[Evaluation the relationship between alleles of+1188 in region of IL-12 with serum level of cytokine in patients with occult HBV infection]

Occult hepatitis B infection [OBI] is a form of hepatitis, which in despite of absence of detectable HBsAg, HBV-DNA is present in peripheral blood of patients. Evaluation the relationship between alleles of+1188 in region of IL-12 with serum level of cytokine in patients with occult HBV infection. In this study, the plasma samples of 3700 blood donors were tested for HBsAg and anti-HBc by ELISA. The HBsAg negative ve and anti-HBc positive samples were selected and screened for HBV-DNA by PCR. HBV-DNA positive samples assigned as OBI cases and PCR-SSP and ELISA were performed to examine the polymorphisms in region of [+1188 and serum level of IL-12] respectively. The results showed that there is a significant difference in CC allele of+1188 region of IL-12 in two groups and no difference in the other evaluated genes. There is not any significant difference in serum level of IL-12 between OBI patients and controls. Our results also showed that there isn't any significant statistically relation between alleles of+ 1188 region of IL-12 with serum level of cytokine. According to the results of this study it could be concluded that OBI patients unable to produce enough quantity of IL-12 and it may be related to different IL-12 gene. CC allele was associated with OBI, hence, it seems that +1188 region of IL-12 gene has an important role in expression of IL-12 gene. Evaluation of relation between polymorphisms in +1188 region of IL-12 gene and its expression. In vitro and under mitogene affect can useful because no association was seen between serum level of IL-12 and alleles of this region

[Effect on alpha hemoglobin chain expression in K562 cell line]

MicroRNAs [miRNA] are small noncoding RNA molecules that transcribed by RNA polymerase II. After biogenesis, these molecules act by incorporation into the RNA-induced silencing complex [RISC]. MiRNAs are involved in multiple physiological and pathological processes such as proliferation, differentiation, apoptosis and cancer. Recently several studies reported down regulation of mir-150 during erythropoesis. Since hemoglobin expression is valuable indicator of erythroid differentiation we evaluated the mir-150 downregulation effect on alpha chain expression by Quantitative RT-PCR. K562cells were grown in RPMI1640 in standard condition. K562 cells were transfected by microRNA 150 Inhibitor using transfection kit .Mir-150 downregulation was confirmed by miRNA Real time PCR, followed by Q-RT-PCR to investigate the alpha chain expression changes. By relative QRT-PCR the alpha chain expression was increased 10 folds in comparison to untransfected and scramble cells. Furthermore, the differences were statistically significant [P<0.05] Elevation of alpha chain expression in our study showed that mir-150 downregulation has a crucial role in erythroid differentiation and can introduce as a novel marker in alpha thalassemia. Further researches to find out the detail mechanism and miRNAs genes target could improve our knowledge about miRNAs potential in management of diseases and their applications in gene therapy and regenerative medicine

Relation of Interleukine-4 [C-589T] and Interferon-Gamma [A+874T] allele polymorphism with Asthma

Asthma is a common respiratory disease caused by acute and chronic bronchial inflammation. Clinical manifestations of the disease are closely related to genetics. IL-4 is a cytokine of TH2 lymphocytes, polymorphism in prompter region, C-589T, is associated with IL-4 production, while IFN-gamma, is a cytokine of TH1, and A+874T polymorphism in interon 1 of IFN-gamma is associated with it.s production and release. Cytokine gene polymorphisms could influence pathogenesis of asthma with TH1/THh2 ratio, being of great importance. 81 unrelated asthmatic patients were selected according to ATS characteristics and separated into two groups of controlled and uncontrolled asthma. 80 normal subjects were chosen as control group. After collection of peripheral blood and DNA extraction, PCR-RFLP method was used for genotyping of IL-4, -589 position. For evaluation of polymorphism in +874 position of IFN-gamma ARMS-PCR method was used. Distribution of frequency of IFN-gamma [A+874T] and IL-4[C-589T] polymorphisms didn.t show any statistically significant difference between two patient groups and healthy control group [p >/= 0.05]. There was neither any significant difference [p >/= 0.05] among other parameters. Studies in field of cytokine polymorphism have had variable results. So many studies have mentioned a relationship between cytokine gene polymorphism and susceptibility and/or severity of asthma and some studies have shown that there is no association between these factors we believe that there may exist factors different from IL-4 and IFN-gamma polymorphism which coner the effects of these genetic vaciants in pathogenesis and severity of asthma

Use of immunoglobulin heavy chain and kappa light chain gene rearrangements by PCR for molecular diagnosis of minimal residual disease in Iranian children suffering from beta-precursor acute lymphoblastic leukemia

Diversity of IgH and IgK molecules is generated during B and T Lymphocyte differentiation through the rearrangement of variable, diversity, junction and constant gene segments. Additionally, random insertion and deletions of nucleotides between gene segments make unique sequences which are cell or clone specific. Similar IgH and IgK genes rearranged in normal cells of lymphoid leukemia cases can be used as a marker of clonality and for evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of IgH chain and IgK gene rearrangements using polymerase chain reaction [PCR] in beta-precursor acute lymphoblastic leukemias [ALL] to follow the MRD at day 14, day 28 [end of remission induction], week 10, 3-6 months and 6-12. month after the initiation of treatment. In our prospective study bone marrow aspirates of 183 children at the mean age of 63.6 months with diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 cases with diagnosis of beta-precursor ALLs were selected for study. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, IgH and IgK [V[K] I-IV / Kde] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were compared and aligned with the sequences homologous for IgH and IgK published by Gene Bank. The follow up specimens were collected at day 14, day 28 [end of remission induction], day 45-month 3, and 3-6 months and 6-12 months after initiation of treatment. After routine cytomorphologic analysis, similar PCR was done on follow up extracted DNAs in parallel with diagnosis DNA. MRD was considered to be approved positive if bands similar to those at the time of diagnosis were present. Statistical analysis using SPSS software [version 11.5] was performed. 90.5% of patients had clonal IgH gene rearrangements. Monoclonal, biclonal and oligoclonal patterns were observed in 57.8%, 34.9% and 5.5% of patients with IgH [CDR III] rearrangement, respectively. Clonal patterns of IgK-Kde were detected in 59 [67%; n: 88] of BP-ALLs. According to cytomorphology about 92% of patients were in complete remission. MRD positivity decreased from more than 90% to 20% using different gene rearrangements in defined time points. Four patients who relapsed during follow up were MRD positive using 1-3 rearrangements and all except one were in clinical remission. Clonal rearrangement of IgH had a pattern similar to other populations. IgK was slightly more frequent than previously reported and the VKI [25%] was the most common type. These differences can be explained by different techniques, DNAs and clonality markers. According to the results, these clonal markers can be used in diagnosis and follow up of MRD

TTV prevalence in Ahwaz blood donors by using semi-nested PCR

Occurrence of new infectious agents threatens access to zero risk in blood transfusion and enhancement of blood safety. Although sensitive methods are available for diagnosis of hepatitis, yet some hepatitis cases do not have a known etiology. In 1997, the novel DNA virus was isolated from post-transfusion serum samples of patients affected by non-A-G hepatitis. Nowadays this novel virus is known as transfusion-transmitted virus. This circular single stranded unenveloped and virucidally resistant virus is the first human circovirus and has universal distribution. It is believed that TTV may cause hepatitis and aplastic anemia. This study was conducted to determine the prevalence of TTV in healthy blood donors in Ahwaz and set up N22 PCR for subsequent first-time viral studies in south region in Iran. In 2003, We studied the presence of TTV DNA by using Okamoto primers with PCR in plasma of blood donors in whom serologic tests for hepatitis A-C and HIV-Ab were negative. Our study showed that the virus prevalence in blood donors was 23.7% [60/253] and there was not any significant differences between prevalence of TTV and background variables. Our findings showed the same prevalence rate as in neighboring countries; however, in comparison with thalassemic patients that were studied in parallel with the present research, the difference was significant [143/250; 57.3%]. It shows the importance of blood transfuison in transmission of the virus

Platelet activation and IL-8 production in platelet concentrates prepared by buffy coat and PRP method

The process of platelet concentrate production by plasma rich [PRP] method could activate the platelet and granules secretion of beta thromboglobulin, LDH and CD62P. Platelets activated during the preparation process do not have sufficient efficiency for hemostasis in vivo. It seems that platelet preparation by buffy coat method has an ability less than PRP to activate the platelet. Measuring platelet activation indices, such as CD62P expression and beta thromboglobulin, is a useful means to evaluate the percentage of activated platelet concentrates and compare the two methods of buffy coat and PRP. In this experimental study, 15 concentrates were prepared via PRP method and 15 via BC method; 15 intact blood units were also considered as control group. The percentages of CD62P expression, soluble CD62P concentrates, IL-8 level, and CD14 positive cells were evaluated. Special monocolonal antibodies that conjugated with flourecence dye in flocytometric method were used for CD62P and CD14. ELISA method was used for evaluation of soluble CD62P and IL-8. The average platelet count in both methods showed no significant difference, but WBC contamination rate in PRP-PCs was more than BC-PCs. In PRP-PCs, we found a little decrease in CD62P expression and increase in soluble form and IL-8 level during reservation time. The level of CD14 showed no significant difference in these components. In BC method during the three day reservation, expression of CD62P, its soluble form, and IL-8 concentrates increased and the level of monocyte surface CD14 showed slight decrease ranging from 0.4 to 0.1. It is concluded that there is a close relationship between IL-8 and WBC count in platelet concentrates. In PRP method in contrary to BC method, high speed centrifuge causes adhesion, aggregation and platelet activation

Detection of N-RAS gene mutations in codons 12, 13 and 61 in patients with acute myeloid leukemia

Acute myeloid leukemia [AML] comprises a heterogenic group of malignant disorders involving cell maturation arrest at an undifferentiated stage in bone marrow. Activation of N-RAS proto-oncogene due to point mutations plays a major role in AML malignancy. Since there was no report on the frequency of N-RAS gene mutations in Iranian AML patients, therefore, we decided to determine its frequency and compare the results with age, sex and FAB subtypes. In this descriptive study, 60 de novo AML patients from Tehran Shariati hospital, hematology-oncology and bone marrow transplantation center were screened for the mutations of N-RAS gene at codons 12, 13 and 61. DNA was extracted from peripheral blood samples before the start of chemotherapy. The above mentioned codons were amplified by PCR and analyzed by restriction endnuclease enzymes. We were able to detect mutations in 12 out of 60 [20%] patients. Most of the mutations were detected in men with an age over 40 years old. The frequency of mutations for codons 12, 13 and 61 were 15%, 11.6% and 5% respectively. Most of the mutations [33.3%] were found to happen in AML-M4 FAB subtype. We could not detect any mutation in AML-MO, M6 and M7. We detected mutations in 20% of our AML patients. In general, the frequency of the mutations we found was in agreement with the results of other studies. However, a study with more patients and a wider range of age using a combination of PCR-RFLP and direct gene sequencing is highly recommended

pattern of cross lineage T-cell receptor delta/gamma gene rearrangements in B-precursor acute lympho-blastic leukemia of Iranian children using polymerase chain reaction

Diversity in heavy chain immunoglobulin [IgH] and T-cell receptor [TCR] molecules occures during B- and T-lymphocyte differentiation through the rearrangement of variable [V], diversity [D], junction [J] and constant [C] gene segments. Lymphoid leukemia cells are similar to normal precursors and have rearranged IgH, IgK and TCR [cross-lineage rearrangement] genes which can be used as a marker of clonality and evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of TCR- delta/gamma gene rearrangements using Polymerase Chain Reaction [PCR] in B-precursor acute lymphoblastic leukemia [ALL] in Iranian children. In our prospective study, bone marrow aspirates of 183 children with early diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 subjects with diagnosis of B-precursor ALLs were selected for study. Sixteen were excluded from our study due to various reasons including cellular degeneration. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, hyper-variable regions TCR-delta [V delta2-D delta3 and D delta2-D delta3] and TCR-gamma [V gamma; V gamma I and V gamma II] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were then compared and aligned to the homologous sequences of Gene Bank for confirmation. T-test, Mann whitney, Fisher exact test and Chi-square were used for data analysis. Clonal rearrangement of TCR-gamma [V gamma] and V gamma l/Il were present in 79.3% and 64.9% of patients respectively and only 5% of cases showed biclonal pattern. The V gamma ll rearrangement was the most common [46.8%] type in TCR-gamma. 47 [45.2%] and 11 [16.6%] of patients had V delta2- D delta3 and D delta2-D delta3 partial gene rearrangements, respectively. Biclonal/oligoclonal patterns were present respectively in 27.7% and 4.3% of cases with Vdelta2-D delta3 rearrangement. Only one patient had biclonal D delta2 D delta3 rearrangement. Clonal rearrangement of TCR-delta [Vdelta2-Ddelta3 and D delta2-D delta3] genes had a pattern similar to other populations. Frequency of TCR- gamma [V gamma I and V gamma II] rearrangements was slightly higher than previous reports, and in contrary to others except for Brazilian report the V gamma II rearrangement was the most common type. We found no significant correlation between presence of different types of rearrangements and quantitative variables. The only significant point was the reduction of Vdelta2Ddelta3 with increase in age. According to preliminary results, these clonal markers can be used in diagnosis and follow up of MRD

[Cross-resistance to anticancer drugs in acute myeloblastic leukemia Pgp expressing K562 cell line]

Drug resistance remains one of the most important clinical obstacles in the treatment of some cancers. This drug resistance referred to as Multidrug Resistance [MDR] induces cross-resistance to many chemotherapy agents such as anthracyclines, vinca alkaloides, epipodophyllotoxins and Taxol. MDR is most likely due to the reduction of drug accumulation with an energy-dependent drug efflux pump. This drug pump is a 170 kDa transmembrane glycoprotein [Pgp]. We developed a resistance subline of K562 by stepwise increase in concentration of Doxorubicin, and Pgp expression was verified by flowcytometry and RT-PCR methods. Cross resistance of the resistant cell line to Etoposide, Vincristine and Taxol was analyzed by MTT assay. IC[50] [the level of drug concentration inhibiting 50% of cell growth] of Doxorubicin, Etoposide and Taxol of parental K562 came out to be 100ng/ml and it was 50 ng/ml for vincristine. IC[50] levels of these drugs on resistant K562 were 500, 500, 450 and 450ng/ml. These drugs also displayed 5-, 5-, 4.5-, and 9- fold resistance respectively. According to the results, expression of Pgp confers MDR phenotype to the K562 cell line and makes it resistant to most of anticancer drugs including anthracyclines, vinca alkaloides, epipodophyllotoxins and taxans. This MDR phenotype is a major obstacle of cancer treatment and in recent years investigators are trying to reverse it by gene therapy

Expression of recombinant human serum albumin in the methylotrophic yeast; Hansenula polymorpha

Human serum albumin [HAS] is the major protein component of human plasma. It plays a very important role in transporting of macro molecules and maintaining the normal osmolarity. It is used as a therapeutical protein in patients with hypoalbuminemia and acute bleeding and burning. Albumin consumption in the world is about 500 ton/year. The aim of this research is to study the production of rHSA in shake flask culture by Hansenula polymorpha. H. polymorpha was used for the production of recombinant human serum albumin [rHSA] in several of shake flask culturing; expression of rHSA was investigated relating several parameters affecting the expression of HSA. To optimize the secretory expression of rHSA under the control of FMD promoter in H polymorpha RB-11 incubation time, culture media temperature and protease inhibitors were analyzed. This study not only established production of rHSA in yeast but also analyzed the correlation between affecting parameters and the level of HSA expression. Comparison of the HSA levels in the culture supernatants showed that the highest HSA yield was 17.6mg/l. The research shows that among three different temperatures [25°C,30°C and 37°C] 37°C was the best temperature and amongst three different incubation times [24h,48h and 72h] 48h was the optimum time and YNB 1% glycerol with buffer was the best derepression medium in comparison with others. Using these optimized conditions, stable production of rHSA of around 17.6mg/l was achieved. Our results suggest that affecting experssion factors improved in this study are suitable for production of recombinant albumin

Evaluation of platelet activation in platelet concentrates during storage in the quality control laboratory of Iranian blood transfusion organization in 2004

Conditions for preparation and storage of platelets for transfusion purposes may lead to platelet activation which in turn contributes to decreased ability of stored platelets to function and to survive in after transfusion as compared with freshly prepared platelets. We investigated platelet membrane expression of CD62P, CD63 in platelet stored for up to 3 days under standard blood banking conditions. Twenty-four platelet units prepared by platelet-rich-plasma and platelet concentrates were evaluated during storage for markers CD2P, CD63 and pH. During storage for up to 3 days platelet units displayed no significant pH [p>0.05]. During storage for up to 3 days [days 1 and 3] platelet units were significant in the CD62P and CD63 expressions as compared with day 0 [p<0.05]. Storage of platelet concentrates causes activated platelets. Moreover, these markers [CD2P and CD63] can act as useful in vitro means in the quality control of platelet components

[Geographical distribution of ABO and Rh [D] blood groups among Iranian blood donors in the year 1361[1982] as compared with that of the year 1380 [2001]]

The distribution of main blood groups vary according to racial, ethnic and geographical differences. Due to their importance in qualitative and quantitative management of safe blood supplies in different geographical regions, due to the relation between a specific blood group with the prevalence of a typical disease, and further due to their significance in kidney transplantation procedure, we decided to analyze the frequency of ABO and Rh[D] blood types among 1,300,000 Iranian blood donors in different provinces of Iran in the year 2001 and compared the results with a similar study that was conducted in 1982. Clotted blood samples were obtained from donors. Then, the samples were tested for A, B, O and Rh [D] blood groups using anti-A, anti-B and anti-D reagents. The ABO blood group was determined by comparing the results of forward typing with that of reverse typing. The final results were collected from 28 different provinces throughout Iran and were then analyzed by Excel program. Our findings are shown in a discending order of frequency: O blood group was detected in 37.62% of population; A blood group in 30.25%; B blood group in 24.36% and AB blood group in 7.77%. The frequency of O and B blood groups has increased 1.3% in comparison to the results obtained in 1982; whereas the frequency of A blood group has decreased by 2%. In some provinces such as Azarbayejan-Gharby, Isfahan, Ilam, Chaharmahal-Bakhtiyary, Khuzistan, Fars, Kordestan, Kohkiloyeh-BoyerAhmad, Mazandaran, Hormozgan and Yazd, the blood group frequencies have shown more alteration. This change in frequency is due to several factors including the modification of provincial borders, migration to other cities during the Iran-Iraq war, as well as the tendency to move to larger, industrialized cities

[Tracing blood units from recipients to their donors: results of a trace back study for hepatitis B]

Post -transfusion hepatitis B may occure if donors donate blood in the window period, in the early convalescence phase and period with very low levels of HBsAg in blood. In December 2003, a case as post- transfusion hepatitis B in a blood recipient was reported to IBTO.The patient had received 2 units of red blood cells.Trace back program was set up to find out the donors possible involvement in viral transmission.Donors and recipients were not positive for HBsAg and Anti-HBc IgM; so current or recent infection during last 6 months had to be excluded. This case report explains a successful trace back , and accurate well- maintained records

cytotoxicity pathway of natural killer cells in cord blood compared to peripheral blood

The cytotoxic activity of natural killer cells is usually tested by radioactive assay [51Cr release assay], which detects the release of cytoplasmic contents after plasma membrane disintegration of dying cells. In contrast to this indirect evaluation of cytotoxicity, the assessment of cell damage by flow cytometry aims to provide a more exact characterization of the death pathway via detection of the percentage of apoptosis and necrotic cells. Annexin V-FITC [Axv -FITC] can be used to label cells in the early apoptopic state, while propidium iodide [PI] indicates late apoptosis or necrosis. The NK cytotoxicity of cord blood [CB] and peripheral blood [PB] was determined after 4 hours of incubation in the absence of cytokines. After 4 hours in vitro incubation, co-staining with Annexin V-FITC [Axv-FITC] and propidium iodide [PI] permitted discrimination between viable, early apoptotic and necrotic cells. As we would expect, the cytotoxicity pathway in PB mononuclear cells [MNCs] consists of both apoptosis and necrosis pathways but in CB MNCs it almost consists of early apoptosis; and necrosis is negligible. With escalating E: T [effector: target] ratio changes in the percentage of apoptotic cells in PB samples were significantly higher than CB samples. The mechanism [s] of the low cytotoxicity of resting cord NK cells is not well understood. Complementary research in this field is recognized to elucidate the phenotypical and functional properties of CB cells and how they relate to maturational stages. CB studies are important for transplantation research and may provide insight to the suppressive mechanism by which the host-recipient could evade GVHD and rejection

Alteration of T-lymphocyte subpopulations in subacute and chronic brucellosis

Human brucellosis is a major health problem in developing countries. A number of patients with human brucellosis do not recover from the acute stage of the disease and pass into the chronic form. Since the pathophysiology of this change is not well understood, we studied the T-cell subsets during the acute, subacute and chronic forms of human brucellosis. In this study we found alterations in T-lymphocyte subsets and in the CD[4]/CD[8] ratio in subacute and chronic brucellosis while no significant change was found in T-cell subsets in acute brucellosis. We believe that this alteration of T-cell subsets is important in the pathophysiology of brucellosis, but it is not clear whether brucella cause this change or a primary immunodysfunction in patients with a disturbance in T-Cell subset regulation prevents recovery from acute brucellosis and leads to the development of chronic brucellosis