Pathobiology Research
Year:2011 | Volume:14 | Issue:1|Papers Count:8

Objective: Nowadays, cord blood Hematopoietic stem cells (HSCs) are known as a valuable source for bone marrow transplantation but unfortunately their insufficient number is a limiting factor for using them in adult bone marrow transplantation. Cord blood HCSs expansion is an approach to overcome this problem, by inducing their self-renewal. TGF-b signaling pathway is a key inhibitory agent for HSCs self-renewal. In this study, we tried to enhance self-renewal of long term culture initiating cell by inhibiting TGFbR2 expression.Materials and Methods: CD34+ HSCs were isolated from cord blood units with MACS column. SiRNA against TGFbR2 was transfected by Lipofectamine™ RNAiMAX as transfection reagent. HSCs were cultured in IMDM medium containing 10% FBS and early acting cytokines (Flt3L, SCF, Tpo) for 8 days. Then we evaluated TGFbR2 expression by QRT-PCR. The CD34+ subpopulation of cultured cells were examined by flow cytometry on the 8th day. Finally the expanded cells were evaluated for the presence of early hematopoietic stem cells by LT-CIC and clonogenic assays.Results: According to our results, TGFbR2 down regulation increases CD34+ subpopulation of HSCs. In addition, LT-CIC assay showed an enhancement in primitive hematopoietic stem cell capable of self-renewal.Conclusion: All in all, it seems that positive regulators have attracted more attention in the field of HSCs expansion while negative regulators have same importance in self-renewal process of HSCs and their inhibition can be a beneficial tool for enhancement of HSCs self-renewa.

Objective: Toxoplasmosis can lead to severe pathological effects in both infected humans and animals. The various DNA vaccines against Toxoplasma compose of single or cocktail antigens have been investigated but they have partial protective against disease. In this study, we used pcROP1 as a DNA vaccine and aluminium phosphate and aluminium hydroxide to compare their efficacy as mineral adjuvants.Materials and Methods: BALB/c mice immunized with pcROP1 alone or with co-administration of Alpo4 or Alum and the effectiveness of these two adjuvants were compared using lymphocyte proliferation assay, cytokine and antibody assay and survival time.Results: The group co-administered alum elicited stronger humoral and Th1-type cellular immune responses than the group co-administered Alpo4, while immune response in group administered with pcROP1 alone is higher than them. When challenged with Toxoplasma gondii RH strain, mice immunized with or without alum had significantly higher survival rates, whereas there was no notable enhancement of survival rate in Alpo4 group (P£0.05).Conclusions: Our result suggest that pcROP1 plus alum and aluminium phosphate not strongly potentiate the efficacy of this DNA.

Objective: Nowadays notable increase in acquired resistance of Candida species to antifungal drugs and necessity of using agents with antifungal properties is unavoidable. In some plants due to presence of components such as polyphenols have antimicrobial properties. In this study antifungal effects of essential oils of Thymus vulgaris, Petroselinum crispum, Cuminum cyminum and Bunium persicum on standard strain of Candida albicans were evaluated.Materials and Methods: 25 grams leafs of the Thymus vulgaris and Petroselinum crispum and seeds of the Cuminum cyminum and Bunium persicum were dried and grinded after that the essential oils of each mentioned plant were prepared by Clevenger system. Serial dilutions of essential oils were made in 96 well microtiter plates. Minimum Inhibitory Concentration (MIC) and Inhibitory zone diameter were assessed by Microdilution broth and Disc diffusion agar techniques, MIC50, MIC90 and MFC were also determined.Results: Minimum inhibitory concentration 90(MIC90) essential oils of Thymus vulgaris, Petroselinum crispum, Cuminum cyminum and Bunium persicum were respectively 25, 72, 412, 130 mg/ml and Minimal Fungicide Concentration (MFC) were 48, 146, 62, 280. Inhibitory zone diameters were 28, 20, 12, 15 millimetres.Conclusion: In this evaluation essential oils of Thymus vulgaris, Petroselinum crispum, Cuminum cyminum and Bunium persicum showed suitable antifungal effects against growth of standard strain of Candida albicans. Thus these herbal essences after supplementary studies possibly can be suitable substitute for chemical medicine on Candida infections especially on mucocutaneous Candidiasis.

Objective: Despite toxic effects of some essential oils, their use is not under control. With a view to increasing trend of utilisation of herbal products, some biological aspects of Thymus daenensis are repoted here for the first time.Materials and Methods: Antimicrobial properties using disk diffusion and dilution tests, nitric oxide radical scavenging by Marcocci et al method and cytotoxic properties employing dimethylthiazolyl diphenyltetrazolium bromide reduction test were carried out with Thymus daenensis and commercial Thyme essential oils and their main chemical compound, thymol.Results: The microbial sensitivity to the oils were in Candida albicans> E. coli> S. aureus> P. aeruginosa order. The minimum inhibitory and microbicidal concentrations were in the range of 0.04-10mg/ml. Nitric oxide radical scavenging was dose dependent with an IC50 of 5, 75, 863 µg, and total phenolics of 644.07±6.79, 16.94±2.55, 10.33±2.31 mg Gallic acid equivalent per mg sample and total flavonoid content of 73.51±1.34, 0.56±0.02, 0.21±0.09 mg Catechin equivalent per gram T. daenensis oil, commercial thyme oil and thymol respectively. The concentrations from T. daenensis oil, commercial thyme oil and thymol required to exert 50% fatal effect (IC50) on healthy human normal lymphocytes and Hela cells were 1455, 12.10, 2867 and 4.95, 3.61, 1730 mg respectively.Conclusion: T. daenensis with its good antimicrobial property can prevent formation of toxic reactive oxygen species and as a good antioxidant, it can directly scavenge NO and O2. With a view to cancerous cells killing properties of the oils at their lowest concentrations without fatal effect on normal healthy cells, feasibility of their application in combating cancerous cells may be promising.

Objective: In this study quantitative expression of MDR1 and hOCT1 genes in CML patients and normal people were measured using Real-Time PCR.Materials and Methods: To study quantitative expression of these genes by real-time PCR, master-mix with syber green was used. Peripheral blood samples from 30 CML patients and 27 normal persons were harvested. Real-time PCR results were analyzed with relative quantification method.Result: This study showed that in the patients group who were under treatment with Imatib, MDR1 gene expression was increased which was statistically significant. This increase has a direct relation with disease progress. Gene expression in AP and BP patients was also higher than CP patients. In contrast, hOCT1 expression in patients group in comparison with normal group was not statistically significant.Conclusion: MDR1 increase in leukemic cell membrane results in the reduction of intra-cellular drug concentration. Thus, optimal concentration of drug for inhibition of BCR-ABL tyrosine kinase is not achieved which culminated in disease progression to AP and BP phases. Moreover changes in hOCT1 gene expression as an influx transporter of Imatib could affect intracellular concentration of drug and finally determine therapy outcome. However, in this study hOCT1 gene expression was variable and was not statistically significant.

Objective: Zoledronic acid as a main treatment for osteoporosis has an important role in differentiation of mesenchymal stem cells. However, mechanism of osteoblastic differentiation induction by zoledronic acid is not well understood until now. In this research we evaluate zoledronic acid effect on methylation status of RUNX2 and DLX5 promoters.Materials and Methods: After isolation and expansion of hMSCs from BM, they were pulse treated with 5 mM ZA for 3h, and incubated in osteogenic differentiation medium for 3 weeks. DNA was extracted after first, second and third weeks of culture and also from undifferentiated MSCs. After SBS treatment, gene specific methylation analysis for RUNX2 and DLX5 were carried out by MSP using methylated and unmethylated primers.Results: In the genes RUNX2 and DLX5, M and U primers of MSP amplified promoter regions of undifferentiated and osteoblastic differentiated MSCs. Therefore, methylation status in RUNX2 and DLX5 promoters present incomplete methylation.Conclusion: Methyltion patterns of RUNX2 and DLX5 don’t change during zoledronic acid osteoblastic differentiation of MSCs. Our findings show that zoledronic acid does not induce osteoblastic differentiation via epigenetic mechanisms. Therefore, zoledronic acid can induce osteoblastic differentiation in a manner independent from DNA epigenetic changes.

Objective: The current methods for bladder cancer diagnosis suffer from low sensitivity and specificity. Therefore, finding a novel tumor markers with high specificity and sensitivity is of great interest. MicroRNAs (miRNA, miR) are small endogenously-produced, non-coding RNAs with an important role in regulating gene expression. Recent studies show that miRNAs expression profiles represent significant tumor-specific changes that are unique for most cancers. The aim of this study was to optimize miRNA containing total RNA extraction from urine and use it as a reliable and repeatable technique for miRNA detection in urine of patients with bladder cancer.Materials and Methods: Total RNA was extracted from the urine of patients with bladder cancer and normal individuals using RNX and Trizol solutions with and without modifications of original protocols. Real-time quantitative RT-PCR was then used to detect miRNAs with a potential link to bladder tumorigenesis.Results: RNX and the modified Trizol are practical methods for RNA extraction from urine samples. The mir-21 amplification of the extracted RNAs using modified Trizol method was more efficient than that of RNX method. It is noteworthy that, the levels of miRNAs expression were much higher in the frozen urines compared to the fresh ones.Conclusion: We have succeeded to set-up a protocol to easily amplify miRNAs in urine samples. Based on the data, microRNAs seem to be good biomarkers for early detection and screening of bladder cancer.

Objective: Shigellosis is one of the most common causes of morbidity and mortality in children with diarrhea in developing countries. It is essential to assess the antibiotic resistance patterns of these bacteria. ipaH gene is one of the virulence factors which can be used for detection of Shigella spp.Materials and Methods: Total of 100 isolates of Shigella were collected from different provinces of Iran. This isolates were characterized by biochemical tests and serological tests using polyclonal antisera for 4 species of S. dysenteriae, S. sonnei, S. boydii and S. flexneri. Antibiotic susceptibility assay for 14 different antibiotics was carried out using agar disc diffusion method. Presence of ipaH gene was investigated by PCR using specific primers.Results: From the results of this study the Shigella isolates were classified as follows: 36 (%73) Shigella sonnei, 9 (%18) Shigella flexneri, 3 (%5) Shigella boydii, 2 (%4) Shigella dysenteriae. Approximately %50 of the Shigella isolates were resistant to Tetracycline and Cotrimoxazole. Shigella sonnei showed more resistance than other serotypes against the studied antibiotics. PCR assays showed that all isolates harbored ipaH gene.Conclusion: The results showed that prevalence of Shigella sonnei is higher than other serotypes. The isolates showed high sensitivity to third generation cephalosporines and aminoglycosides. PCR detection of ipaH gene as a reliable marker for identification of Shigella species could be recommended.