Static electromagnetic field and recombinant human fibroblasts encoding miR-451 and miR-16 increased cell trans-differentiation to CD71+ and CD235a+ erythroid like progenitor.

Introduction: Ex vivo blood production is an urgent need of most countries, and creating production protocols can save the lives of many patients. Despite the recent advances in blood production in ex vivo conditions, its high-scale production is not yet possible, and requires further studies. Therefore, by transfecting fibroblast cells with miR-16, and miR-451 genes, as well as applying low frequency electromagnetic fields (ELF-EMF) treatment, we tried to increase the differentiation of these cells into CD71+ and CD235a+ erythroid like progenitors. Methods: After preparation, and cultivation of human dermal transgenic fibroblast cells, they were transfected by Plenti3-hsa-miR451, Plenti3-hsa-miR16 and Plenti3-backbone inserted into E. coli Stbl4 genome. Then, transgenic fibroblast cells were treated with 10mT ELF-EMF every day for 20 minutes for 7 days. Using a flow cytometer, the expressions of CD71, and CD235a were studied in these cells, and the expressions of genes involved in hematopoiesis were studied using the RT-PCR technique. Results: The results indicated an increase in the differentiation of fibroblast cells treated with 10mT ELF-EMF to erythroid like progenitors. Furthermore, the percentage of CD71+ and CD235a+ cells was the highest in irradiated cells encoding miR-16 and miR-451, which indicates their differentiation into erythroid like progenitors. Also, in the transgenic cells treated with ELF-EMF, an increase in the expressions of α-chain, ß-chain, γ-chain and GATA1 genes was observed, which indicates the potential of these cells for hematopoiesis. However, there was no significant difference in the expression of CD34 and CD38 genes in these cell lines. Conclusion: Both ELF-EMF and upregulations of miR-16 and miR-451 lead to improved differentiation of fibroblast cells into erythroid like progenitors.

The Effect of the Persian Gulf Jellyfish (Cassiopea andromeda) Venom on the Expression of P15, P21, P53, DNMT1, and Bcl-2 in Acute Lymphoblastic Leukemia Jurkat Cells.

Background: One of the acute hematologic malignancies is acute lymphoblastic leukemia (ALL), which is formed in B or T lymphocyte stem cells. Regarding the increasing tendency to herbal and marine studies and unclear characteristics of Cassiopea andromeda Venom, this study was performed to determine its effects on Jurkat cells as a model for T-ALL. Materials and Methods: In this experimental study, the cells were treated with a variety of concentrations of Cassiopea andromeda venom at different periods and times. Growth inhibition and toxic effects of Cassiopea andromeda Venom were evaluated by methyl thiazole tetrazolium salt reduction (MTT test). The flow cytometry analysis was carried out using 7-aminoactinomycin D (7AAD) and Annexin V stains to evaluate the venom's effect on apoptotic pathways. Besides, Real-Time PCR was performed to evaluate the relative gene expression. Results: Cassiopea andromeda venom inhibited the growth of Jurkat cells in a concentration and time manner. Jurkat cell growth was inhibited by 48.9% after 72 hours of treatment with 250µg/mL Cassiopea andromeda venom. The venom increased the apoptotic process through the upregulation of p15INK4b and P53 proteins and downregulation of Bcl-2, p21 WAF1/CIP1, and DNMT1 in the Jurkat cell line. Conclusion: Considering the growth inhibitory property of Cassiopea andromeda Venom, we recommend it as a part of combinational medication for treating ALL in animal trials and for other leukemias in vitro studies.

A Pleurocidin-Like Peptide from Poecilia Mexicana Fish Induces Selective Cytotoxicity in Leukemia Jurkat Cells Through The Apoptosis Pathway.

OBJECTIVE: Some cationic anti-microbial peptides show a wide range of cytotoxic action versus malignant cells, which may lead to developing a novel group of antitumor medications. In the present study, the anticancer activity of pleurocidin-like peptide WF3 isoform X2 (AMP-WF3), from the Poecilia Mexicana fish, against leukemic cell line Jurkat was evaluated, and the cytotoxicity compared with the effects on normal cells, including peripheral blood mononuclear cells (PBMCs) and human dermal fibroblast (HDF) cells. MATERIALS AND METHODS: In this experimental study, cells were treated with various dosages of AMP-WF3 for 24 hours. Using methyl thiazole tetrazolium salt reduction (MTT test), the effects of the AMP-WF3 on cell viability and toxicity were evaluated. The impact of this peptide on apoptotic pathways was examined using flow cytometry and Annexin V-PI stains. Additionally, the relative expression of the P53, P21 and BCL-2 genes was evaluated using a real-time polymerase chain reaction. RESULTS: The Jurkat cell line was more susceptible to AMP-WF3 cytotoxicity [half-maximal inhibitory concentration (IC50)=50 µM], while normal cells (PBMCs and HDF) were less susceptible. Flow cytometry verified that the apoptotic activity of AMP-WF3 on Jurkat cells was significantly higher than that of HDF and PBMCs. Peptide-treated Jurkat cells were associated with increased expression of P21, and P53 genes. In contrast, the changes in P21, P53, and BCL-2 genes differed in PBMCs and HDF cells. In HDF cells, simultaneous increase of P21, P53, and BCL-2, and in PBMCs, only the increase of BCL-2 was observed. CONCLUSION: Our research showed that AMP-WF3 could be developed as a novel treatment agent with minimum side effects for ALL patients.

Overexpression of MiR-506 in Jurkat (Acute T Cell Leukemia) Cell Line.

BACKGROUND & OBJECTIVE: Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from various mutations in B or T-lymphoid progenitors. MicroRNAs (miRNAs) regulate gene expression by binding to the 3' untranslated region of protein-coding genes. Dysregulation of miRNA expression may result in the development of cancerous phenotypes. Therefore, for the first time in this field, the present study aims to investigate the effect of overexpression of miR-506 in Jurkat (acute T cell leukemia) cell line. METHODS: In this study, Jurkat cell lines were cultured in RPMI-1640 medium. Next, miR-506 was transfected with concentrations of 50 and 100 nM with Lipofectamine 2000. The accuracy of the transfection was confirmed by the transfection of siRNA conjugated with FITC. 48 h after transfection, the cells were prepared for other tests (flow cytometry, MTT assay, and RNA extraction). The expression level of miR-506 in the cells was analyzed using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Finally, SPSS 21 software was used for the data analysis. RESULTS: According to our results, the viability of cells in concentrations of 50 and 100 nM was significantly higher than the control group. By overexpression of miR-506, the expressions of pro-apoptotic genes (p53, p21) and anti-apoptotic gene B-cell lymphoma-2 (BCL-2) are decreased and increased, respectively. CONCLUSION: This study showed that miR-506 may function as an oncogenic miRNA in the T- ALL cell line. In conclusion, overexpression of miR-506 leads to an increase in viable cancer cells.

Direct Differentiation of Human Primary Fibroblast into Hematopoietic-Like Stem Cells; A New Way without Viral Transduction.

OBJECTIVE: The aim of this study was to investigate the possibility of producing safe hematopoietic stem cells without the use of viral infectious agents that can be used in stem cell transplantation. MATERIALS AND METHODS: In this experimental study, after single layer cell formation, human primary fibroblast cells were treated with static electromagnetic fields of 10 and 15 milli Tesla (mT) for 20 minutes each day for seven consecutive days. On the seventh day and immediately after the last radiation, the cells were added to the wells containing specific hematopoietic stem cell expansion media. After 21 days and colony formation, the cells belonging to each group were evaluated in terms of the expression of CD34, CD38, and GATA-1 genes using quantitative real-time polymerase chain reaction (PCR), as well as surface marker expression of CD34 by flow cytometry. RESULTS: Exposure to 10 mT and 15 mT electromagnetic field increased the expression of CD34 and CD38 genes (P<0.05). This increase in gene expression levels were 2.85 and 1.84 folds, respectively, in the 10mT group and 6.36 and 3.81 folds, respectively, in the 15 mT group. The expression of the GATA-1 gene in the 10 mT and 15 mT groups was not significantly different from that of the control group (P<0.05). Electromagnetic waves caused a marked increase in the expression of the CD34 marker at the surface of reprogrammed cells. The rate of expression was about 42.3% in the 15 mT group and 23.1% in the 10 mT group. CONCLUSION: The presence of human primary fibroblasts exposed to electromagnetic fields can increase the expression of specific hematopoietic genes. This method can be suitable for reprogramming cells differentiated into hematopoieticlike stem cells and does not pose the risks of retroviral use.

Simultaneous regulation of miR-451 and miR-191 led to erythroid fate decision of mouse embryonic stem cell.

OBJECTIVES: Various microRNAs (miRNAs) are expressed during development of mammalian cells, when they aid in modulating gene expression by mediating mRNA transcript cleavage and/or regulation of translation rate. miR-191 and miR-451 have been shown to be critical regulators of hematopoiesis and have important roles in the induction of erythroid fate decision. So, the aim of this study is investigation of the miR-191 and miR-451 roles in the controlling mouse embryonic stem cell (mESC) differentiation toward the erythroid lineage. MATERIALS AND METHODS: mESCs were infected with either pCDH-miR-Off-191 viruses in pCDH-miR-Off-191 group or simultaneously with pCDH-miR-Off-191 and pCDH-miR-451 lentiviruses in simultaneous group. Then, the expression profiles of erythroid specific transcription factors and globin genes were analyzed using QRT-PCR on day 14 and 21 of differentiation. Flow cytometry analysis was used to evaluate of TER119 and CD235a erythroid specific surface markers. RESULTS: Gata-1, Klf-1, Epor and globin chains were found to be expressed in pCDH-miR-Off-191 and in simultaneous groups. The majority of globin chains showed changes in their expression levels with progression of differentiation from day 14 to day 21. Flow cytometry results showed that miR-451 up- regulation and miR-191 down-regulation is associated with the expression of TER119 and CD235a. Of these two groups analyzed, simultaneous group was most significantly potent in stimulation of erythroid fate decision of mESCs. CONCLUSION: Together, present data demonstrate that down-regulation of miR-191 alone can enhance the differentiation of mESCs. However, the simultaneous effect of miR-451up-regulation and miR-191 down-regulation is much stronger and can have more practical use in artificial blood production.

The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. MATERIALS AND METHODS: In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, ß, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14thand 21thafter transduction. RESULTS: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, ß, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). CONCLUSION: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines.

Immunological Testing Reveals Exposure to Malaria in the Hypoendemic Region of Iran.

Background. South eastern parts of Iran remain endemic for malaria infection. There is some concern that malaria infection may spread into Bushehr, which is located in the south western part bordering the Persian Gulf and at the periphery of the declared endemic region Hormozgan province due to frequency of visitors from eastern endemic areas and from neighboring malaria endemic countries. We investigated malaria prevalence in Bushehr. Methods and Results. Attempts were made to identify malaria active infection in blood smears and malaria specific antibody and antigens in serum samples. Traditional blood smears prepared from 1955 blood specimens yielded no definitive malaria positive case by microscopic technique. A total of 270 (13.8%) serum samples were positive for malaria antibodies. Using specific ELISA kits, presence of histidine rich proteins and lactate dehydrogenase antigens were investigated in serum samples. No histidine rich proteins specific for P. falciparum were detected amongst 270 antibody positive samples. However, six samples representing 0.3% of total population, were found to be positive for plasmodium pan specific lactate dehydrogenase antigens. This suggested the possibility of low level exposure to malaria in Bushehr community. Conclusions. Out of a total of 1955 samples tested, 270 (13.8%) were positive for malaria antibodies and six (0.3%) of these were positive for plasmodium-specific lactate dehydrogenase antigen suggesting a low level exposure to malaria in a hypoendemic region based on immunological testing. Since none of the 270 antibody samples were positive for histidine rich protein antigens, there is scope for further testing of blood samples by molecular methods such as polymerase chain reactions to confirm the plasmodium species and provide information valuable for future investigations. Our testing strategy for hypoemdemic malaria can be used as a template for investing malaria in 32 eliminating countries for testing ongoing transmission. This approach may be useful as a method in epidemiological studies.

Oncogenic human papillomavirus genital infection in southern Iranian women: population-based study versus clinic-based data.

BACKGROUND: Epidemiological studies on genital human papilloma viruses infection (HPVs) in general population are crucial for the implementation of health policy guidelines for developing the strategies to prevent the primary and secondary cervical cancer. In different parts of Iran, there is a lack of population-based studies to determine the prevalence of HPV in the general population. The aim of this population-based study is to compare the prevalence rate of genital HPV infection among reproductive women with our previous clinic-based data, which showed a prevalence rate of 5% in women in southern Iran. RESULTS: Using general primers for all genotypes of HPV, of 799 randomly selected women, five (0.63%, 95% CI 0.23-1.55%) tested positive for HPV DNA. Overall, seven different HPV genotypes were detected: six types (16, 18, 31, 33, 51 and 56) were carcinogenic, or "high risk genotypes" and one genotype (HPV-66) was "probably carcinogenic." CONCLUSIONS: In a population-based study, the prevalence of HPV infection among southern Iranian women was lower than that observed worldwide. However, our gynaecological clinic-based study on the prevalence of HPV infection showed results comparable with other studies in the Middle East and Persian Gulf countries. Since gynaecological clinic-based data may generally overestimate HPV prevalence, estimates of prevalence according to clinic-based data should be adjusted downward by the population-based survey estimates.

Relationships among serum receptor of nuclear factor-kappaB ligand, osteoprotegerin, high-sensitivity C-reactive protein, and bone mineral density in postmenopausal women: osteoimmunity versus osteoinflammatory.

OBJECTIVE: The aim of this study was to investigate the correlations among circulating osteoprotegerin (OPG), the receptor activator of nuclear factor-kappaB ligand (RANKL), high-sensitivity C-reactive protein (hsCRP), and bone mineral density (BMD) in healthy postmenopausal women. METHODS: In a population-based study, highly specific enzyme-linked immunosorbent assay methods were used to evaluate the sera of 382 healthy Iranian postmenopausal women (mean age +/- SD, 58.7 +/- 7.5 y) for RANKL, OPG, hsCRP, degradation products of C-terminal telopeptides of type I collagen, and osteocalcin. BMD was determined for the lumbar spine (L2-L4) and the proximal femur using dual-energy x-ray absorptiometry. RESULTS: Circulating levels of OPG (r = 0.30, P < 0.001) and the RANKL/OPG ratio (r = -0.17, P < 0.001) were significantly associated with age. The geometric mean of hsCRP was 1.89 mg/L (SE, 1.05) in the population studied. There was a significant correlation between log(hsCRP) levels and body mass index (BMI; r = 0.36, P < 0.001). Multivariate linear analyses revealed that age (beta = -0.295, P < 0.001), BMI (beta = 0.464, P < 0.001), RANKL (beta = -0.105, P = 0.014), and OPG (beta = 0.098, P = 0.029) were the independent determinants for lumbar BMD (R(2) = 0.35). Age (beta = -0.250, P < 0.001), BMI (beta = 0.486, P < 0.001), and RANKL (beta = -0.110, P = 0.009) were independently correlated with femoral neck BMD (R(2) = 0.36). Age- and BMI-adjusted analysis by quartiles of log-transformed hsCRP did not reveal an association with BMD, serum levels of biochemical markers of bone turnover, RANKL, or OPG. CONCLUSIONS: The circulating levels of the RANKL/OPG osteoimmunity system have an association with BMD, but subclinical systemic inflammation may not be involved in bone mass in healthy postmenopausal women.

High sensitivity C-reactive protein is associated with the metabolic syndrome independent to viral and bacterial pathogen burden.

AIM: To investigate the influences of bacterial or viral pathogen burden in the relationship of high sensitivity C-reactive protein (hs-CRP) and the metabolic syndrome in a population-based study. METHODS: Data from 1754 men and women aged >or=25 years, from the Persian Gulf Healthy Heart Study were analyzed. The definition of the metabolic syndrome according to the Adult Treatment Panel III was used. Sera were analyzed for IgG antibodies to Chlamydia pneumoniae, Herpes simplex virus type 1, Helicobacter pylori and cytomegalovirus using ELISA. Measurement of CRP by a high-sensitivity CRP assay was done. RESULTS: The subjects with the metabolic syndrome had a higher geometric mean of CRP levels than the normal persons (p<0.0001). A linear relationship between an increase in the number of metabolic syndrome components and CRP concentrations was observed (p for trend<0.0001). In multiple logistic regression models, hs-CRP showed significant associations with the metabolic syndrome after controlling for cardiovascular risk factors and infectious burden divided into 2, 3 and 4 pathogens [OR=2.06, CI (1.32-3.21), p=0.001; OR=1.75, CI (1.26-2.42), p=0. 001; OR=2.12, CI (1.46-3.08), p<0.0001; respectively]. CONCLUSION: There was a strong association between inflammation and the metabolic syndrome independent to viral and bacterial infectious burden.