Expression of IL-17 and COX2 gene in peripheral blood leukocytes of vitiligo patients

Vitiligo is a pigmentation disorder in which inflammatory mediators such as cytokines have a pivotal role in disease's pathogenesis. Interleukin 17 [IL-17A] is a proinflammatory cytokine which is involved in the induction of several proinflamatory mediators such as cyclooxygenase 2 [COX2]. The aim of this study was to investigate the gene expression of IL-17 and COX2 in peripheral blood leukocytes of vitiligo's patients. Peripheral blood leukocytes from 15 patients with vitiligo and 15 healthy controls were separated using a gradient density centrifugation method. After total RNA isolation and cDNA synthesis, IL-17 and COX2 gene expression were quantified by real-time polymerase chain reaction [PCR]. There were no significant differences in IL-17 and COX2 gene expression in lymphocytes of patients with vitiligo compared with control group [p<0.05]. However there was an increased IL-17 and COX2 gene expression in neutrophils of patients compared to controls, but it did not reach statistical significance [p=0.05]. We could not find any differences in IL-17 and Cox2 gene expression between clinical diseases subtypes, sex and age. There was a significant correlation between IL-17 and COX2 genes expression in the neutrophils of patients [p=0.00, r=0.80]. Our results showed an increased expression in IL-17 and Cox-2 genes in neurophils of patients with vitiligo. This suggested that these two factors are involved in the inflammatory process. Further studies with a larger sample size might help to establish the role of these factors in the pathogenesis of diseases

[Study of cytotoxicity effect of total saffron extract on hepatocarcinoma cells line [HepG2]]

The anti-tumor effect of saffron extract has been proved in both in vitro and in vivo in recent years. In this study, cytotoxicity effect of total saffron extract on human liver carcinoma cells [HepG2] investigated in in vitro condition. In this experimental study, the effects of total saffron extract on quantitative proliferation of all cell lines were determined by using 3- [4,5-dimethyl thiazol-2yl] -2.5-diphenyl tetrazolium bromide [MTT] colorimetric assay. Also, effects of extract on mouse fibroblast cells [L929] were evaluated as the control. MTT assay is a fast, sensitive and quantitative method for all kind of cells' proliferation by spectrophotometry. Amount of required extract for occurring 50% cytotoxicity to the cells [IC 50] was achieved by administering different concentration of extract, and comparing the left alive cells with cells that no drug was used on them. 50% inhibition of tumor cells growth was achieved at 400 microg/ml. Extract had no inhibitory effect on normal cells growth. Total saffron extract can be used as cytotoxic agent against Cancer in vivo by producing cytoplasmic and nuclear changes

Association of HLA-DQB1 allelic sequence variation with susceptibility to systemic lupus erythematosus

Systemic lupus erythematosus [SLE] is an autoimmune disease in which polymorphisms within the human leukocyte antigen [HLA] region have been associated to its etiology. We conducted this study to compare the HLA-DQB1 allelic sequence variation among SLE patients and controls in the northeast of Iran. Genomic DNA of 40 SLE patients and 83 healthy controls were amplified by Polymerase Chain Reaction with Sequence-Specific Primers technique [PCR-SSP]. Seven serological subclasses of the HLA DQB1 were detected. Allele distribution comparison showed in the SLE group a significant increase of HLA DQ6 [*0601-*0609] [p=0.006]; whereas alleles HLA DQ7 [*0301-*0304] were significantly decreased [p=0.005]. Combination of DQ5 [*0501-*0504]-DQ6 [*0601-*0609] was increased in patients. These results suggest that DQ6 is the dominant HLA DQB1 allele probably associated with genetic susceptibility to SLE in the northeast of Iran. The association supports the importance of ethnic background and indicates the importance of various genes that has been observed in different SLE populations

Cytotoxic effect of a new endodontic cement and mineral trioxide aggregate on L929 line culture

The aim of this study was to compare the cytotoxicity of Mineral Trioxide Aggregate [MTA] and a New Endodontic Cement [NEC] on L929 mouse fibroblasts. Different dilutions [Neat, 1/2, 1/10, 1/100] of fresh and set materials placed adjacent flasks of L929 in DMEM medium. Cellular viability was assessed using MTT assay in three time intervals [24, 48, and 72 h after mixing]. Differences in mean cell viability values between materials were assessed by using the One-way ANOVA and Bonferoni post-test. Optical microscopic analysis of morphology of the untreated control and the cementtreated cell cultures were carried out in all experimental periods. It was indicated that there was not a significant difference in cytotoxicity among the materials of test and between them and the control group. However, there was a statistically significant difference between different time intervals within each group [P

effect of periodontal treatment on IL-6 production of peripheral blood monocytes in aggressive periodontitis and chronic periodontitis patients

Several cytokines, including IL-6 have been implicated in the pathogenesis of periodontal disease. It is established that monocytes from periodontitis subjects show an increased production of IL-6 as compared to healthy subjects. However, little is known about the effect of periodontal treatment on IL-6 production by monocytes in subsets of periodontitis patients. The aim of the present study was to evaluate the effect of surgical periodontal treatment on IL-6 production of peripheral blood monocytes [PBM] in aggressive periodontitis patients [AP] and chronic periodontitis patients [CP] before and after stimulation by E.coli LPS. Fifteen AP patients, 15 CP patients and 15 periodontally healthy subjects [PH] took part in the study. PBM IL-6 production was measured, using ELISA, before and after stimulation of cultured PBM cells by 0.1 jig/mi LPS of Emit. Following full-mouth non-surgical and surgical periodontal treatment of the AP and CP groups, the same measurements were repeated for these two groups. LPS-stimulated lL-6 production was significantly greater than non- stimulated IL-6 for all 3 groups. Before periodontal treatment, LPS-stimulated TL-6 production of the AP group was significantly greater than the other 2 groups. Periodontal treatment did not result in a significant decrease in unstimulated or LPS-stimulated IL-6 production by PBM cells in AP and CP patients. No correlation was detected between TL6 levels and baseline clinical parameters or changes in clinical parameters. PBM cells in AP patients might be hyper-responsive in terms of IL-6 production. This hyper-responsiveness does not seem to return to that of healthy subjects even after a successful periodontal treatment. Moreover, the regulation of host inflammatory mechanisms upon LPS challenge might be different between AP and CP patients

Identification of leishmania species causing cutaneous leishmaniasis by RAPD-PCR

Leishmaniasis, especially cutaneous leishmaniasis is considered as an important health problem in many parts of Iran such as Neishabour, in Razavi Khorasan Province. Since DNA of every parasite such as every organism is specific, this facilitates extensive use of DNA for diagnosis and identification of parasite species. Among these methods, RAPD-PCR seems to be a useful method. Since Neishabour is an important focus for cutaneous leishmaniasis and so far molecular researches haven't been done, this study was done to identify different species of leishmania parasites causing cutaneous leishmaniasis by RAPD-PCR. In this study a total of fifty-seven patients, whom their disease confirmed by direct smear, were recruited and samples isolated and cultured in NNN medium, followed by sub-culturing in RPMI-1640. This study was approved by the local ethics. Then DNA was extracted by proteinase k and desirable samples were amplified by RAPD-PCR method, using four oligoprimers. Electrophoresis patterns from each isolate were compared with the reference strains of L. major, L. tropica and the marker. The results of this study indicated that L. tropica is the responsible parasite for causing cutaneous leishmaniasis, in Neishabour, Northeast of Iran. It seems that Leishmania tropica is the only causative agent of Cutaneous Leishmaniasis in this study area. RAPD-PCR technique is a suitable tools for Leishmania characterization the in epidemiological studies

Association of the expression of IL-4 and IL-13 genes, IL-4 and IgE serum levels with allergic asthma

Immune and inflammatory responses mediated by cytokines, play important roles in the pathophysiology of asthma. These responses are associated with overexpression of Th2 cytokines such as IL-4 and IL-13. These two cytokines use common receptors for signaling that lead to identical immunological effects and regulation of the Th1/Th2 balance. The aim of this study was to determine whether patients with allergic asthma display overexpression of IL-4 and IL-13 genes. Using RT-PCR, we examined the expression of IL-4 and IL-13 genes in twenty asthmatic cases and twenty normal individuals. Total levels of serum IgE and IL-4 were also determined by ELISA method. Expression of IL-13 gene in 70% of patients with allergic asthma was higher than controls [P=0.01]. There was no correlation between the expression of IL-13 gene and total level of serum IgE [P=0.07]. Expression of IL-4 gene was detected in 30% of the patients and none of the normal individuals as determined by RT-PCR [P=0.01]. Mean of serum IgE levels in patients and controls were 84.9 IU/ml and 62.2 IU/ml, respectively. Level of serum IgE was more than 100 IU/ml in 30% of patients [P=0.03]. Mean of serum IL-4 levels in patients and controls were 15.73 pg/ml and 13.07 pg/ml, respectively. There was a relation between levels of serum IgE and IL-4 in 73% of cases. The results showed that there was a correlation between the expression of IL-4 gene and the level of serum IL-4. Levels of serum IgE and IL-4 were considerably higher in asthmatics than nonasthmatic controls

Association between the polymorphisms of IL-4 gene promoter [-590C>T], IL-13 coding region [R130Q] and IL-16 gene promoter [-295T>C] and allergic asthma

Allergic asthma is a multifactorial disease, influenced by genetic and environmental factors. Recent family-based studies have revealed evidence for linkage of human chromosomes 5q31-33, 12ql5-24, Ilql3 and 15q23.6 as regions likely to contain genes related to asthma. Among the candidate genes in these regions are the genes encoding for human interleukin-4, interleukin-13 and interleukin-16. To evaluate this linkage, we examined an Iranian population of patients with asthma. A total of 30 patients with allergic asthma and 50 normal subjects were studied. Allergic asthma was confirmed using skin prick test and spirometry. DNA was extracted from blood cells and IL-4 [-590C>T], IL-13 [R130Q] and IL-16 [-295T>C] polymorphisms were determined by PCR-RFLP method. Out of 30 patients with allergic asthma, the following genotypes for IL-4, IL-13 and IL-16 cytokines were found: IL-4 genotypes consisted of 17 [56.7%] CC, 8 [26.7%] CT and 5 [16.7%] TT; IL-13 genotypes consisted of 11 [36.7%] GG, 13 [43.3%] GA and 6 [20%] AA; IL-16 genotypes consisted of 23 [76.7%] TT and 7 [23.3%] CT. No patient showed CC genotype for IL-16. A higher proportion of case subjects with the C allele for the IL-4, G allele for the IL-13 and T allele for the IL-16 polymorphisms was found compared with the T, A and C alleles, respectively. These results suggest an influence of genetic variability at the promoter of IL-4 gene [-590C>T] and a coding region of IL-13 gene [R130Q] on the occurrence of allergic asthma and no relationship between IL-16 promoter polymorphism [-295T>C] and this disease

Molecular cloning and expression of human Gamma Interferon[IFN-Lambda] Full cDNA in Chinese Hamster Ovary [CHO] Cells

IFN-gamma is mostly secreted by activated CD4+, CD8+ T cells and NK cells. This cytokine has immunomodulatory, anti-cancer and anti-microbial effects and is important for prophylaxis, diagnosis and treatment of chronic infections and cancers. The purpose of this study was to clone the full cDNA of human IFN-gamma and express it on CHO cell line. Lymphocytes from a healthy individual were isolated and activated by phytohaemagglutinin [PHA] in vitro. After 4 hours, total RNA extracted and first cDNA str and was synthesized. cDNA was amplified with primers containing EcoRI and NotI sites. The amplified fragment and the PcDNA3.1 vector were cut by EcoRI and NotI and ligated. The construct [pcDNA3.1-IFN-gamma] was transferred into E.coli [strain: DH5 alpha] using CaCl2 method and selected by plating on a medium containing ampicillin. The construct sequence was confirmed by PCR and sequence analysis. Construct expression was achieved by performing a calcium phosphate-mediated transfection into CHO cells and followed by selection of stable drug [G418] resistant clones by limiting dilution assay [LDA]. The IFN-gamma production by transected CHO cells was measured using ELISA technique. Out of 33 grown transformed bacterial colonies, only 6 had the entire sequences of the insert and one of them was used for the transfection experiment. Out of 768 wells, 5 clones produced more than 100 ng/ml/10[6] cells of IFN-gamma. Among the 5 clones, one with the maximum production of INF-gamma [143 ng/ml/10[6] cells] was selected and used for propagation

Association between the polymorphism of TGF- beta 1 gene promoter [-509C>T] and idiopathic chronic Urticaria

Idiopathic Chronic Urticaria [ICU], the most common form [70-80%] of chronic urticaria is supposed to have immune basis causes. It is speculated that the promoter polymorphism of TGF- beta 1 gene may be involved in ICU. This condition is thought to affect at least 0.1% of the population and often can be severe and difficult to treat. A total of 40 patients with ICU and 41 normal subjects were studied. DNA was extracted from whole blood and TGF- beta 1 promoter -509C>T polymorphism was determined by PCR-RFLP method. Out of the 40 patients with ICU, 11 [27.5%] had CC, 26 [65%] had CT and 3 [7.5%] had TT genotypes. A higher proportion of case subjects with the C allele [CT type or CC type] was found compared with the T allele. These results do suggest an influence of genetic variability at the promoter of TGF- beta 1 gene [-509C>T] on the occurrence of ICU. This polymorphism has been shown as a useful genetic change in our study. Further work is required to confirm this result

Evaluation of specific purified TCR effect on the immunoregulatory potential of TGF-Beta

Transforming growth factor beta [TGF-beta] is a mediator released by nearly all cell types. It has suppression activity on the immune system, but exactly how this effect is carried out is not clear. Previous experiments showed that IgG interacts with or carriers active TGF-P, that could suppresses cytotoxic T-cell responses to an immunogenic tumor in mice. Since T cell receptor [TCR] has structural similarities with IgG, we asked the question-whether a specific TCR could interfere with and enhance the suppressive effect of TGF-P on T-cell proliferation. T-cell lines were established by limiting dilution and specific TCR were extracted and purified. Mixed lymphocyte reaction [MLR] was carried out using DA [RT1a] vs. LEW [RT11] lymph node cells and DA vs. PVG [RT1u] lymph node cells. DA cells were used as responder cells and PVG/LEW as stimulator cells. Proliferation of DA cells was examined with different concentration of TGF-beta by adding 1 micro ci 3 H-thymidine 24 hours prior to harvesting the cells. The results showed that the presence of a specific TCR does not have any effect on the percentage of suppression when already fully suppressed by TGF-P. However, it does have an effect on TGF-beta stimulated suppression under certain conditions. When TCR was added at the same concentration as TGF-beta [1-2 ng/ml], inhibited TGF-P stimulated suppression of proliferation, but when added at higher concentration than TGF-P, this effect disappeared, and the proliferation was suppressed in the same way, as TCR was absent. Thus, TCR interaction with TGF-beta could play an important role in the homeostasis of immunity by augmenting the proliferation of activated dominant lymphocyte clones. This would promote suppression of activation/proliferation of new specific antigen- reactive clones that may arise during ongoing immunity, and suppressing some autoimmune diseases