal immune activation. We compared serum soluble interleukin-2 receptor to classical inflammatory markers and other immune parameters in the assessment of clinical disease activity and prediction of relapse in patients with Crohns disease. Soluble interleukin-2 receptor serum levels correlated well with the Crohns disease activity index, and multivariate analysis showed that this correlation was independent of the other inflammatory and immune markers. The correlation was not greater, however, than that between some inflammatory markers, such as ESR, and Crohns disease activity index. Longitudinal follow-up showed that a high soluble interleukin-2 receptor serum level was highly predictive of relapse. Multivariate analysis showed that the soluble interleukin-2 recepteur serum level was complementary to other inflammatory, and clinical markers in the prediction of relapse of disease. We conclude that soluble interleukin-2 receptor is of use in monitoring Crohns disease, particularly in prediction of relapse.

Acute renal toxicity of doxorubicin (adriamycin)-loaded cyanoacrylate nanoparticles.

Acute doxorubicin-loaded nanoparticle (DXNP) renal toxicity was explored in both normal rats and rats with experimental glomerulo-nephritis. In normal rats, 2/6 rats given free doxorubicin (DX) (5 mg / kg) died within one week, whereas all control animals and all rats having received free NP or DXNP survived. A 3 times higher proteinuria appeared in animals treated with DXNP than in those treated with DX. Free NP did not provoke any proteinuria. Two hr post-injection, DXNP was 2.7 times more concentrated in kidneys than free DX (p<0.025). In rats with immuneexperimental glomerulonephritis, 5/6 rats given DX died within 7 days, in contrast to animals treated by DXNP, NP, or untreated, which all survived. Proteinuria appeared in all series, but was 2-5 times more intense (p>0.001) and prolonged after doxorubicin treatment (400-700 mg / day), without significant difference between DXNP and DX. Rats treated by unloaded NP behaved as controls. These results demonstrate that, in these experimental conditions, DXNP killed less animals than free DX, despite of an enhanced renal toxicity of the former. Both effects (better survival and nephrosis) are most probably related to an enhanced capture of DXNP by cells of the mono-nuclear phagocyte system, including mesangial cells.

Tissue Culture of Normal Rat Glomeruli

The biosynthesis of glycosaminoglycans (GAG) by cultivated rat glomerular epithelial and mesangial cells was studied by incorporation of [35S] sulfate or [14C] glucosamine for 48

Evidence that the interaction between circulating IgA and fibronectin is a normal process enhanced in primary IgA nephropathy

A solid-phase ELISA was set up to measure the direct binding capacity (BC) of different, commercially available, purified human IgA preparations to plates coated with human fibronectin (FN). It was found that secretory, polymeric, and, to a much lesser extent, monomeric IgA exhibited elevated FN-BC as compared to their BC to plates coated with bovine serum albumin. This binding was specific since not observed with human IgG or IgM antibodies. In addition, we noted that this interaction was dose dependent, Ca2+ dependent, saturable, and not covalent, was inhibited by soluble FN, but not by a prior incubation of FN-coated plates with anti-human fibronectin antibodies, and appeared to involve on the dimeric FN other structures than its heparin-binding, collagen-binding, or C1q-binding domains. Similar experiments conducted with normal plasma indicated that plasma IgA, but not plasma IgG or IgM, was also capable of significant binding to FN-coated plates. In contrast, serum IgA did not significantly bind to those plates under otherwise identical experimental conditions. Thus, the coagulation process induces a strong decrease in the FN-BC of circulating IgA, which implies the necessity of using plasma rather than serum to study such interactions. The apparent molecular weight of plasma IgA interacting with FN-coated plates ranged between 450 and 900 kd, and its major binding characteristics were quite similar to those observed with purified polymeric IgA. The FN-BC of plasma IgA was then measured by the same ELISA in 30 patients with primary IgA nephropathy (IgAN) and in 23 healthy controls. The mean FN-BC of plasma IgA was significantly higher in patients than in normal controls. This enhancement was due mainly to the augmentation in the concentration of circulating “macromolecular” IgA and was significantly correlated with the plasma levels of IgA-FN complexes. However, the pathogenetic role of these findings was probably not determinant since similar observations were made in alcoholic liver cirrhosis without urinary abnormalities and since the FN-BC of plasma IgA or the plasma levels of IgA-FN complexes were not correlated with the various biological parameters of evolutivity of primary IgAN. In conclusion, these studies suggest that the ability of polymeric IgA to directly bind to FN is involved in the formation of circulating IgA-FN complexes and that this normal binding process, although enhanced in IgAN, is probably not responsible for kidney injury, at least in the patients studied.

Decrease in systemic tolerance to fed ovalbumin in indomethacin-treated mice.

The oral administration of non-steroidal anti-inflammatory drugs (NSAID) to animals induces a quick increase in intestinal permeability and secondary inflammatory lesions of the intestine. The mechanisms leading to the inflammatory lesions are hypothetical. The increased intestinal permeability could allow a greater mucosal and systemic penetration of fed antigens and bacterial products leading to an abnormal mucosal and systemic immune and inflammatory response toward these materials. We examined the effect of oral dosing with indomethacin on ovalbumin serum levels and the systemic immune response to ovalbumin in mice fed with ovalbumin. The ovalbumin serum level was higher in indomethacin-treated mice and the increase was proportional to the dose of indomethacin. It was associated with epithelial and subepithelial lesions. Moreover, the systemic humoral and, to a lesser extent, the cellular tolerance were partially abrogated in the treated mice. These findings suggest that the oral administration of indomethacin in mice induces an increased passage of fed antigen through the intestinal epithelium associated with a decrease in systemic tolerance to this antigen. The reason for this decrease remains unclear. Besides a disequilibrium between systemic and mucosal immune responses, a loss of integrity of the intestinal epithelial cells and a direct immunomodulating effect of indomethacin may also be involved. This decrease in systemic tolerance to luminal antigen could be involved in the development of NSAID enteropathy.

Effects of methylprednisolone on the Fc-receptor function of human reticuloendothelial system in vivo.

Abstract. To determine whether the Fc‐receptor function of reticuloendothelial system (RES) is modified by corticosteroid administration, we studied the spleen to liver uptake ratios of autologous, 99Tc‐labelled heatdamaged or IgG‐coated erythrocytes, injected intravenously into 10 normal volunteers, 4 h after receiving a single dose of 32 mg of methylprednisolone by mouth.