Association of +3179G/A insulin-like growth factor-1 receptor polymorphism and insulin-like growth factor-1 serum level with systemic lupus erythematosus.

The insulin-like growth factor (IGF) system plays a prominent role in the regulation of immunity and inflammation. Inappropriate balance of IGF-1 signaling has been reported in autoimmune disorders. This study was designed to compare +3179G/A IGF-1R genotype distribution in 148 systemic lupus erythematosus (SLE) patients with a group of 240 healthy donors. We also investigated serum IGF-1 levels in SLE patients and healthy controls in an association to genotype. IGF-1 serum levels were measured by enzyme-linked immunosorbent assay and genotyping for the +3179G/A polymorphism was performed by restriction fragment length polymorphisms (RFLP)-polymerase chain reaction (PCR) assay. The higher frequency of homozygous genotype AA (22% vs. 17% with OR 1.319, 95% CI 0.71--2.44) and lower frequency of heterozygous genotype AG (42% vs. 46% with OR 0.698, 95% CI 0.38-1.27) were seen in cases versus controls. Serum IGF-1 levels were comparable between SLE patients and age- and sex-matched healthy donors, even when the groups was stratified according to +3179G/A IGF-1R genotypes. However, when patients were sub grouped according to the disease activity index (SLEDAI score), serum IGF-1 levels were increased in patients with severe disease activity. These results indicated that systemic lupus erythematosus activity is affected by a modulation of the insulin-like growth factor-1 signal pathway and +3179G/A IGF-1R polymorphism.

Enhanced expression of IL-10 in contrast to IL-12B mRNA in poultry with experimental coccidiosis.

Interleukin (IL)-12 and IL-10 are immunoregulatory cytokines with an antagonistic effect on the T-helper (Th)1/Th2 cytokine balance and they provide a functional link between innate resistance and the adaptive immune response. This investigation was conducted to determine the expression of IL-10 and IL-12B mRNA levels in chickens' gut mucosa infected with Eimeria tenella and in sulfachlorpyrazine-sodium treated animals after infection. Broiler chickens were randomly allocated in three groups: healthy untreated control; infected untreated animals and infected, treated with sulfachlorpyrazine sodium chickens 6 days after the challenge with an E. tenella. Quantitative real time PCR analysis was performed using specific primer pairs and probes for IL-10 and IL-12B. The expression of IL-10 mRNA was greater in the duodenum then in the caecum and the liver of healthy chickens. E. tenella infection led to significant up-regulation of IL-10 mRNA in the caecum, followed by mRNA in the liver. A significant down regulation was observed mainly in the caecum after the treatment with sulfachlorpyrazine. In contrast, IL-12B expression in all investigated tissues remained insignificantly affected in the studied groups of animals. Distinct up-regulation of IL-10 mRNA, after the challenge with E. tenella, in the caecum can be attributed to the tissue tropism of Eimeria spp. The production of IL-12 is regulated by negative feedback through IL-10 which explains lack of increase in IL-12B mRNA. Sulfonamide treatment resulted in clinical improvement and restoration of IL-10 mRNA to the levels observed in healthy chickens.

Cytokine production in thromboangiitis obliterans patients: new evidence for an immune-mediated inflammatory disorder.

OBJECTIVE: To study IL-6, IL-10, IL-12 levels and circulating immune complexes (CIC) containing IgG, IgM or IgA in sera of 14 TAO patients and 12 healthy blood donors. To evaluates the ability of TAO PBMC to produce IL-6, IL-10, IL-12, as well as to detect PBMC apoptosis after stimulation with different stimuli. METHODS: In vitro stimulation of PBMC with lypopolysaccharide (LPS), phytohemagglutinin (PHA), C3 binding glycoprotein from uscuta europea (C3bgp), pokeweed mitogen (PWM), and dexamethasone (DM) were performed. The quantities of the secreted cytokines in sera and in culture supernatants, as well as CIC were detected by ELISA. The apoptosis was assessed according to nuclear morphology, after acridine orange staining, by fluorescence microscopy. RESULTS: Significantly higher IL-6 levels in the patients', than in the controls' sera was found. An increased production of IL-6 and IL-12 in TAO PBMC supernatants was detected, regardless of the stimuli used. A hyporeactivity of TAO PBMC toward IL-10 production was found after C3bgp, LPS, PHA and PWM stimulation, compared to the controls' PBMC. The spontaneous and induced apoptosis was significantly higher in TAO compared to the control group. Increased CIC quantities were detected in 75% of the patients tested. According to the CIC isotype, the IgG CIC positives (75%) prevailed over IgA CIC positives (50 %). CONCLUSION: The altered production of IL-6, IL-12 and IL-10, the increased apoptosis as well as the elevated levels of CIC could be a reason for the persisting immune inflammation in TAO.

Comparative study of circulating immune complexes quantity detection by three assays--CIF-ELISA, C1q-ELISA and anti-C3 ELISA.

The assessment of the soluble immune complexes (IC) in human sera is traditionally performed by the C1q binding assay. In the present study, a novel method for the quantity of immune complexes was reported. The methodology was based on measuring their deposition on solid-phase C3 binding glycoprotein (CIF), using an enzyme-linked immunosorbent assay. We also used ELISA that employed anti-C3 antibodies to determined the quantity of immune complexes. The three assays were evaluated for their performance characteristics on the same specially prepared samples: 55 normal sera, 99 sera from RA, 88 sera from SLE, and 27 sera from PSS. The results were compared by reference to a common standard-heat aggregated IgG that possesses many activities of immune complexes. Three of the tests used displayed almost the same specificity (over 95%), while their relative sensitivity varied depending on the disease sera tested. The sensitivity of the assays used was recorded highest for C1q ELISA-28.97% of positive sera, followed by CIF-ELISA-19.63% and lowest for anti-C3 ELISA-17.29%. A well-expressed correlation was found between CIF-ELISA and anti-C3 ELISA data (r=0.42), and a week correlation was noted when comparing CIF-ELISA and C1q ELISA IC levels detected (r=0.28). When the correlation coefficients were calculated individually for each disease category, they were clearly different, and that reflected indirectly in different sensitivities of the test for various disease categories. We also found that the results from the simultaneous performance of the tests demonstrated low percentage positive results when three or two assays were used. This is most probably due to the different assay abilities to detect IC with different sizes and composition, which shows that a small part of IC in the tested sera can be detected simultaneously by more than one assay. On the basis of the results obtained, we concluded that optimal screening for IC could be achieved by parallel application of several different methods.

Preliminary studies on the immunomodulatory effect of the C3 binding glycoprotein isolated from Cuscuta europea.

This study investigates the immunomodulatory effect of a C3 binding glycoprotein (C3bgp), isolated from the parasitic plant Cuscuta europea. When BALB/c mice, immunized with sheep red blood cells (SRBC), were given a single intraperitoneal injection of C3bgp a dose-dependent immunostimulation was observed. The stimulation was assessed by an increase in the number of haemolytic plaque forming cells (PFC) and haemaglutination titres. The induction was time dependent in respect to the administration of both the C3bgp and SRBC. When C3bgp was applied 24 h before SRBC at a dose of 30 microg per mouse (1.2 mg/kg), a well expressed immunostimulation was found. It was also found that giving C3bgp to mice, which had previously been treated with the immunosuppressive drug cyclophosphamide (CY), produced an increase in PFC. The immune response was also restored in vitro experiments were performed using human whole blood cultures stimulated with 30 microg/ml C3bgp in the presence or absence of egg albumin (OVA) as antigen for 72 to 168 h. In C3bgp stimulated cultures it was found that after 120 h there was a high expression of the CD 19+ subset of the activation antigen CD25 (IL-2R) as assessed by flow cytometric phenotype analysis. Supernatants from cultures with different stimuli were assayed by a solid phase ELISA for the determination of OVA-specific IgM at 120, 144 and 168 h. It was found that C3bgp application alone, failed to enhance OVA specific IgM, but significantly high levels of IgM in cultures containing C3bgp and OVA, were detected. Overall it has been shown that the C3 binding glycoprotein, as obtained from the parasitic plant Cuscuta europea, has strong immunostimulatory properties both in vivo and in vitro.

Isolation, partial characterisation and complement inhibiting activity of a new glycoprotein from Cuscuta europea.

A new complement inhibiting factor (CIF) was isolated from the seeds of Cuscuta europea parasitic plant. When activated via both classical and alternative pathway, the complement activity was completely depleted by CIF at a concentration of 0,25 mg per ml serum. Studies concerning the precipitation showed that CIF developed one or two precipitin bands against human sera. It was established that the precipitation is as a result of the specific association of CIF to the C3 component of complement. A partial characteristic of the CIF was carried out. It is a glycoprotein with molecular weight between 27000 and 28000 Da. Its molecule consists of one polypeptide chain.