Influence of genetic variants of gamma interferon, interleukins 10 and 12 on Visceral Leishmaniasis in an endemic area, Iran.

Visceral leishmaniasis (VL) is a life threatening disease in which a variety of cytokines regulating the immune responses can determine its outcome. As based on their region in the gene, some single nucleotide polymorphisms (SNP) can influence the expression of their corresponding proteins, this study aimed to investigate the association between SNP in the IL-10, IL-12, IFN-γ genes and susceptibility to VL. The study was carried out on 120 patients with VL, 67 patients' families (family group), and 102 healthy individuals with positive leishmanin skin test as positive control group. SNPs in IL-10 (-592, -819, -1082), IL-12 (+1188) were analyzed using PCR-RFLP and allele specific polymerase chain reaction (ASPCR) was used to analyze SNPs in IFN-γ (+874 A/T). The results showed that at position +874 of IFN-γ, AT genotype was significantly more frequent in patients than that in families and controls, but TT genotype was significantly more frequent in families than in patients. Distributions of IFN-γ alleles were not significantly different between the study groups. As for IL-12 and IL-10 genotypes and alleles, no significant difference was observed between the groups. Although a strong linkage disequilibrium was observed between alleles -592, -819 and -1082 of IL-10, distributions of the most common haplotypes and haplogenotypes reconstructed from IL-10 alleles were not significantly different between the study groups. It could be suggested that heritage of AT genotype at position +874 of IFN-γ may predispose and TT genotype can resist individual to VL in an endemic area in the southwest of Iran.

Anti-cancer Parasporin Toxins of New Bacillus thuringiensis Against Human Colon (HCT-116) and Blood (CCRF-CEM) Cancer Cell Lines.

Bacillus thuringiensis is one of the most important microorganisms used against cancer cell lines in latest studies all over the world. This study aims to perform the isolation, molecular identification, and to identify novel B. thuringiensis strains that specifically targeting human cancer cell-killing activities in Iran. A total of 88 B. thuringiensis isolates were recovered from Iran. Upon the treatment of the non-hemolytic crystal proteins by proteinase K, five isolates belonging to three biotypes, thuringiensis, kurstaki and sotto of B. thuringiensis are found to have different cytotoxicity toward HCT-116 and CCRF-CEM cell lines. Digested inclusions of the isolates consisted of one major poly peptide of 34-kDa, as estimated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The structure, molecular identification, and functionality of five isolates inclusion proteins have shown to be closely like to parasporin-2 but their size of activated protein is not similar to this parasporin. It is unclear that discovered damaging proteins are parasporin-2. This 34-kD protein exhibited varying degrees of cytocidal activity toward human colon and blood cancer cells and caused cell swelling and the formation of blebs in the surface of the cells or alteration in cytoskeleton. The soil in the humid and temperate climates of Iran is a good reservoir for parasporin producing B. thuringiensis. The isolated B. thuringiensis strains exhibit specific and different cytocidal activities against human colon and blood cancer cells. Parasporin is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis and these toxins appeared to attack an identical target on human cancer cells.

The importance of TH22 and TC22 cells in the pathogenesis of Helicobacter pylori-associated gastric diseases.

BACKGROUND: An association exists between Helicobacter pylori (H. pylori), peptic ulcers, gastritis, and sometimes gastric carcinomas. Th22 cells have protective and inflammatory roles in defense against microbes. AIM: We investigated the frequencies of Th22, Tc22, Th22/17, and Tc22/17 cells in addition to the changes in levels of cytokines IL-22, IL-6, IL-23, TNF-α, IL-1ß, and TGF-ß in sera from patients with H. pylori-associated gastritis, and peptic ulcer, and in uninfected patients. METHODS: A total of 76 patients with H. pylori-associated disorders formed the studied group. Frequencies of T-cell subsets were determined by flow cytometry. Levels of cytokines IL-22, IL-6, IL-23, TNF-α, IL-1ß, and TGF-ß in the sera and supernatants of patients were measured by ELISA and flow cytometry. RESULTS: The study participants included 32 males and 44 females with a mean age of 38.5±15.3 years. We divided the infected group into peptic ulcer and gastritis (mild, moderate, active chronic, and chronic). The frequencies of Th22, Tc22, and Tc22/17 increased significantly in the peptic ulcer, moderate, active chronic, and chronic gastritis groups compared to the uninfected group. Th22/17 only increased significantly in the chronic gastritis group. We observed significant increases in IL-22 in the moderate and active chronic gastritis, IL-23 in the active chronic and chronic gastritis, and TNF-α in the peptic ulcer and moderate gastritis groups. Following in vitro antigenic stimulation, we observed significantly higher levels of IL-1ß, IL-23, and IL-6 in the active chronic gastritis group, as well as IL-6 and IL-1ß in the chronic gastritis group compared to the uninfected group. CONCLUSION: Increased Th22, Tc22, and Tc22/17 cells and IL-22 levels appear to be influential in progression and severity of H. pylori infection. Th22/17 can be an interesting therapeutic target for chronic H. pylori infections where eradication is more difficult.

Molecular Diversity between Salivary Proteins from New World and Old World Sand Flies with Emphasis on Bichromomyia olmeca, the Sand Fly Vector of Leishmania mexicana in Mesoamerica.

BACKGROUND: Sand fly saliva has been shown to have proteins with potent biological activities, salivary proteins that can be used as biomarkers of vector exposure, and salivary proteins that are candidate vaccines against different forms of leishmaniasis. Sand fly salivary gland transcriptomic approach has contributed significantly to the identification and characterization of many of these salivary proteins from important Leishmania vectors; however, sand fly vectors in some regions of the world are still neglected, as Bichromomyia olmeca (formerly known as Lutzomyia olmeca olmeca), a proven vector of Leishmania mexicana in Mexico and Central America. Despite the importance of this vector in transmitting Leishmania parasite in Mesoamerica there is no information on the repertoire of B. olmeca salivary proteins and their relationship to salivary proteins from other sand fly species. METHODS AND FINDINGS: A cDNA library of the salivary glands of wild-caught B. olmeca was constructed, sequenced, and analyzed. We identified transcripts encoding for novel salivary proteins from this sand fly species and performed a comparative analysis between B. olmeca salivary proteins and those from other sand fly species. With this new information we present an updated catalog of the salivary proteins specific to New World sand flies and salivary proteins common to all sand fly species. We also report in this work the anti-Factor Xa activity of Lofaxin, a salivary anticoagulant protein present in this sand fly species. CONCLUSIONS: This study provides information on the first transcriptome of a sand fly from Mesoamerica and adds information to the limited repertoire of salivary transcriptomes from the Americas. This comparative analysis also shows a fast degree of evolution in salivary proteins from New World sand flies as compared with Old World sand flies.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Alterations in Th17 and the Respective Cytokine Levels in Helicobacter pylori-Induced Stomach Diseases.

BACKGROUND: Infections by Helicobacter can cause the stimulation of sophisticated immune response in mucosal immunity. Among the different lymphocytes, Th17 plays an important role in the defense against H. pylori and may cause gastritis and peptic ulcer due to the increased activation of Th17 and cytokine changes. AIM: To find a relationship between Th17 and IL-17A, IL-21, IL-22, IL-23, TGF-ß in the patients with H. pylori infection having signs including gastritis and peptic ulcer. METHODS: A total of 36 samples from the patients [24 Hp+ and 12 Hp- cases] with dyspepsia symptoms were collected. The percentage of Th17 was measured by flow cytometry. The levels of Th17-associated cytokines in the sera and supernatants of peripheral blood mononuclear cells (PBMCs) which were stimulated with the H. pylori antigen, phytohemagglutinin (PHA), or Dynabeads were measured by ELISA. RESULTS: Patients were divided into two groups of having either H. pylori infected (peptic ulcer, gastritis (mild, moderate)) or being uninfected. The percentage of Th17 in the patients with peptic ulcer and gastritis was significantly higher than their uninfected counterparts (p ≤ .001). The serum levels of IL-17A, IL-23, and TGF-ß in the peptic ulcer and gastritis groups were significantly higher compared with the corresponding levels in the uninfected population (p < .05). A significant association of TGF-ß, IL-21, and Th17 was observed with low levels of IL-17A in the mild gastritis patients (p < .05). Significantly higher levels of IL-22, IL-17A, IL-23, and higher Th17 frequencies were detected in the moderate gastritis patients, as compared with the uninfected patients (p ≤ .001). CONCLUSION: It can be concluded that among the cytokines associated with Th17, the two cytokines of IL-21 and TGF-ß play a more critical role in peptic ulcer and gastritis in the individuals infected with H. pylori. Furthermore, inflammation varies depending on the type of the cytokine and its secreted level.

Downregulation of Th17 cells and the related cytokines with treatment in Kawasaki disease.

Given the inflammatory nature of Kawasaki disease (KD) and the pro-inflammatory properties of Th17, this study aimed to determine the frequency of Th17 cells and the levels of corresponding cytokines in acute phase of KD and to evaluate their alterations one and eight weeks after treatment. Th17 and the related cytokine levels were measured in 21 KD patients and 42 positive and negative controls, using flow cytometry and ELISA, respectively. Th17, IL-17, IL-22 and IL-23 were significantly higher (P<0.05) in patients compared with negative controls, but no significant differences (P>0.05) with the positive controls. Furthermore, Th17, IL-17, IL-22 and IL-23 were significantly higher in patients before treatment than those one and eight weeks after. Considering the downregulation of Th17 and its related cytokines with aspirin and intravenous immunoglobulin therapy implies the probable role of Th17 in KD pathogenesis.

FoxP3+ regulatory T cells in peripheral blood of patients with epithelial ovarian cancer.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Immunostimulatory effects of Leishmania infantum HSP70 recombinant protein on dendritic cells in vitro and in vivo.

BACKGROUND: Activation of dendritic cells (DCs) has an important role in immunity against Leishmania. AIM: We investigated the effect of Leishmania infantum (L. infantum) heat shock protein 70 recombinant protein (rHSP70) as a vaccine on DC maturation and function. MATERIALS & METHODS: BALB/c mouse splenic DCs were isolated and treated with different concentrations of rHSP70. Maturation markers, cytokine production and capability of DCs to proliferate allogeneic T cells were evaluated. Furthermore, this recombinant protein was injected into BALB/c mice, and expression of CD86, CD40 and MHC class II molecules by their splenic DCs were evaluated. RESULTS: rHSP70 significantly increases the production of IL-12p70 by DCs. It had no effect on allogeneic T-cell proliferation in mixed lymphocyte reaction. It increased IFN-γ and decreased IL-4 cytokine level in mixed lymphocyte reaction supernatant. The in vitro study showed that rHSP70 had no significant effect neither on the percentage of CD40(+), CD86(+) and MHC class II(+) DCs nor on the mean fluorescent intensity. However, in vivo results showed that rHSP70 increases the percentage of CD86-, CD40- and MHC class II-expressing cells as well as mean fluorescent intensity of CD40 and MHC class II. CONCLUSION: This study demonstrated the capability of L. infantum-derived rHSP70 in maturating BALB/c mice splenic DCs and in vivo polarization of immunity to a Th1 response.

Interleukin-8 but not interleukin-6 variant may affect susceptibility to brucellosis.

BACKGROUND: 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. OBJECTIVE: To investigate the probable association between IL-6 (-174 C/G) and IL-8 (-251 A/T) gene polymorphisms and susceptibility/resistance to brucellosis. METHODS: 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. RESULTS: 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. CONCLUSION: This study indicates that the IL-8 -251 AA genotype may be considered as a genetic susceptibility factor for brucellosis.

New immunological investigations on Helicobacter pylori-induced gastric ulcer in patients.

Although Helicobacter pylori (Hp) plays an important role in the pathogenesis of chronic gastritis and gastric ulcer, little is known about the probable mechanisms of these types of gastrointestinal damage. To determine the precise mechanisms involved in ulcer formation, immune responses in patients with gastric ulcer (GUP) caused by Hp infection (Hp(+)) were compared with those of other gastritis patients (GP). The sensitivity and proliferation of peripheral blood mononuclear cells (PBMNCs) obtained from patients were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against exposure with complex Hp crude antigen (HPCA) and mitogen (phytohemagglutinin, PHA). Production of inflammatory cytokines, including interleukin (IL)-1ß and IL-8, in serum and supernatants of PBMNCs were then measured by ELISA. It was found that, after stimulation with PHA, both IL-8 and IL-1ß concentrations in sera and supernatants as well as proliferation and sensitivity were statistically greater in GUP Hp(+) than GP Hp(-) . Furthermore, HPCA inhibited the proliferation of PBMNCs dose-dependently; however, it stimulated IL-8 and IL-1ß production in supernatants of mononuclear cells. Therefore, the up-regulated concentrations of IL-8 and IL-1ß may have been caused by increase in the size of mononuclear cell subpopulations or in their cytokine secretory activity, indicating the greatest cell responsiveness in GUP Hp(+) patients. These results suggest that tissue damage and ulcers occur in patients who produce more IL-8 and IL-1ß than patients who do not develop ulcers; the former consequently have more activated immune cells at the site of infection. Therefore, both host responses and Hp virulence factors may be involved in the development of gastric ulcers.

The role of IL15 gene variants in visceral leishmaniasis among Iranian patients.

The role of IL-15 in the protection against Leishmania (L) parasites has been clarified in previous studies, in which IL-15 similar to IFN-γ induces IL-12 production and stimulates the leishmaniacidal activity of the macrophages infected with L. infantum. Furthermore, the increased level of IL-15 in acute visceral leishmaniasis patients (VL) can suppress Th2 cytokines such as IL-4. Since different single nucleotide polymorphisms (SNPs) in the IL15 gene have been described, this study aimed to investigate the association of the SNPs at the positions 267, 367, 13,687 and 14,035 with VL. The IL15 gene variants were compared between two groups consisting of 117 VL patients and 146 healthy individuals using polymerase chain reaction-restriction fragment length polymorphism. The results showed that the frequencies of the alleles 267C (83.9 vs. 73.5%, P=0.0035), 13687A (22.4 vs. 12.8%, P=0.032), genotype 267CC (68.5 vs. 55.6%, P=0.031), haplotypes CGCA (16 vs. 8.3%, P=0.02) and TACA (11.2 vs. 4.8%, P=0.02) were significantly higher in the controls than those in the patients, while the genotypes 267TT (8.5 vs. 0.7%, P=0.0016), 13687CC (78.6 vs. 65.5%, P=0.015), the haplotypes TGCT (10 vs. 2.5%, P=0.00002) and TGCA (5.7 vs. 0.35%, P=0.000001) were significantly more frequent in the patients. In conclusion, it may be speculated that these gene variants with probable effects on the IL-15 production can serve as the factors influencing VL among Iranian population. However, to clarify the association of these variants with the level of IL-15, further studies are recommended.

Effect of nonsurgical periodontal treatment on C-reactive protein levels in maintenance hemodialysis patients.

OBJECTIVE: C-reactive protein (CRP) has been implicated as a possible mediator of the association between periodontitis and several systemic diseases. This study evaluated the impact of nonsurgical periodontal treatment on the serum levels of CRP in chronic kidney disease (CKD) patients on hemodialysis. METHODS: A total of 77 CKD patients on hemodialysis were included in this study. At baseline, periodontal examination was assessed for all the patients, and chronic periodontitis was defined through clinical attachment level and probing pocket depth, according to the American Association of Periodontology. Nonsurgical periodontal treatment was performed and serum levels of CRP were evaluated at baseline and 8 weeks after periodontal treatment. RESULTS: Periodontal treatment resulted in significant reductions in CRP levels (p < 0.001). The difference between pre- and posttreatment CRP concentrations did not show any significant relationship with the severity of periodontitis. CONCLUSIONS: Periodontitis is an important source of systemic inflammation in CKD patients. Nonsurgical periodontal treatment can effectively reduce the serum level of CRP in these patients.

Association of interleukin-18 gene variants with susceptibility to visceral leishmaniasis in Iranian population.

Host resistance to Leishmania infection is mediated by cellular immune responses leading to macrophage activation and parasite killing. Interleukin-18 (IL-18) known as interferon-γ (IFN-γ) inducing factor, stimulates IFN-γ production by T cells. Taking into account the important role of IL-18 in the defense against visceral leishmaniasis (VL) and the known effect of IL-18 gene polymorphisms on its production, the aim of this study was to investigate the probable relationship between IL-18 gene polymorphisms and the susceptibility to VL. The study groups included 118 pediatric patients who suffered from VL and 156 non-relative healthy people as the controls from the same endemic area. IL-18 gene polymorphisms at the positions -656 G/T, -137 G/C and +105A/C (codon 35/3) were analyzed by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). The results showed that the frequency of T allele at the position -656 was significantly higher in the controls, compared with that in the patients (P = 0.047), but it couldn't tolerate Bonferroni correction. Regarding the IL-18 genotypes, there was no significant difference between the patients and controls. Although the frequencies of ATG single haplotype and AGG/ATG double haplotype were significantly higher in the controls (P = 0.043) and the patients (P = 0.044), respectively, the two P values couldn't tolerate Bonferroni correction. Furthermore, a strong linkage disequilibrium was observed among the -656, -137 and +105 single nucleotide polymorphisms of IL-18 gene (all Ps < 0.001). In conclusion, this study suggests that the inheritance of T allele at the position -656 may be considered as a genetic factor for resistance to VL.

Interleukin-17A genetic variants can confer resistance to brucellosis in Iranian population.

On the subject of brucellosis, it seems that Th1/Th2 cytokines balance may be involved in the resistance or susceptibility to Brucella infection. In this respect, Th1 cytokines confer resistance, while Th2 cytokines predispose brucellosis. It is also clarified that IL-17 is required for the induction of IFN-γ and IL-12 in macrophages and dendritic cells. Then, it seems that IL-17 can affect the induction of Th1 immunity which is necessary for controlling Brucella. In the present study, we tried to investigate probable relationship between IL-17A genetic variants and susceptibility to the human brucellosis. One hundred and seventy six patients with brucellosis and 84 healthy animal husbandmen, who consumed contaminated raw milk and dairy products from animals with brucellosis, were included in this study. All individuals were genotyped for 9 single nucleotide polymorphisms (SNPs) (rs4711998AG, rs8193036CT, rs3819024AG, rs2275913AG, rs3819025AG, rs8193038AG, rs3804513AT, rs1974226AG and rs3748067AG) being selected by using NCBI SNP database and literature using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The distribution of IL-17 rs4711998, rs8193038, rs3748067 AA genotypes and AAGAA haplotype were significantly more frequent in the patients than in the controls (P=0.008, 0.0019, 0.003 and 0.002, respectively) while IL-17 genotypes rs3819024GG and rs3819025AA were more frequent in the controls than the patients (P=0.001 and 0.0035, respectively). Based on the results, IL-17 rs4711998, rs8193038, rs3748067 AA genotypes and AAGAA haplotype could be considered as susceptibility factors for brucellosis while the inheritance of IL-17 rs3819024GG and rs3819025AA genotypes might be resistance factors against the disease.

Toll-like receptor 4 (TLR4) polymorphisms in Iranian patients with visceral leishmaniasis.

The role of Toll-like receptor (TLR) 4 in visceral leishmaniasis (VL), a disease caused by an obligate intracellular protozoan parasites belonging to the genus Leishmania, has been shown in the recent leishmaniasis experimental studies. As genetic host factors play an important role in the susceptibility and/or resistance to VL, the association between TLR4 gene mutations [A896G and C1196T single nucleotide polymorphisms (SNPs)] and VL was investigated. Genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) SNPs was performed in the patients with VL (N = 122) and ethnically matched controls (N = 155) using polymerase chain reaction-restriction fragment length polymorphism method. When VL patients and the controls were compared, no statistically significant differences were observed in A896G and C1196T alleles and genotypes (P > 0.05). The TLR4 A896G and C1196T were in moderate linkage disequilibrium in the controls and patients (r (2) = 0.497, 0.548 and D' = 0.705, 0.808, respectively), and haplotypes reconstructed from these SNPs were not significantly different between the aforementioned study groups. In conclusion, based on the results, TLR4 gene polymorphisms at the positions 896 and 1196 cannot be regarded as the major contributors to VL susceptibility among the Iranian population.

IL-1ß (-511T/C) gene polymorphism not IL-1ß (+3953T/C) and LT-α (+252A/G) gene variants confers susceptibility to visceral leishmaniasis.

Lymphotoxin-α (LT-α) and interleukin-1beta (IL-1ß) are proinflammatory cytokines playing important roles in immunity against Leishmania infection and the outcome of the disease. As cytokine productions are under the genetic control, this study tried to find any probable relationship between these cytokine gene polymorphisms and the susceptibility to visceral leishmaniasis in Iranian pediatric patients. Ninety-five pediatric patients involved with visceral leishmaniasis and 128 non-relative healthy people, from the same area as the patients, were genotyped for LT-α (+252A/G) and IL-1ß (+3953T/C and -511T/C) gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There was not found any significant differences in allele and genotype frequencies of LT-α (+252A/G) and IL-1ß (+3953) among the study groups. However, the frequency of IL-1ß -511TT genotype was higher in the controls (P = 0.0004) while the frequency of IL-1ß -511CC genotype and C allele were higher in the patients (P = 0.008 and P = 0.00006, respectively). Furthermore, IL-1ß CC (-511/+3953) haplotype was more frequent in VL patients compared with the controls (P = 0.0002) and the distribution of TT haplotype was higher in the controls compared with the patients (P = 0.003). In conclusion, based on the results, IL-1ß -511C allele, CC genotype and CC (-511/+3953) haplotype could be considered as the susceptibility factors for visceral leishmaniasis while IL-1ß -511TT genotype, T allele and TT haplotype (-511/+3953) might be counted as the influential factors for resistance to the disease.

Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System.

BACKGROUND: Various prokaryotic and eukaryotic expression systems have been 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Interleukin-18 single nucleotide polymorphisms contribute to the susceptibility to brucellosis in Iranian patients.

It seems that IL-18 has a crucial role in immunity against Brucella infection. Since the expression of IL-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relationship between the six different IL-18 gene polymorphisms and brucellosis. A total of 193 patients with brucellosis and 83 healthy farmers who consumed contaminated raw milk and dairy products from the animals with brucellosis, were included in this study. All the individuals were genotyped for six IL-18 polymorphisms at positions -656, -607, -137, +113, +127 and codon 35/3, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The distributions of alleles for IL-18 polymorphisms at positions -137G/+113T/+127C/codon 35/3A (correlated with higher production of IL-18) were significantly higher in healthy controls than in patients (P=0.012, 0.012, 0.012 and 0.0018, respectively). It could be suggested that individuals who inherited the aforementioned genotypes/alleles are able to produce higher levels of IL-18 at the onset of infection, and it leads to more IFN-gamma production and control Brucella infection before the emerging brucellosis.

Early diagnosis of systemic candidiasis in bone marrow transplant recipients.

OBJECTIVES: Systemic candidiasis, are common infections during the neutropenic phase. The aim of this study was to identify quantitative Candida species ribosomal DNA using TaqMan technology for diagnosing candidiasis and monitoring them during hospitalization. MATERIALS AND METHODS: During the prospective, cross-sectional study, from September 2006 to September 2007, a total of 375 clinical blood specimens were collected from 35 patients with hematologic disorders once a week pretransplant and posttransplant. Patients were evaluated for systemic candidiasis during hospitalization. Cultures from the throat, urine, feces, and sputum, along with sonography and computerized tomographic scans, were done when patients were febrile and not having a response to antibiotics. All samples were cultured on Sabouraud dextrose agar with chloramphenicol, and direct, microscopic examination was performed. Blood samples were cultured by bedside inoculation into BACTEC medium at 35 degrees C for 7 days. Clinical blood specimens were evaluated for Candida infections using the TaqMan-based PCR assay. RESULTS: Of the 35 recipients, 6 had multiple samples that were TaqMan-positive with Candida species probe, 3 had 1 PCR positive-result in their blood samples, and the 26 recipients showed negative results. Fungal rDNA was found in 2 patients before and after transplant. All 6 patients with systemic candidiasis had microbiologic and/or radiologic evidence of Candida infections. CONCLUSIONS: It seems that TaqMan-based PCR assay can serve as an accurate method for diagnosing and monitoring Candida infections. This is the first report of its kind that shows Candida infections can be present in the blood of the bone marrow transplant candidates, so closer observation of the recipients who are neutropenic and receive immunosuppressive drugs seems warranted to improve their chances for survival.