فهرست مطالب manoochehr rasouli

Interleukin-17A (IL-17A) gene can be a potential candidate gene implicated in visceral leishmaniasis (VL), a disease caused by an infection with Leishmania parasite. The aim of this study was to explore whether there is an association between IL-17A polymorphisms and VL in the Iranian population. A total of 202 participants (55 VL patients and 125 healthy controls) were investigated in the present case-control study. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The frequencies of IL-17A rs3819024, rs3819025, and rs8193038 A alleles, and haplotype AGAG were significantly higher in the controls than patients (P = 0.0006, 0.017, 0.0003 and 0.001, respectively), while IL-17A rs3748067 A allele distribution was higher in patients than controls (P = 0.00004). Also, the frequencies of AA genotypes of rs3819024, rs3819025 and rs8193038 were higher in the controls (P = 0.0048, 0.014, and 0.018, respectively) while rs3748067 AA genotype was of greater distribution in the patients (P = 0.000048). The findings highlighted the role of IL-17A in the pathogenesis of the VL in humans.

Bacillus thuringiensis, is a Gram-positive spore-forming bacterium that produces crystalline parasporal protein (Cry) during sporulation. Some of these Cry toxins do not show cytotoxicity against insects but they are capable to kill some human and animal cancer cells. The aim of this study was to verify whether cytocidal parasporal of B thuringiensis strains have immunostimulatory activity on human peripheral blood mononuclear cells (PBMNC) and to evaluate the ability of IL-2 and IL-5 production.
B. thuringiensis toxin with cytocidal activity was isolated and treated with proteinase K. PBMNC was cultured and treated with activated crystal proteins. We evaluated the ability of different cytokines production with Flow Cytometry.
In this study, immune stimulatory toxins Cry1 were distinguished. This toxin can stimulate production of cytokines IL-2 and stop production of IL-5.
Discussion and According to anti-cancer effect of B. thuringiensis toxins and also immune stimulatory effect, with more research these toxins can be introduced as immunotherapy drug in cancer treatment.

Brucella is an intracellular Gram-negative bacterium. Previous reports showed that gene polymorphisms of cytokines can affect resistance or susceptibility to Brucella infection. Interleukin-13, a cytokine secreted by Th2 lymphocytes, has an important role in immune responses against established infections. In this study, we investigated the association of three polymorphic sites of IL-13 with susceptibility to brucellosis in Iranian population. In this study 169 patients with brucellosis and 71 healthy controls were included. DNA was extracted and genotyped for three bi-allelic polymorphisms of IL-13 gene at positions -1512A/C, -1055C/T, and +2044G/A by polymerase chain reaction-restriction fragment length polymorphism method. None of the studied alleles and genotypes of IL-13 gene (-1512A/C, -1055C/T, and +2044G/A) showed significant relationship with susceptibility to brucellosis. However, among eight haplotypes, the distribution of TCG and CAA haplotypes were significantly higher in the patients compared with those in the controls (P=0.002 and P=0.034, respectively). Although, the later did not tolerate Bonferroni correction. On the contrary, the distribution of TCA haplotype was higher in the controls compared to that in the patients (P=0.01). Furthermore, TAG/TCA haplogenotypes were significantly higher among controls compared to the brucellosis patients (P=0.025). P value resulted from TCA and TAG/TCA did not tolerate Bonferroni correction. There is no association between the inheritance of different alleles and genotypes of interleukin-13 gene and susceptibility to brucellosis. However, it seems that the inheritance of some haplotypes and haplogenotypes of IL-13 can impact the susceptibility to brucellosis.

The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method. In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+) plasmid. The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing. The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.

Ovarian cancer is the fifth leading cause of death from malignancy in women. CD4+CD25+FoxP3+ regulatory T (Treg) cells are a subset of T lymphocytes with great inhibitory impact on immune response. To investigate the percentage of CD4+CD25+FoxP3+ regulatory T cells in the peripheral blood of the Iranian patients with epithelial ovarian cancer compared to healthy women and to evaluate the correlation of the Treg cell percentage with clinicopathological characteristics including cancer stage and CA-125 serum level. Seventeen women with epithelial ovarian cancer and 20 healthy subjects were enrolled in the study. Peripheral blood mononuclear cells were stained at the surface, for CD4 and CD25 molecules, followed by fixation, permeabilization and intracellular staining for FoxP3 molecule. After processing and flowcytometry analysis, prevalence of Treg cells was determined as the percentages of CD25+FoxP3+ cells among CD4+ lymphocytes. Despite no difference in the percentage of total CD4+ lymphocytes, analysis indicated that Treg cell percentage was significantly higher in ovarian cancer patients than controls (5.7 ± 3.1% versus 2.8 ± 1.4%, p=0.002). A trend toward higher Treg cells was observed in higher stages of ovarian cancer (III+IV) in comparison to lower stages (I+II) (6.5 ± 3.2% vs. 4.44 ± 2.7%, p=0.2). Higher percentage of Treg cells was also observed in the patients with high CA125 (CA-125 >100 U/mL) in comparison to those with low CA-125 serum level (CA-125 ≤ 100 U/mL) although the difference was not significant (6.44 versus 4.18%, p=0.19). Increased frequency of Tregs in ovarian cancer might participate in immune suppression in these patients. The findings collectively suggest the likely impact of Treg cell–targeted immunotherapy in ovarian cancer.

Increased levels of interleukin-8 (IL-8) and interleukin-6 (IL-6) in acute human brucellosis have been reported. Previous studies have shown that the production and level of IL-6 and IL-8 cytokines are associated with the polymorphism of the encoding genes. To investigate the probable association between IL-6 (-174 C/G) and IL-8 (-251 A/T) gene polymorphisms and susceptibility/resistance to brucellosis. The patient group included 196 patients suffering from Brucella infection and the control group consisted of 82 healthy animal husbandmen from the same geographical area. IL-8 (-251 A/C) and IL-6 (-174 C/G) gene polymorphisms were analyzed by PCR-RFLP and Allele Specific PCR (AS-PCR) respectively. The frequency of -251 IL-8 AA genotype was significantly lower in the controls compared with that of the patients (p=0.0051), while the frequencies of other genotypes (AT and TT) and alleles (A and T) were not significantly different among the participants. No association was found between IL-6 (-174 C/G) polymorphism and brucellosis. This study indicates that the IL-8 -251 AA genotype may be considered as a genetic susceptibility factor for brucellosis.

Host resistance towards Leishmania infection is mediated by cellular immune responses leading to macrophage activation and parasite killing. According to the important role of IL-13 in the defense against visceral leishmaniasis (VL) and the known effect of the IL-13 gene polymorphisms on its production، the aim of this study was to investigate the probable relationship between IL-13 gene polymorphisms and susceptibility to VL. The patient group included 52 patients who had suffered from VL infection and the control group consisted of 104 non-relative healthy people from the same endemic areas the patients were from (southern part of Fars Province). IL-13 (position -1512 A/C) gene polymorphism was determined by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). There was no significant association between the frequencies of IL-13 (-1512) alleles and genotypes in the patients with VL compared to the thenormal population. This study indicated that the IL-13 (position -1512 A/C) genotypes cannot be considered as a genetic susceptibility factor for leishmaniasis.

Various prokaryotic and eukaryotic expression systems havebeen developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis.

TTV is the first human circoviridae that was isolated from Japanese patients with unknown hepatitis in 1997. Since then, several studies have been done on different aspects of TTV pathogenesis. The aim of this study is to determine the prevalence of TTV in patients with chronic hepatitis using two different primer sets. In this descriptive study, blood samples from 240 patients with chronic hepatitis C at Professor Alborzi Clinical Microbiology Research Center were assessed in terms of the presence of TTV DNA in plasma through the nested polymerase chain reaction using two primer sets. Of the 240 patients, TTV-DNA was detected in 220 (92%) patients with chronic hepatitis C using 5΄-UTR primer and in 12 (5%) patients using N22 primer. According to the demographic data, there was not a significant difference between male female patients in prevalence of TTV infection. The prevalence of TTV DNA in plasma samples from patients with chronic HCV by using 5΄-UTR primer was high and it was congruent with studies done in other countries; however, N22 primer showed a lower prevalence of viral DNA in the samples. Overall, there was not a significant correlation between sex and the presence of viral DNA in patients. Controversial or high prevalence of this virus in HCV infected people necessitate further studies for determining the relationship between HCV and TTV infection.

Previous studies imply that IL-1 and IL-8 gene variations may play a crucial role in the genetic predisposition to different gastric disorders upon H. pylori infection. The aim of this study was to determine the potential association between the prevalence of certain polymorphic sites and the risk of gastric disorders inIranian population. One hundred and forty three unrelated individuals withdifferent gastric disorders and 374 normal individuals with no gastric disorders and witha negative serology test for H. pylori (control group) were studied for the associationbetween IL-1β (+3953 C/T) and IL-8 (-251 A/T) gene polymorphisms and H. pylorimediated gastritis and gastric ulcer. An analysis of genotype frequency for these genes was performed using RFLP-PCR. Based on the data obtained from culture and pathologic findings, the patients were classified into three subpopulations: H pylori+non-ulcerative gastritis+, H. pylori+ ulcerative gastritis+ and H. pylori- non-ulcerativegastritis+. A significantly higher frequency of TT genotype (p=0.02) in IL-1β +3953 inH. pylori+ ulcerative gastritis+ was revealed compared to the control group. There wereno significant differences among other subpopulations. No significant differences in alleleand genotype frequencies of IL-8 (-251A/T) were found among the patients. The data suggest that TT genotype in IL-1β +3953 may be a major contributing genetic risk factor for H. pylori induced gastric ulcer. Moreover, the role of other bacterial and host response factors, such as bacterial adherence peptides, host chemokines, and genes involved in gastric acid secretion, must be further investigated in different ethnic populations.

Heat shock protein 70 (HSP70) is present in all organisms studied so far,and is a major immunogen in infections caused by pathogens including Leishmania spp. The aim of this study was to clone and express HSP70 from L. infantumstrain MCAN/IR/96/LON-49 and evaluate antibody response against HSP70 in visceralleishmaniasis (VL). The L. infantum HSP70 gene segment was amplified byspecific primers. It was cloned into pTZ57R vector and subcloned into pET32a (+) expressionvector. The new construct was transformed in the E.coli Rosetta strain, andHSP70 protein was expressed in the presence of 1 mM IPTG and purified using a Hi-Trap chelating column. Antibody responses against HSP70 were determined by ELISAin 37 patients with visceral leishmaniasis and 63 healthy controls. Expressionof HSP70 protein was confirmed using SDS-PAGE electrophoresis and dot blot with ananti-His tag antibody. There was no difference between the sequence of nucleotides ofthe HSP70 gene in the present study and other reported sequences. The ELISA resultsindicated that the sera of 81.1% (30/37) of the patients and 6.3% (5/63) of controls reactedto L. infantum HSP70. The conservative nature of the HSP70 moleculeis an advantage in vaccine studies, because of minor differences (6%) between thenucleotide sequences and consequently the similarity in amino acid sequences in variousstrains of L. infantum. It could therefore be used in vaccine research againstleishmaniasis and also as a tool for serodiagnosis.

Brucella is a gram-negative bacterium, causing acute and chronicinfection in humans and animals. Cell-mediated immunity is the main protective immune response against Brucella spp. Activation of macrophages by IFN-γ and generation of reactive oxygen intermediates and nitric oxide are the main immunologic mechanisms responsible for control of Brucella infection. To investigate the correlation between IFN-γ gene polymorphism and brucellosis. 195 patients with brucellosis, 186 healthy patients'' family members and 82 healthy farmers who kept infected animals and consumed their contaminated dairy products were selected to take part in the study. IFN-γ genotyping at position +874 (T→A) was carried out by allele specific polymerase chain reaction (AS-PCR) method. The frequency of AT and TT genotypes significantly increased in farmers compared to patients with brucellosis (P=0.03) while there was no significant difference in genotype distribution between patients and their healthy family members. IFN-γ (+874) AA genotype is probably a genetic factor that contributes to the susceptibility of the individuals to brucellosis.

There is a growing concern about the application of molecular methods in epidemiological studies of infectious diseases. The objective of this study was to determine the genetic stability of methicillin resistance genes in Staphylococcus aureus for the evaluation of resistance strain distribution. One hundred and fifteen S. aureus isolates from patients with staphylococcal infection were collected. The isolates were screened for methicillin resistance by minimum inhibitory concentration (MIC) examination. The stability of methcillin resistance genes was examined by physical curing and PCR screening methods. The results showed that methicillin resistance Staphylococcus aureus (MRSA) had risen up to 43% in Nemazi Hospital (Shiraz, Iran). Indeed, the incidence of MRSA in our hospital was 10% during the last four years. The ability to lose (curability) of methicillin resistance genes (mecA) was examined by physical curing method in 49 isolates with MIC ³ 16 μg ml-1. No sign of curability of mecA gene was observed where 500 colonies from each strain have been studied and exhibited by the same MIC values before and after curing test. Positive PCR results for isolates with MIC ³ 16 μg ml-1 before and after curing experiment have been achieved. These data confirm the results of curing method, indicating that stable genetic determinants confer methicillin resistance. These results support the hypothesis that resistance isolates may be selected due to clonal selection under antibiotic pressure used in clinics rather than transmission of mobile genetic determinant. The high prevalence of MRSA immerged in our Hospital could be originated due to antibiotic pressure and poor control measures.

In order to improve simultaneous detection and identification of Helicobacter genus in general and Helicobacter pylori specifically and reduce the number of amplifications needed, we established a multiplex PCR. In this study, two pairs of primers: Hcom1 and Hcom2 specific for Helicobacter genus, Hicd1 and Hicd2 specific for Helicobacter pylori species were used. To determine the sensitivity of our multiplex PCR, the lower limits of DNA detection from pure culture were established using phenol-chloroform method. To evaluate the specificity of our protocol, we tested DNA extracted from various Gram-negative and -positive bacteria. A study was subsequently undertaken on stomach tissue samples from 18 patients to evaluate this protocol for detection of Helicobacter in tissue samples. In our optimized PCR, two fragments of 389- and 1200-bp were produced using Hcom1-Hcom2 and Hicd1-Hicd2 primers, respectively. Amplification of Helicobacter pylori genomic DNA was achieved at concentration as low as 0.03 pg equivalent to 150 bacteria. No DNA amplification of other Gram- positive and -negative bacteria was seen. Twelve specimens, positive in culture and rapid urease test for Helicobacter pylori were positive for DNA of this organism using this multiplex PCR. Our results demonstrate that this protocol represents a specific and sensitive assay for simultaneous detection of Helicobacter genus members, in general, and Helicobacter pylori species, specifically, in clinical samples yielding no false-positive

Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of Staphylococcus aureus and 50 isolates of Coagulase Negative Staphylococci (CNS) was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with MIC>8 mg/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when Iran. Biomed. J. 8 (3): 161-165, 2004