Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34 (+) Stem Cells.

Objective(s) : Stem cell differentiation into different cell lineages depends upon several factors, cell cycle control elements and intracellular signaling elements, including P15INK4b and P16INK4a genes. Epigenetics may be regarded as a control mechanism which is affected by these factors with respect to their promoter structure. Materials and Methods : The CD34 + cord blood stem cells were purified, isolated and then expanded. The undifferentiated day genome was isolated from part of the cultured cells, and the seventh day differentiated genome was isolated from the other part after differentiation to erythroid lineage. The procedure was followed by a separate Real-Time PCR for the two genes using the obtained cDNA. The processed DNA of the former stages was used for MSP (Methylation Specific PCR) reaction. Finally, pre- and post differentiation results were compared.  Results : After performing MSP for each gene, it became clear that P15INK4b gene has undergone methylation and expression in predifferentiation stage. In addition, its status has not been changed after differentiation. P15INK4b gene expression was reduced after the differentiation. The other gene, P16INK4a, showed no predifferentiation methylation. Itwas completely expressed methylated and underwent reduced expression after differentiation. Conclusion : Specific predifferentiation expression of P15INK4b and P16INK4a genes along with reduction in their expression after erythroid differentiation indicated animportant role for these two genes in biology of CD34+ cells in primary stages and before differentiation. In addition, both genes are capable of epigenetic modifications due to the structure of their promoters.

Evaluation of H3 histone methylation and colony formation in erythroid progenitors treated with thalidomide and sodium butyrate.

OBJECTIVES: ß-thalassemia and sickle cell disease are hemoglobinopathies with reduced/absent ß chains in the former and dysfunctional ß chains in the latter. In both conditions, up-regulation of hemoglobin F through demethylation can alleviate the symptoms. This can be attained with drugs such as thalidomide and sodium butyrate. MATERIALS AND METHODS: This study was performed on erythroid progenitors derived from CD133+ cord blood stem cells. Erythroid progenitors were treated with thalidomide and sodium butyrate in single and combined groups. Colony-formation potential in each group was evaluated by the colony assay. Real-time polymerase chain reaction (RT-PCR) was used to evaluate the effect of these drugs on histone H3 lysine 27 (H3K27) methylation patterns. FINDINGS: Compared to other treatment groups, CD133+ cells treated with thalidomide alone produced more hematopoietic colonies. Thalidomide alone was also more effective in decreasing H3K27 methylation. CONCLUSIONS: Thalidomide shows superiority to sodium butyrate as a hypomethylating agent in this cell culture study, and it has the potential to become conventional treatment for sickle cell disease and ß-thalassemia.

The prevalence of Chlamydia trachomatis in patients who remained symptomatic after completion of sexually transmitted infection treatment.

BACKGROUND: Sexually transmitted infections (STIs) are among the most common causes of illness in the world and have far-reaching health, economic and social consequences for many countries. Failure to diagnose and treat STIs at an early stage may result in serious complications and sequels. OBJECTIVE: This study aimed to determine the prevalence of Chlamydia trachomatis infection in patients who remain symptomatic after completion of their first episode of treatment for STI. MATERIALS AND METHODS: We conducted a cross-sectional study on 49 patients suffering from symptoms or signs of sexually transmitted infections despite their first complete anti STI treatment. Conducting physical exam and smear preparation from vaginal discharge, diagnosis was confirmed by Polymerase chain reaction (PCR) method on every patient's first-voided urine sample. RESULTS: Among the etiologic factors investigated in this study, Chlamydia was reported in 17 patients. Trichomoniasis, Candidiasis, Gonorrhea and nonspecific germs were next organisms with 11, 9, 6 and 6 patients, respectively. Sixteen specimens were PCR positive (32.65%), while 33 patients had negative PCR results (67.34%) for Chlamydia trachomatis. CONCLUSION: Gonorrheal infection was the most prevalent infection in patients with completed treatment (6/10), which must be remembered in patients follow ups, because this prevalence warrants empirical therapy for Gonorrheain similar clinical conditions. Chlamydia trachomatis was the responsible organism in approximately a quarter of patients (17/75) who despite their full compliance on anti-Chlamydial treatment still suffered from signs and symptoms of STI. This rate also recommends empirical therapy for Chlamydia trachomatis in the similar clinical signs and symptoms.

The effect of aligned and random electrospun fibrous scaffolds on rat mesenchymal stem cell proliferation.

OBJECTIVE: The development of combining mesenchymal stem cells (MSCs) with surface modified three-dimensional (3D) biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL) and unaltered PCL scaffolds were compared, and then p-PCL scaffolds were evaluated for MSC culture. MATERIALS AND METHODS: Aligned and random PCL nanofibrus scaffolds were fabricated by electrospining and their surface modified with O2 plasma treatment to enhance MSC proliferation, adhesion and interaction. Chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance (ANOVA). RESULTS: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide(MTT) assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber. CONCLUSION: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs.