In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-gamma/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

Evaluating the effect of four extracts of avocado fruit on esophageal squamous carcinoma and colon adenocarcinoma cell lines in comparison with peripheral blood mononuclear cells

Most patients with gastrointestinal cancers refer to the health centers at advanced stages of the disease and conventional treatments are not significantly effective for these patients. Therefore, using modern therapeutic approaches with lower toxicity bring higher chance for successful treatment and reduced adverse effects in such patients. The aim of this study is to evaluate the effect of avocado fruit extracts on inhibition of the growth of cancer cells in comparison with normal cells. In an experimental study, ethanol, chloroform, ethyl acetate, and petroleum extracts of avocado [Persea americana] fruit were prepared. Then, the effects if the extracts on the growth of esophageal squamous cell carcinoma and colon adenocarcinoma cell lines were evaluated in comparison with the control group using the MTT test in the cell culture medium. Effects of the four extracts of avocado fruit on three cells lines of peripheral blood mononuclear cells, esophageal squamous cell carcinoma, and colon adenocarcinoma were tested. The results showed that avocado fruit extract is effective in inhibition of cancer cell growth in comparison with normal cells [P<0.05]. Avocado fruit is rich in phytochemicals, which play an important role in inhibition of growth of cancer cells. The current study for the first time demonstrates the anti-cancer effect of avocado fruit extracts on two cancers common in Iran. Therefore, it is suggested that the fruit extracts can be considered as appropriate complementary treatments in treatment of esophageal and colon cancers

[ relationship between T CD4[+] cells count and IL-17, IL-11 serum level in idiopathic thrombocytopenic purpura]

Immune Thrombocytopenic Purpura [ITP] is an acquired autoimmune disorder characterized by a low platelet count; because of anti platelet auto-antibodies. ITP patients have auto antibodies against platelet antigens. T CD4[+] lymphocytes are effective cells in immune system that has an important role in auto reactive antibody production and class switching. The pathophisiology and mechanism of ITP is complex and unknown. Numerous studies have difference results about role of T cells in ITP patients. T lymphocytes have been characterized to different subsets. To further investigate about the pathogenesis of ITP, we studied the role of T CD4[+] cells and cytokines attributed with platelet count. Therefore, in this research, we evaluated T CD4[+] lymphocytes count and interleukin 17 [IL-17], interleukin 11 [IL-11] levels in ITP in comparison with control. In a case-control study, we have studied 60 patients with ITP and 50 normal individuals as the control group. Peripheral blood mononuclear cells were isolated by ficoll histopaque 1.077. T CD4[+] cells count in ITP patients and control subjects were studied by flow cytometry method and serum interleukin 17 [IL-17], interleukin 11 [IL-11] concentration were measured by enzyme-linked immunosorbent assay [ELISA] test. All data were expressed as mean +/- SD. Differences between means were considered significant at the P<0.05. Tests were performed using SPSS software version 16. This study showed, T CD4+ cells and plasma IL-17 concentration were not significantly different between patients with ITP and the control group. But plasma IL-11 levels were significantly increased in immune thrombocytopenic purpura patients in comparison with controls [P=0.031]. In summary, our study indicated a role of IL-11 in ITP patients, also showed that ITP may not be associated with changes of plasma IL-17 levels and T CD4[+] cells count relative to control population. Therefore, measurement of plasma IL-11 levels may be important criteria in development of ITP. In addition, it is concluded that determination of IL-11 can be a diagnostic marker to recognize thrombocytopenic purpura patients

Vaccination of diffuse large B- cell lymphoma patients with antigen-primed dendritic cells

Dendritic cells [DCs] are professional antigen presenting cells that have a potential role in the initiating of immune responses. The cell vaccination is a new strategy in treatment of infectious diseases and cancers. In this study, we have generated monocyte-derived dendritic cells of lymphoma patient's peripheral blood mononuclear cells then; these cells were used as vaccine in lymphoma patients. We generated dendritic cell vaccine from lymphoma patient's blood monocytes with human interleukin-4, granulocyte monocyte colony stimulating factor and then, antigen-primed DCS were administrated subcutaneously close to the inguinal lymph nodes after maturation of dendritic cells. After 7 days, we analyzed immune response in lymphoma patients with determining of LDH, Beta 2 Microglobulin, CD4+T cell percent, CD8+ Tcell percent and Tumor size before and after vaccination. Furthermore, phenotypic and functional analysis of dendritic cells was performed using anti CD83-FITC monoclonal antibodies. Before vaccination, the mean +/- SD of LDH was 530.62 +/- 140.65 but after vaccination it was 459 +/- 109.45 that significantly different between experimental groups [P=0.002]. In addition, the CD8+ T cells percentage significantly different between two groups [MX002]. We concluded that the use of dendritic cell probably is one of the suitable noninvasive treatments for lymphoma patients that they have not response to chemical drugs

[Evaluation of caspase 3 and 9 gene polymorphisms in gastric cancer patients in Mazandaran province: a brief report]

Gastric cancer is the most prevalent cancer with poor survival in gastrointestinal tract. Caspase 3 and 9 play an important role in the development and progression of cancer. Polymorphisms in the genes for these enzymes can affect gene activity and thus may influence susceptibility to gastric cancer. In this study, caspase 3 and 9 genes polymorphisms in patients with gastric cancer were examined. In a case - control study, 100 patients with gastric cancer and 100 healthy individuals were evaluated in the region rs4647601: G> T for caspase-3 and -1263 A> G gene promoter for caspase 9. DNA extraction was performed from whole blood according to manufacture protocol. RFLP-PCR method was carrying out for detection of caspase 3 and 9 genes genotype in two groups. In this study, 143 men and 57 women were evaluated. All of them were selected from the same race and geographical area. The results indicated an increase of the mutant G allele in the control group, which leads to a decreasing in the incidence of gastric cancer [P<0.0001, OR: 0.096, [%0.95CL] =0.04-0.23]. It seems that screening of -1263 A> caspase 9 polymorphism could be a useful marker in personal sensitivity to gastric cancer and help to cancer treatment and prevention process. It is concluded that caspase gene variation may be a diagnostic factor in the gastric cancer.

Production of cyclin D1 specific siRNAs by double strand processing for gene therapy of of esophageal squamous cell carcinoma

[RNA interference] is a new strategy in gene therapy and biotechnology which provides new viewpoints in treatment of different diseases such as cancer and viral diseases. CCND1 which is a key gene in cell cycle is amplified and over expressed in esophageal cancer. The aim of this study was to produce siRNAs for CCND1, the key gene in cell cycle. dsRNA digestion method was applied by using recombinant human dicer enzyme to cleave in vitro transcribed dsRNA into a pool of 22bp siRNA. Total RNA was extracted and cDNA was produced using RT-PCR. T7 promoter was added to both ends of the DNA template by PCR. RNA was produced from both strands of the DNA using T7 RNA polymerase. After annealing both strands, dsRNA was prepared. Finally siRNA pool was produced by dicer treatment. RNA extraction yield from HN5 cell line was 14.69 micro g/106 cell. The results from beta actin control gene confirmed the cDNA integrity. After optimization, T7 promoter adding was confirmed using gel electrophoresis and DNA sequencing. After optimization dsRNA yield was improved. The best incubation condition was 18h. Each microgram of dsRNA yielded 0.5 micro g siRNA. dsRNA digestion method includes several steps in which the product of each step is used as the precursor for the next step. So optimization and increasing the specificity and product yield should be the most important goals of the study, because the yield of each step has a direct relationship with the final product yield namely; siRNA. Optimizing and increasing the yield, dsRNA digestion method could be a rapid, available and profitable method for siRNA generation, and providing large amounts of siRNA

Alterations in salivary IgA levels in infectious and inflammatory disorders of upper respiratory tract

The airway surfaces are one of the most common ways of entry of infectious agents. The impact of upper respiratory tract diseases on salivary IgA production has not been fully understood. Therefore, in this study, we investigated the salivary IgA levels in patients suffered from upper respiratory tract diseases to indicate the effect of these diseases on salivary IgA production. In this study, salivary IgA level of 156 patients with inflammatory diseases of the upper respiratory tract including chronic rhinosinusitis, ear and pharynx diseases have been evaluated by direct immunoenzymatic determination. In pharynx disorders 11.8% of patients were IgA deficient, 76.2% were normal and11.8% had elevated level of IgA .In patients with chronic rhinosinusitis IgA deficiency was observed in 9.2%, 75.9% were normal and there was an elevation in 14.8% of patients .In ear disorders 11.6% were IgA deficient ,76.7% normal and 11.6% had elevated IgA level. This study provided evidence for the first time that changes in salivary IgA level are almost the same in different sites of infectious and inflammatory diseases of upper respiratory tract. Our investigation revealed that local up regulation of salivary IgA is not particular interest in majority of patients with upper respiratory tract infections

[Tyrosine kinase domain gene polymorphism of epidermal growth factor receptor in gastric cancer in northern Iran]

Gastric cancer is one of the most common diseases of digestive system with a low 5-year survival rate and metastasis is the main cause of death. Multi-factors, such as changes in molecular pathways and deregulation of cells are involved in the disease development. Epidermal growth factor receptor pathway [EGFR] which is associated with cell proliferation and survival can influence cancer development. EGFR function is governed by its genetic polymorphism; thus, we aimed to study the tyrosine kinase domain gene mutations of the receptor in patients with gastric cancer. In this experimental study, 123 subjects [83 patients with gastric cancer and 40 normal subjects] were investigated in north of Iran for EGFR gene polymorphisms during 1 year. Genomic DNA was extracted by DNA extraction kit according to the manufacture's protocol. Polymerase chain reaction single-stranded conformation polymorphism [PCR-SSCP] and silver staining were performed for investigating EGFR gene polymorphisms. The participants included 72 men and 44 women. Gene polymorphism in exon 18 was present in 10% of the study population but SSCP pattern in exon 19 did not show different migrate bands neither in patients nor in normal subjects. It seems that screening for tyrosine kinas gene polymorphism of epidermal growth factor receptor in patients with gastric cancer and use of tyrosine kinas inhibitors could be useful in the prevention of disease progress and improvement of treatment process for a better quality of life in these patients

Effect of lithium chloride on the luteal steroidogenesis in gonadotropin-stimulated rat

Main function of corpus luteum is progesterone synthesis that is significantly accompanied with an increase in levels of mRNA encoding of steroidogenic enzymes known as luteal markers. This study was designed to evaluate effects of lithium chloride on the release of steroid hormones and steroidogenic enzymes in gonadotropin-stimulated rats. Immature 23 days old Wistar rats were divided into 10 groups; each group comprised of 8 rats, and induced with single injection of pregnant mare's serum gonadotrophin [PMSG] and followed by single injection of human chorionic gonadotropin [hCG]. Then, rats were given lithium chloride [LiCl] or saline at 12 hours post-hCG injection. Ovaries were collected in 4-hour interval from 8-24 hour post-hCG injection. Expression pattern of steroidogenic acute regulatory protein [StAR], side-chain cleavage cytochrome P450 [P450scc] and 3beta-hydroxysteroid dehydrogenase [3beta-HSD] genes were determined by semi-quantitative RT-PCR. In addition, serum levels of progesterone and 17beta-estradiol were measured by ELISA. Our results showed that hCG stimulation of progesterone was markedly diminished and transcript levels of key steroidogenic enzymes were altered in the hormone-stimulated rats following LiCl treatment. These results suggest that critical steps in the function of corpus luteum are disrupted by lithium. It is concluded that LiCl is an effective factor for suppressing of steroid genes expression

[Risk factors for tuberculosis infection: a brief report]

Tuberculosis is one of the most important diseases with annually 8 million new cases worldwide. The purpose of this study was to investigate the risk factors for tuberculosis [TB] infection. In this descriptive study performed in Health center of Maznadaran province during 2010-2011, 183 patients with pulmonary and extrapulmonary TB infection were recruited. After measuring fasting blood sugar, and human immunodeficiency virus [HIV] antibodies, history of smoking was taken by using a questionnaire. The mean age of the participants was 46.8 +/- 19.8 years. The most common risk factor was diabetes and the lowest was HIV infection. Moreover, the prevalence of diabetes in women compared with that of men [OR=0.19, 95% CI=0.07 +/- 0.46] and smoking in men compared with women [OR=12.4, 95% CI=2.8 +/- 54.4, P<0.05] had statistically significant differences [P<0.05]. The results of this research show that diabetes and smoking could be risk factors for tuberculosis infection. It is concluded that, in case of respiratory symptoms in patients with diabetes and smoking, tuberculosis can be considered as an important differential diagnosis

Immunohistochemical expression of vascular endothelial growth factor and its correlation with tumor grade in breast ductal carcinoma

Breast cancer is the most common cause of cancer-related death in female, after lung cancer. Angiogenesis is essential for tumor growth and metastasis; therefore, antiangiogenesis strategies for treatment of cancer are currently an issue of interest. The role of vascular endothelial growth factor that assumed to be most potent angiogenesis factor is ambiguous in breast cancer. This study described the correlation between vascular endothelial growth factor expression and tumor grade, to define the breast cancer patients who responder to anti-vascular endothelial growth factor therapy. In this research, 200 cases of histological proved invasive ductal breast carcinomas analyzed for vascular endothelial growth factor expression by immunohistochemical staining via cross-sectional descriptive study. Vascular endothelial growth factor expressed in 72.54% of the breast cancers. The VEGF was more detectable in grade I [78.5%] than grade II [77.4%] and grade III [56.2%]. There is a significant correlation between tumor grade and VEGF expression [P<0.05]. According to this study, VEGF often expressed in invasive ductal breast carcinomas and inversely correlated with tumor grade. Anti-vascular endothelial growth factor postulated more convenience for tumor progression suppression in low grade tumor than high grade tumor

Determination of trace elements in patients with chronic hepatitis B

Chronic Hepatitis B virus [HBV] infection is a major liver disease worldwide and its clinical manifestations are linked to immune response. The purpose of this study was to evaluate the relationship between selenium, copper, and zinc in comparison with transaminase level in chronic HBV patients. Serum samples of the HBV infected patients were obtained from Tooba medical center, Sari, Iran. Sixty patients were enrolled in this study [36 men and 24 women], mean age: 39.6 +/- 12.2 years. The concentration of zinc, selenium, copper and transaminases were determined using an autoanalyzer system. Concentrations of selenium [0.273 +/- 0.056 micro g/dl] and zinc [2.1 +/- 0.037] was elevated in patients with low transaminase levels as were significantly different in comparison with patients with high transaminase level [P<0.05]. Serum copper concentration was similar in two groups of patients. Elevated levels of transaminase concentrations were independently associated with low zinc and selenium concentrations in chronic HBV patients. It is concluded that serum zinc and selenium levels are associated with less hepatic damage in chronic HBV patients and might have a protective role during liver injury

MICB gene expression on peripheral blood mononuclear cells and susceptibility to multiple sclerosis in north of Iran

Multiple sclerosis [MS] is an autoimmune multifactorial degenerative disease with detrimental affliction on central nervous system. MHC class I chain- related geneA,B [MICA and MICB] are nonclassical human leukocyte antigens that can affect on some diseases and also on transplantation. The purpose of this study was to evaluate the MICA and MICB MRNA expression in multiple sclerosis patients. In this study, we evaluated MICA and MICB MRNA expression in the peripheral blood mononuclear cells by reverse transcryptase-polymerase chain reaction [RT-PCR] in MS patients and normal controls. The results of this study showed that 32.6% of patients with progressive clinical outcome over expressed MICB genes in comparison with controls [p=0.002]. It is concluded that the high expression of MICB gene in MS patients is an important criterion of MS disease that it may be due to the interaction between MICB and its receptor on CD8+T or NK cells

Human Leukocyte Antigen-G Expression on Dendritic Cells Induced by Transforming Growth Factor-beta1 and CD4[+] T Cells Proliferation

During antigen capture and processing, mature dendritic cells [DC] express large amounts of peptide-MHC complexes and accessory molecules on their surface. DC are antigen-presenting cells that have an important role in tolerance and autoimmunity. The transforming growth factor-beta 1 [TGF-beta1] cytokine has a regulatory role on the immune and non-immune cells. The aim of this study is to evaluate the effect of TGF-beta1 on the induction of human leukocyte antigen-G [HLA-G] expression on the DC which is derived from monocyte. Methods: In this study, we evaluated the effect of TGF-beta1 in induction HLA-G expression on the monocyte-derived DC by flowcytometry and then CD4[+] T cell proliferative responses in the presence of DC-treated TGF-[beta1] was studied. Results: The results of this study showed that DC bearing HLA-G down-regulated activation of CD4[+] T cells and production of IL-6 and IL-17 in comparison with control [P<0.05]. Conclusion: It is concluded that TGF-beta1 has an important regulatory role in CD4[+] T cell proliferation by increasing HLA-G on DC and these cells can probably prevent unexpected immune responses in vivo

FOXP3 and TGF-beta gene polymorphisms in allergic rhinitis

Regulatory CD4[+]T [Treg] cells are effective in maintaining immune tolerance. To investigate single nucleotide polymorphisms [SNPs] of Transforming Growth Factor beta-1 [TGF-P 1] and Forkhead Box Protein 3 [FOXP3] genes in Iranian patients with allergic rhinitis [AR]. Variations at codons 10 and 25 of TGF-p 1 and FOXP3 at positions -3279 A>C and -924 A>G were evaluated in AR patients and compared with controls. In a case-control study, 155 AR patients and 163 allergy-free controls were genotyped using polymerase chain reaction sequence-specific primer [PCR-SSP] technique. The analysis of the frequency of these SNPs showed that the haplotype formed by FOXP3 -3279 A allele occurred significantly more frequently in patients than controls [odds ratio=1.44, 95% CI=1.312-2.66; p=0.001]. Our results suggest that polymorphism in FOXP3 gene is associated with susceptibility to AR

Comparison of several maturation including factors in dendritic cell differentiation

Dendritic cells [DCs] are professional antigen presenting cells that have an important role in the initiation of immune response. The use of maturation factors in dendritic cell differentiation provides a promising approach in immunotherapy. In this study, we compared tumor necrosis factor-alpha, polyribocytidylic acid, lipopolysacharide and CpG oligonucleotides in inducing dendritic cell maturation. We generated immature dendritic cells with GM-CSF in combination with IL-4 from peripheral blood mononuclear adherent cells and used tumor necrosis factor-alpha, polyribocytidylic acid, lipoplysacharide and CpG for the induction of dendritic cell maturation. CD83 maturation marker on the dendritic cells was analyzed by flowcytometry after 7 days. In addition, mixed leukocyte reaction between dendritic cells and T cells was performed by MTT proliferation assay. Flow cytometry results demonstrated a comparable high level of CD83 expression on the mature dendritic cells generated by TNF-alpha, CpG, Poly I:C, and LPS treatment of the immature dendritic cells. However, a significantly poorer proliferation of lymphocytes cocultured with the Poly I:C-treated DCs was observed compared to the CpG-treated DCs in mixed leukocyte reaction [p=0.025]. Our results indicated that all of studied maturation inducing factors can be used in DC maturation but TNF-alpha and CpG were the preferred in vitro maturation factors. It is concluded that maturation of dendritic cells by CpG motif and TNF-alpha can be used to regulate immune responses

Generation of immune inhibitory dendritic cells and CD4+T regulatory cells inducing by TGF-beta

Variety of positive as well as negative regulatory signals are provided by antigen presenting cell in particular by dendritic cells. In this research, we studied the capacity of dendritic cells to expand antigen-specific T regulatory cells.We also investigated the role of TGF-beta in induction inhibitory functions of dendritic cells in mixed leukocyte reactions. Dendritic cells were generated from blood CD14+ monocytes with granulocyte-Monocyte colony stimulating factor and interleukin-4 with or without TGF-beta [TGF- Beta-GM-DC or GM-DC]. CD4+ T cell were isolated to assess lymphocyte proliferation by lymphocyte transformation test assay and the ratio of CD4+FOXp3+ CD25+ T cells were determined by fluorescene-activated cell sorter. T cell proliferation responses in GM-DC showed a significance antigen-specific proliferative response comparing with TGF Beta-GM -DC. T Cell proliferation was inhibited in co-culture system containing DC-treated TGF- Beta. It can be suggested that the expsansion of T regulatory by TGF- Beta-GM-DC provides a means for antigen specific control of unwanted immune reactions

effect of beta interferon on dendritic cells and cytokine synthesis by CD4+ T cells

Dendritic cells [DC] are a key regulator of the immune response, and interferon-beta [IFN-beta] is considered an immunomodulatory molecule for DC. The purpose of this study was to evaluate the ability of IFN-beta treated DC to induce cytokine secretion by CD4+ T cells Dendritic cells were generated from blood monocytes with granulocyte-monocyte colony-stimulating factor and interleukin-4 with or without IFN-beta. We analyzed the production of CD4+ T helper cytokines [IL-17, IFN- y and IL-10] in the supernatant of the dendritic cell-T cell co- cultures by ELISA. We also studied the effects of HLA-G and costimulatory molecules on immature and mature DC. IFN-? and IL-17 decreased significantly in the presence of HLA-Gbearing DC compared to control cultures [p < 0.05]. Using the mixed leukocyte reaction, we found that DC treated with IFN- beta mediated the inhibition of T cell activation via cytokine production. We conclude that this is important for preventing overactivation of the immune system

Dendritic cells bearing HLA-G inhibit T-cell activation in type 1 diabetes

HLA-G is normally expressed on human trophoblast cells. It is a non-classical MHC molecule class I b. The role of HLA-G in diabetic type 1 is not known. We investigated the role of IFN-beta in induction HLA-G expression on the monocyte derived dendritic cells [DC] in diabetes type 1. Treatment of dendritic cell with IFN-beta in vitro from diabetic patients [n=20] and normal subjects [n=20] resulted to the production and expression of HLA-G on these cells from both groups. However, comparison of DC from the diabetic patients with DC from the controls revealed lower levels of HLA-G molecules in DC from diabetic patients. Using mixed lymphocyte reaction [MLR], it was found that DC expressing HLA-G mediated the inhibition of autologous T cell activation. It is concluded that IFN-beta can increase HLA-G in DC from diabetic patients; subsequently it may prevent the immune regularly pathway in the diabetic pathogenesis