Analysis of IL-6, IL-10 and IL-17 genetic polymorphisms as risk factors for sepsis development in burned patients.

Infection risk, sepsis and mortality after severe burn are primarily determined by patient age, burn size, and depth. Whether genetic differences contribute to otherwise unexpected variability in outcomes is unknown. We sought to determine whether there was an association between IL-6, IL-10 and IL-17 polymorphisms with cytokine production and development of sepsis. We evaluated 71 patients with burns ≥15% TBSA and 109 healthy subjects. The genotypes of IL-6 (-174C/G), IL-10 (-819C/T and -1082A/G) and IL-17 (7488T/C) polymorphisms were identified applying polymerase chain reaction protocols. The cytokine levels in serum were determined with enzyme-linked immunoabsorbent assays. Our results demonstrated no significant differences in the genotype frequencies studied between burn patients and healthy subjects. No significant associations were found among IL-6 and IL-17F genotypes and the related cytokine serum levels. Only IL-10 promoter -1082GG genotype was related to an increased IL-10 production in burned patients. In addition, septic subjects bearing -1082G/G genotype have shown the highest and non-septic bearing -1082A/* genotypes the lowest IL-10 serum levels. All together these data seem to indicate that genetically determined individual difference in IL-10 production might influence the susceptibility to septic complications in burned patients and suggest that these markers might be useful in burned patient management.

Differential regulation of Fas-mediated apoptosis in both thyrocyte and lymphocyte cellular compartments correlates with opposite phenotypic manifestations of autoimmune thyroid disease.

Several mechanisms are probably involved in determining the evolution of autoimmune thyroid disease (AITD) towards either hypothyroidism and the clinical syndrome known as Hashimoto's thyroiditis (HT) or toward hyperthyroidism and the symptoms of Graves' disease (GD). To gain further insight into such mechanisms we performed an exhaustive comparative analysis of the expression of key molecules regulating cell death (Fas, Fas ligand [FasL], Bcl-2) and apoptosis in both thyrocytes and thyroid infiltrating lymphocytes (TILs) from patients with either GD or HT. GD thyrocytes expressed less Fas/FasL than HT thyrocytes, whereas GD TILs had higher levels of Fas/FasL than HT TILs. GD thyrocytes expressed increased levels of the antiapoptotic molecule Bcl-2 compared to the low levels detected in HT thyrocytes. The opposite pattern was observed in GD (low Bcl-2) and HT (high Bcl-2) TILs. The patterns of apoptosis observed were consistent with the regulation of Fas, FasL, and Bcl-2 described above. Our findings suggest that in GD thyroid the regulation of Fas/FasL/Bcl2 favors apoptosis of infiltrating lymphocytes, possibly limiting their autoreactive potential and impairing their ability to mediate tissue damage. Moreover, the reduced levels of Fas/FasL and increased levels of Bcl-2 should favor thyrocyte survival and favor the thyrocyte hypertrophy associated with immunoglobulins stimulating the thyrotropin (TSH) receptor. In contrast, the regulation of Fas/FasL/Bcl2 expression in HT promotes thyrocyte apoptosis, tissue damage, and a gradual reduction in thyrocyte numbers leading to hypothyroidism. These findings help define key molecular mechanisms contributing to the clinical outcome of thyroid autoimmunity.

Tetracycline inhibits the nitric oxide synthase activity induced by endotoxin in cultured murine macrophages.

Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation. For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM. The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events. The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription. The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds.

Effect of partially modified retro-inverso analogues derived from C-reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages.

1. The ability of three modified tetrapeptides, representing fragments of the C-reactive protein (CRP) sequence and stabilized in the first peptide bond by retro-inverso modification, to affect the secretion of nitric oxide (NO) was studied in macrophages of BALB/c mice. 2. These tetrapeptides, resembling the aminoacid sequence of tuftsin (CRP 1, H-gThr-(R,S)mLys-Pro-Leu-OH, ITF 1192; CRP II, H-gGly-(R, S)mLys-Pro-Arg-OH, ITF 1127; CRP III, H-gThr-(R,S)mLys-Pro-Gln-OH. ITF 1193), were able to induce NO synthesis by peritoneal macrophages in a dose-dependent manner; the most stimulating dose was 1000 ng ml-1 for CRP II and 100 ng ml-1 for CRP I and CRP III. NO synthesis was not strictly dependent on lipopolysaccharide (LPS) activation. 3. The enhanced effect of retro-inverso CRP-related analogues on the expression of iNOS (inducible NO synthase) was confirmed by higher levels of iNOS activity in the cytosol and by the increase in iNOS protein, as evaluated by Western blot analysis, in macrophages stimulated by CPR compared with untreated ones. 4. The production of NO by retro-inverso CRP-peptide analogues was significantly inhibited by dexamethasone (20 microM), NG-monomethyl-L-arginine (L-NMMA) (500 microM) and pyrrolidine dithiocarbamate (PDTC) (100 microM). 5. Retro-inverso CRP-peptide analogues stimulated macrophages to produce high levels of interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) in the presence of LPS. 6. Retro-inverso CRP-peptide analogues stimulated NO synthesis by the enhancement of endogenously produced IL-1 and TNF-alpha, as the treatment of peritoneal macrophages with LPS in the presence of neutralizing anti-IL-1 and anti-TNF monoclonal antibodies (mAbs) reduced retro-inverso analogue-induced NO secretion. Data indicate a predominant role for IL-1 alpha in the induction of NO secretion by retro-inverso analogues. 7. These results suggest that retro-inverso CRP derived analogues act as costimulators of NO and cytokine synthesis in macrophages. The mechanisms by which they cause iNOS induction appear to be strongly dependent on the activation of nuclear factor-kappa B (NF-kappa B).

HLADR5 and C4BQO high frequency and antinuclear antibodies positivity in patients with 21 hydroxylase deficiency from Campania region.

HLA haplotypes, complement C4 factor and factor B immunochemical concentrations and autoantibodies titer have been studied in six patients with mild congenital adrenal hyperplasia (MC-AH), in two patients with classical congenital adrenal hyperplasia (CCAH) and in their parents. A high frequency of DR5 and C4BQO alleles have been found in MCAH patients. Moreover, C4BQO allele is carried out in three out of four cases associated with DR5. In the two CCAH patients we found a B51 and a B14 allele, the last one usually described in the non classical form of the disease in population of different ethnic origin. Signs of autoimmunity in some patients and parents have been found. C4 null alleles were several-fold more frequent among our patients with respect to the same ethnic control group and the autoantibody positivity could be the result of an altered immune regulation. The presence of a positive correlation between cortisol basal levels and C4 and Bf concentrations in the six MC-AH patients suggests an interrelationship between hormonal factors and immunological findings in this disease. Our finding about HLA antigens not previously described in this syndrome may stimulate more profound studies by genomic and cDNA probes.