SERUM LEVELS OF INTERLEUKIN - 23 AND INTERLEUKIN - 17 IN HASHIMOTO'S THYROIDITIS.

CONTEXT: Overproduction of proinflammatory cytokines plays a significant role in the pathogenesis of Hashimoto's thyroiditis (HT). Recent studies revealed a prominent role of newly discovered Th17 subset in the induction of autoimmune disorders and that the signaling induced by IL-23 on Th17 cells is crucial to obtain a pathogenic and sustained phenotype. The objective of this study was to provide the involvement of interleukin IL-23/IL-17 axis in pathologic processes. DESIGN: Serum levels of IL-23 and IL-17 in controls and HT patients were studied in different stages of disease activity. SUBJECTS AND METHODS: We investigated 93 patients with HT: 33 patients with newly diagnosed euthyroid HT (Group I), 11 patients with newly diagnosed hypothyroid HT (Group II), and 49 subjects treated with Levothyroxine (Group III). Thirty healthy subjects were included as controls. Concentrations of IL-23 and IL-17 in the serum samples of patients and controls were evaluated by enzyme-linked immunosorbent assay. RESULTS: Serum level of IL-23 was significantly higher in all HT patients (p<0.0001) as well as in subgroups of patients in comparison with controls (p<0.01). Serum concentrations of IL-17 were statistically increased in the group of HT patients (p=0.014); the differences in IL-17 levels between groups I and III in comparison to healthy controls were also significant, but not for group II. CONCLUSIONS: Our results highlight the involvement of the IL-23/IL-17 axis in the development of HT and its severity. Moreover, upregulated secretion of IL-23 could be a biomarker for progression and monitoring of HT.

IL-12B and IL-10 gene polymorphisms in the development of Hashimoto's thyroiditis.

Functional genetic polymorphisms that altered gene expression of cytokines are candidate genetic factors that could modulate the development and progression of Hashimoto's thyroiditis (HT). IL-12B gene encoded the IL-12p40 subunit, which is included in the pro-inflammatory heterodimeric cytokines IL-12p70 and IL-23. IL-10 is an important Treg cytokine suppressing inflammatory cytokine production and autoimmunity. This study was designed to compare -1082A/GIL-10 and +1188A/C3'UTRIL-12B genotype distribution in 130 patients with HT to a group of 157 healthy controls in attempts to determine an association with HT development. Genotyping for the 3'UTRA/C IL-12B polymorphism was performed using RFLP-PCR and genotyping for -1082A/G IL-10 by ARMS-PCR assay. Patients with HT were divided into euthyroid and hypothyroid stages. There were no significant differences in the genotype and allele frequencies of the IL-12B polymorphism between patients with HT and controls. We observed higher euthyroid HT risk for individuals with CC genotype, unlike to develop hypothyroidism with OR = 1.68. Regarding the polymorphism rs1800896, it was shown the significantly higher frequency of homozygous genotype GG in cases vs controls (OR = 2.19; P = 0.024). Moreover, the combination of genotype AA of 3'UTRIL-12B with GG of -1082IL-10 was associated with a threefold increasing risk (OR = 3.188; P = 0.022) of developing HT compared to individuals with the presence of 3'UTR allele C (AC+CC) simultaneously with AA genotype of -1082IL-10. Our data raise the possibility that the combined effect of polymorphisms from proinflammatory and anti-inflammatory cytokines may be more decisive to HT development.

Adipogenic potential of stem cells derived from rabbit subcutaneous and visceral adipose tissue in vitro.

Rabbits are considered as appropriate animal models to study some obesity-associated abnormalities because of the similarity of their blood lipid profile and metabolism to humans. The current study was focused on comparison of adipose differentiation ability in rabbit adipose-derived stem cells (ADSC) in vitro. Subcutaneous and visceral stromal vascular fractions (SVF) were isolated from three 28-d-old New Zealand rabbits by collagenase digestion. Supernatants from both isolates were collected 24 h after the initial plating. On the fourth passage, all isolated cell types undergo triplicate adipogenic induction. The adipose induction potential was calculated as percentage of increasing optical density (OD) values. The data revealed that with increasing the number of induction cycles, the induction tendency in visceral ADSC decreased in contrast to the subcutaneous ones. Although the supernatants did not reach induction levels of their relevant precursors, they follow the same pattern in both subcutaneous and visceral ADSC. All cell types successfully passed osteogenic and chondrogenic differentiation. In conclusion, the best adipose induction ability was observed in directly plated subcutaneous cell population. The increase of induction numbers depressed adipose induction ability in cell populations derived from visceral fat depots.

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.

[Development of multiplex PCR for fast detection of Paenibacillus larvae in putrid masses and in isolated bacterial colonies].

The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies' DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies.

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.

[Role of IL-12P40 and IL-10 in progression of colorectal cancer].

The aim of study was to investigate the association between serum levels of IL-12p40 and IL-10 and progression of the colorectal cancer (CRC). Be observed an association between severity of CRC and serum levels of investigated cytokines. IL-12p40 was in the highest level in stage-I (423.6 +/- 224.7 pg/ml) compared to more severe stages. In the opposite direction were the data for immunosuppressive cytokine, IL-10. Patients with stage-IV had the highest level of IL-10 (36.02 +/- 9.5 pg/ml). On the basis of our results we could conclude that IL-12p40 and IL-10 serum levels were associated with tumour progression. More severe stages of CRC are characterized by low IL-12p40 and high IL-10 serum levels.

Association of polymorphisms in regulatory regions of interleukin-12p40 gene and cytokine serum level with colorectal cancer.

The present study investigates the association between IL12Bpro and +16974A/C in 3' -untranslated region (UTR) polymorphisms of IL12B and IL-12p40 serum level related to development of colorectal cancer (CRC). Our results showed similar distribution of both the investigated polymorphisms among CRC patients and healthy donors, which suggests that these polymorphisms in IL12B were not associated with the development of CRC. However, we found an increased IL-12p40 level in sera from patients compared to healthy donors and the highest level was observed in stage I compared to more advanced stages of CRC. These findings demonstrated that IL-12p40 serum level has an association with the progression of CRC.

IL-12Bpro and GSTP1 polymorphisms in association with silicosis.

To assess the role of IL-12Bpro and GSTP1 polymorphisms on induced interleukin (IL)-12p40 production in respect to silicosis development, we examined their distribution in 63 silicosis patients and 165 healthy donors. Decreased frequencies of IL-12Bpro-2 allele (42% vs 62.5%, P = 0.0042) and genotype 22 (19% vs 40%, P = 0.02) in patients compared with exposed healthy donors suggested their protective role in silicosis susceptibility. The GSTP1 A/G polymorphism was also associated with silicosis susceptibility. The allele G and genotype GG were overrepresented among patients than among healthy men (36% vs 23%, P = 0.013; 14% vs 2%, P(c) = 0.012). Also, patients' peripheral blood mononuclear cell (PBMC) produced higher IL-12p40 than healthy donors' depending on both IL-12Bpro and GSTP1 polymorphisms. In conclusion, homozygosity of high producer IL-12p40 genotype IL-12Bpro-11 and GSTP1-GG had the highest genetic risk of silicosis development in Bulgarian miners.

Interleukin-12B-3'UTR polymorphism in association with IL-12p40 and IL-12p70 serum levels and silicosis severity.

Susceptibility to silicosis and to disease severity is in part genetically determined. In this study, the role of IL-12B-3'UTR polymorphism in susceptibility and severity of silicosis and its influence on IL-12p40 and IL-12p70 serum level were investigated. The quantity of IL-12p40 and IL-12p70 was detected by enzyme-linked immunosorbent assay and the genotype of IL-12B was determined using the polymerase chain reaction-restriction fragment-length polymorphism method. We observed elevated IL-12p40 in contrast to IL-12p70 serum levels in a group of 62 silicosis patients compared with both control groups. In severe silicosis patients, we detected the highest IL-12p40 serum levels (129.1 +/- 67.7 pg mL(-1)); lower in patients with the moderate (94 +/- 41.6 pg mL(-1)), whereas in mild silicosis, the IL-12p40 levels (67 +/- 23.5 pg mL(-1)) was similar to these in healthy donors. According to IL-12B polymorphism, increased serum levels were observed in subjects with AA genotype (103.2 +/- 46.9 pg mL(-1)) compared to silicosis patients with AC genotype (82.7 +/- 38.3 pg mL(-1)). No significant differences of genotype and allele frequencies of the 3'UTR polymorphism were observed between silicosis patients and healthy controls. However, the heterozygous genotype was found approximately five times more frequently in patients with mild and moderate (48% and 52%) silicosis compared to patients with severe silicosis (11%), and that IL-12B polymorphism may contribute to silicosis severity rather than to susceptibility. Our data demonstrated that elevated serum IL-12p40, independently of IL-12p70 levels, is associated with severity of silicosis, and suggested that IL-12p40 profibrotic activity may contribute to silicosis severity.

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.

Taq-I polymorphism in 3'UTR of the IL-12B and association with IL-12p40 production from human PBMC.

A wide array of studies has demonstrated differences in genotype and allele frequencies of cytokine gene polymorphisms depending on ethnicity and race. In this study, the frequency of Taq-I polymorphism in 3' untranslated region of IL-12B was investigated in two Bulgarian ethnic groups-Bulgarians and Turkish minority. No significant differences of genotype and allele frequencies were observed between these groups. Genotype distribution in the total group of Bulgarian citizens was: AA (61%), CA (32%) and CC (7%), and the allele frequency of 16974 A allele was 0.77. We also evaluated whether this polymorphism affects IL-12p40 production from human PBMC after stimulation. We demonstrated that association between genotype and IL-12p40 production by stimulated PBMC depends on the stimuli used. Our results indicated a significantly decreased IL-12 p40 secretion for the following order of genotypes: AA>CA>CC, after stimulation of PBMC with C3-binding glycoprotein (C3bgp) in contrast to lipopolysaccharide, phytohaemagglutinin and pokeweed mitogen.

Influence of antigen stimulation on the oxidative stress parameters in outbred and inbred rabbits.

The influence of antigen stimulation on the oxidative stress parameters in two groups of rabbits-inbred and outbred were explored by evaluation of the level of lipid peroxidation products (MDA) in the plasma membrane, and the activity of erythrocyte antioxidant defense enzymes superoxide dismutase (SOD) and catalase (CAT). There was not a significant difference between levels of MDA in inbred and outbred rabbits before immunization. However, SOD activity in inbred rabbits was significantly increased in comparison with that of outbred (p = 0.006). Significantly higher plasma levels of lipid peroxidation products were detected in both inbred and outbred rabbits during immune response in comparison to the corresponding groups before immunization (p = 0.008 and p = 0.002). SOD and CAT activities in erythrocytes of rabbits during immune response were also significantly increased compared to that before immunization. In addition, during immune response SOD and CAT activities were found to be positively correlated to each other in both inbred and outbred rabbits (r = 0.727 and r = 0.916). In conclusion, our results suggest the presence of an increased oxidative stress during the antigen stimulation accompanied by an adaptive increase of SOD and CAT activities. 30 days after immunization, the plasma levels of MDA and the activities of SOD and CAT in erythrocytes decreased and reached values close to the controls.

Changes in serum levels of cytokines in mice injected with an immunostimulator C3bgp isolated from Cuscuta europea.

The effect of C3 binding glycoprotein (C3bgp), isolated from Cuscuta europea seeds on induction of in vivo cytokine synthesis was investigated. Different groups of mice were stimulated with 30 microg C3bgp per mouse, injected intraperitoneally. The quantitative determination of IL-1alpha, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma was performed in mouse sera by ELISA. The quantities of these cytokines were measured at different hours: 1, 2, 3, 4, 5, 6, 7, and 24 h after injection. No significant changes in serum level of IL-2, IL-4 and TNF-alpha in experimental animal groups were found. A little increase of IL-1alpha, moderate elevation of IL-10 and IFN-gamma (5- to 6-fold more) and strong release, more than 10-fold of IL-6 in sera of C3bgp-treated mice were detected. The results obtained from C3bgp stimulated cultures of mouse peritoneal macrophages and mouse splenocytes suggest that C3bgp binds to mouse peritoneal macrophages and induces production mainly of IL-6, followed by IFN-gamma and in a very low degree of IL-1alpha and IL-10. Based on the results presented, we conclude that the increased level of IL-6 was the basic after injection of C3bgp and that the mouse macrophages were the major cell targets for the C3bgp effect.

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.

Two glycine containing 2-chloroethylnitrosoureas--a comparative study on some physicochemical properties, in vivo antimelanomic effects and immunomodulatory properties.

Physicochemical properties such as alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of the spin labeled (nitroxyl free radical containing) glycine nitrosourea (SLCNUgly) and its nonlabeled analogue (ChCNUgly), synthesized in our laboratory are studied and compared to those of antitumour drug 3-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU). We have demonstrated that introducing of glycine moiety in the nitrosourea structure in practice does not affect either alkylating or carbamoylating activity. On the other hand replacement of cyclohexyl moiety in ChCNUgly structure with nitroxyl free radical leads to a decrease in carbamoylating activity and an increase in alkylating activity. Compound ChCNUgly showed in vivo a higher antimelanomic activity against B16 melanoma in comparison with CCNU and SLCNUgly. It completely inhibited B16 melanoma growth (TGI=100%) at a dose 64.0 mg/kg. Moreover, we established that joint i.p. application in normal mice of SLCNUgly plus a new immunostimulator (C3bgp) formerly isolated in our laboratory led to a 75% restoration in immune function with respect to antibody production measured by Jerne hemolytic plaque assay. In contrast, no immunostimulation was found after joint application of C3bgp plus ChCNUgly or CCNU at the same experimental conditions. Based on these preliminary results, a possibility for developing of new combination immunochemotherapy schemes for treatment of human cancers is discussed.

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.