Cell Journal (Yakhteh)
Year:2006 | Volume:8 | Issue:2 (30)|Papers Count:12

Introduction: Decidua as the nearest mother's tissue layer to the fetus which is in direct contact with the embryo has an important role in composing the appropriate microenvironment for protection of allogenic fetus from mother's immune response. In this study the effect of supernatant from culture of decidual cells on dendritic cells regarding the induction of T-cell allogenic proliferation response and Indoleamine 2, 3 dioxygenase (IDO) enzyme presentation was evaluated.Material and Methods: Decidua of allogenic pregnant mice (C57BL/2 x Balb/c) were isolated at mid gestation and after their enzymatic digestion, the obtained cell suspensions were cultured. The cell supernatants were collected after 48 hours of culture and frizzed (-70  °c) until use. Dendritic cells were obtained from the spleen of C57BL/6 mice by enzymatic digestion and Nycodenz gradient centrifugation. The adherent cells obtained after 2 hours of culturing were incubated with various concentrations of decidual supernatant for 12-15 hours, during this time dendritic cells maturated and floated.T lymphocytes were isolated from brachial and axillary lymph nodes of Balb/c mice by nylon wool method. Decidual supernatant of treated dendritic cells were cultured with allogenic T cells after  Y-irradiation (3000 rad) in MLR condition and cell proliferation was measured by 3H-thymidine incorporation. For identification of suppressive mechanism of IDO enzyme its specific inhibitor (L-Methyl-Tryptophan) was used.Results: Flowcytometric analysis showed that dendritic cells and Tcells were %93±2 and %91±2 pure, respectively. MLR results showed a decrease in T cell proliferation response when dendritic cells were treated with decidual supernatant of pregnant mice. While treatment of dendritic cells with culture supernatant of none pregnant mice uterus didn't show such effect. This suppression was adjusted by addition of L-Methyl tryptophan.Conclusion: Decidual supernatant may suppress the T-cell responses via IDO induction in dendritic cells.

Introduction: Pronuclear size, morphology and orientation are known to affect embryo quality and implantation. The present study was aimed at evaluation of the possible relationship between protamine deficiency with pronuclear morphology and also cleavage and embryo quality.Material and Methods: Semen sample of 160 candidate ICSI and IVF couples were analyzed for sperm concentration, and motility. A portion of semen was used for Papanicolaou and CMA3 staining for assessment of sperm morphology (WHO criteria) and protamine deficiency, respectively. Around 18-20 hours after sperm insemination or ICSI ‚ oocytes were scored for presence and orientation of pronuclei‚ nuclear precursor bodies' distribution and also the axis of second polar body and were therefore categorized into & groups accordingly. Embryos were evaluated in the third day after fertilization for percentage of cleavage and embryo quality. The relationship between percentages of CMA3 positive sperms and pronuclei morphology ‚ cleavage coefficient and embryo quality were determined.Results: Significant correlation was observed between semen parameters, percentage of fertilization and percentage of CMA3 positivity (p<0.01). Assessment of zygote morphology revealed a significant correlation between unequal pronuclei and abnormal sperm morphology (p<0.01). However no significant correlation was observed between CMA3 positivity and pronuclei morphology, cleavage and embryo quality score (p>0.05). The only significant correlation observed during zygote assessment was between nucleolar precursor body polarity‚ distribution and percentage of cleavage on day 3. (r=0.237 p<0.001).Conclusion: The results revealed that protamine deficient sperms have lower fertilization potency. However‚ protamine deficiency does not effect embryo development and cleavage rate post ICSI until day 3 .However pronuclei morphology is considered valuable for selection and transfer of embryo but assessment of cleavage could be used as a complementary criteria.

Introduction: To study the prevalence of most common  b-thalassemia mutations in Lorestan province and use the results for epidemiologic study and prenatal diagnosis of b_thalassemia major.Material and methods: 130 chromosomes from 65 unrelated homozygous b_thalassemia patients from Lorestan province of Iran (west-central) were investigated for b globin gene mutations by ARMS PCR.Results: Most common mutations of the Mediterranean region were examined in this study. We found a different mutation spectrum in Iran compared to the data obtained by other authors. Our results showed that codons 36/37 (-T) with a frequency of 33.84% represented the most common mutation followed by the following four mutations in the Mediterranean region, IVS-II-1 (G-->A), IVS-I-110 (G-->A), frameshift codons (FSC) 8/9 (+G) and IVS-I-5 (G-->C) with frequencies 27.69%, 11.53%, 10.76% and 4.47% respectively. The less frequent alleles, IVS-II-745 (C-->G), Codon 5(-CT), IVS-I (25 bp deletion) and Frameshift CD44 (-C) with the following frequencies 1.59%, 0.76%, 0.76% and 0.76%, respectively, accounted for only 3.87% of the mutations. No mutations in Codon 30 (G-->C), Codon 39 (C-->T), IVS-I-6(T-->C) and IVS-I-1(G-->A), were detected. The unknown alleles were 7.63%.Conclusion: These data suggest that the pattern of mutations in Lorestan province differs from those reported for the Mediterranean and other thalassemic regions in Iran.

Introduction: The aim of this study was to setup, optimize and introduce a sensitive and specific PCR detection method for identification of Neisseria meningitidis DNA in clinical samples.Material and Methods: Capsular transport gene A (ctrA) was selected as a specific target sequence. This primer pair amplifies 101bp of the target gene. Neisseria meningitidis strain: ATCC; 13090 and Neisseria meningitides serogroup C were used as a standard organism for optimization experiments. A range of bacterial pathogens were used for specificity testing, including, Haemophilus influenzae Type b: ATCC; 49766, Escherichia coli: ATCC;35218, Enterobacter, Klebsiella pneumoniae,  Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus group D.Phenol – chloroform method was used for DNA extraction. Amplified product was detected by gel agarose electrophoresis, stained by ethidiome bromide. Results: Our results confirmed amplification of the expected product. Specificity test proved no cross reaction with tested organisms. Sensitivity test detected 500fg of Neisseria meningitidis DNA as a final detection limit.Conclusion: As a conclusion, PCR is a method with high sensivity and specificity and specificity which can be performed within 3 hours, and therefore utilization of this test in the clinical laboratories can help rapid diagnosis of Neisseria meningitides in clinical samples.

Introduction: Beta thalassemias are a heterogenous group of autosomal recessive disorders, characterized by reduced or absent production of the b-Globin chain by the affected allele. Transplantation of allogenic hematopoietic stem cells (HSC) is a curative approach but this therapeutic option is not available to the majority of patients. Transplantation of genetically corrected autologous HSC is an attractive approach for the cure of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy because it can precisely correct the mutant genes and restore their normal expression. In the present study a specific gene construction for b-Globin gene targeting was designed and constructed. This construction consists of: two homologous arms including, upstream and downstream regions of b-Globin gene, b-Globin gene as the target gene, hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase gene as negative selection marker.Material and Methods: All segments were amplified by PCR and cloned in pTZ57T/A cloning vector and then sub cloned in pBGGT. The authenticity of cloning and sub cloning steps was checked by PCR, restriction analysis and finally by sequencing. The final plasmid was named pFBGGT. The mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing.Results: The results of PCR, restriction analysis and sequencing during all cloning and sub cloning steps confirmed the authenticity of these steps. The results of sequencing of PCR products on selected cells, confirmed the occurrence of homologous recombination.Conclusion: In this novel strategy, gene replacement was achieved in one step and by a single construct.

Introduction: The aim of this study was to investigate the effect of the HSP-70 (Heat Shock Protein-70) induction in the lysate of heat shocked tumor cells on splenocyte proliferation, nitric oxide production by peritoneal macrophages, and fibrosarcoma tumor size reduction in BALB/c mice.Material and Methods: WEHI 164 cells (mouse fibrosarcoma cell line) were injected subcutaneously into the right flank of syngeneic BALB/c mice to establish a tumor model. Then the lysate of heat shocked (42oC, 1 h) and non heat shocked WEHI 164 cells, were prepared by 5 times freezing and thawing. Then we immunized test group mice with lysate of heat shocked tumor cell (at days 0, 7 and 14). In the control groups we injected the lysate of cells without heat shock and PBS. In this study we detected HSP-70 expression by immunoblot. Tumor volume was measured every 5 day. We detected the proliferation of mouse spelenocyte by using MTT test. We also detected the nitric oxide (NO) production by mouse splenocytes and normal macrophages.Results: In this study we detected increases in HSP-70 expression in the lysate of heat shocked cell in comparison with non heat shocked cells, by immunoblot. Tumor volume was significantly decreased and the proliferation of splenocytes increased in test group. We also observed that immunizing with heat shocked tumor cell lysate resulted in a significantly increased NO production.Conclusion: These results indicated that the lysate of heat shocked tumor cell is more potent than non heat shocked tumor cell in inducing anti tumor immunity.The dual roles of HSPs as molecular vehicles for antigen cross-priming and as activation signals for the innate immune system cell, make them particularly useful for tumor immunotherapy. These findings provide a useful therapeutic model for development of novel approaches to cancer treatment.

Introduction: The purpose of this study was to cultivate bone marrow cells from two different mouse strains (NMRI and Balb/c) and to examine them in terms of expression of ten surface antigens from primary culture to passage 3.Material and Methods: 6-8 weeks old either NMRI or Balb/c mice were sacrificed and their bone marrows were harvested and cultivated .Two weeks after culture initiation, the cells were tripsinized and about half of them subcultured in a new culture dish and the other half was prepared for flow cytometry in order to examine the expression of ten hematopoietic and endothelial markers including CD135, CD44, CD31, Thy1.2, CD11b, CD45, CD34, Vcam1, Sca-1and c-Kit. The cell cultures were examined till passage 3 for the given markers and there after the passage-3 cells from each strain were investigated for differentiation into bone and fat cells in vitro.Results: In primary culture, the cell population was heterogeneous in which different morphology including flat, spindle and polygonal cells were observed. In passage 3 the culture mainly consisted of spindle cells. According to our result passage-3 cells from Balb/c mice seemed to be slightly longer than that of NMRI cells. Flow cytometric analysis showed that CD 44 was expressed in more than 90% of the cells in all examined passages. Although Thy1.2 was not expressed in primary culture, it was observed in about half to one fifth of the cells in passage 3.CD31 was not expressed on cells throughout the culture periods and the expression of CD135, CD45 and CD11b were gradually decreased from primary culture to passage C-kit, Sca1 and CD34 expression was slightly increased during the culture period. The two mice strains showed some differences in expressing certain markers. In contrast to Balb/c cells, some cells of NMRI strain expressed VCAM antigen. Furthermore, the two mice strains were different in that they showed varying pattern of sca-1 and Thy1.2 expressions during cultivation period.Passage-3 cells from both strains were easily differentiated into bone and fat cells in appropriate culture conditions.Conclusion: Our results showed that mesenchymal cell cultures never become homogenous in terms of the markers expressed on the cell surfaces at least from primary culture to passage 3, although it gradually becomes morphologically homogenous during this period. The two mouse strains were somewhat different in terms of their morphology in culture and certain surface antigens.

Type 1 diabetes is the result of an autoimmune attack against the insulin-producing β cells of endocrine pancreas. Current therapies for type 1 diabetes, including fastidious blood glucose monitoring and multiple daily insulin injections, are not sufficient to prevent complications of the disease. The only real cure for type 1 diabetes is replacement of the beta cell mass, currently being accomplished through ecto-pancreatic transplantation and islet transplantation. But the chronic shortage of donor organs limits widespread application of these approaches. Understanding the mechanisms of b cell mass expansion by the power of pancreatic developmental knowledge as well as the means to exploit these pathways have enabled researchers to develop new strategies to differentiate, expand and maintain islet cell mass. The characteristics of stem cells in self renewal and differentiation to different cell types has stimulated the interest in using stem cells as a starting material from which to generate insulin secreting cells in vitro. Insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models. In the present review, we discuss pancreas development and some of the recent advances, focusing primarily on the differentiation and genetic manipulation of embryonic and adult stem cells to functional b cells of endocrine pancreas that may form the basis for future treatment.