simple and rapid method for the detection of HIV-1/HCV in co-infected patients

Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 [human immunodeficiency virus] and HCV [hepatitis C virus], it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. The current research recruits a multiplex nucleic acid sequence base amplification [NASBA] in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 [20 samples] and HCV [20 samples] were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

[TGF-b downregulation by RNAi technique in ex vivo-expanded HSCs on 3D DBM scaffold]

Bone Marrow Transplantations [BMT] are limited by low CD34+ cell counts in umbilical cord blood [UCB] and these cells need to be expanded for success in such procedures. To achieve this goal, ex vivo expansion of hematopoietic stem cells [HSCs] by enhancing their self-renewal activity on demineralized bone matrix [DBM] scaffold coated with mesenchymal progenitor cells [MPCs] and unrestricted somatic stem cells [USSCs] was recommended. TGF-b pathway is a key inhibitory factor for HSCs self-renewal. In this study ex vivo expansion and downregulation of TGF-b pathway were simultaneously performed. USSC cells were isolated from UCB and then coated on DBM scaffold as a feeder layer. UCB CD34+ cells were isolated from UCB by magnetic activated cell sorting [MACS] method and were transfected by siRNA against TGFbR2 in two-dimensional [2D] and three-dimensional [3D] cultures by co-cultivation with USSC. TGFbR2 expression levels were evaluated by quantitative real-time PCR. Cell count and flow cytometry were performed and clonogenic activity was evaluated. Ex vivo expansion of CD34+ cells was significantly enhanced [41 +/- 0.7 folds] by TGFbR2 downregulation, especially in 2D than 3D cultures. Finally, 2D culture showed less TGFbR2 expression levels and higher increase in the percentage of CD34 markers by flow cytometry assay. The 3D siRNA delivery system would be of lower efficiency in contrast to 2D settings where the cells have less freedom and are in more contact with the feeder layer

[Detection and genotyping of human-associated microsporidia in pigeon Columba livia of Tehran in 2010]

Microsporidia are ubiquitous opportunistic pathogens infecting all animal phyla. The purpose of this study was to characterization human-associated microsporidia in pigeons of Tehran by staining and molecular methods. In the year 2010 a total of 147 pigeon's fecal samples were randomly collected from bird stores and public parks of Tehran and screened for the existence of human pathogenic microsporidia by staining method and multiplex/Nested-PCR and RFLP techniques. Nineteen [12.92%] of the studied samples were positive by microscopic examination, and 31 [21.08%] isolates were detected with specific primers. Genotyping based on the ITS regions of the rRNA gene were done for the Entrocytozoon bieneusi, Encephalitozoon intestinalis, Enc. hellem and Enc. cuniculi, respectively. The genotypes of Ent. bieneusi were identical to the D, M and J; genotypes of Enc. hellem were similar to the genotype 1 and 3 and genotypes of Enc. cuniculi were equal to I and II genotypes which previously characterized in human and animal origins. These results revealed that there is no limits to microsporidia transmission between pigeons of Tehran and humans for human infective species. This study points to the hygienic importance of this bird, because feces of pigeons are one of the sources of infection with microsporidia in human and easily pollutes our environment; on the other hand, children and elderly people comprise the principal visitors of public parks and they are the populations at risk for microsporidiosis

Knocking down of nucleostemin gene expression in human bladder cancer cell line by RNA interference strategy

Nucleostemin [NS] is a recently identified gene that is expressed mainly in the nucleoli of neuronal, embryonic and mesenchymal stem cells which plays a role in their self renewal. Over expression of this gene has been reported in several cancer cell lines tumoral tissues including gastric, hepatic and prostate cancer. In this study, we investigated the effects of suppression of this gene by RNAi technique in a human bladder cancer cell line. After confirmation of expression of nucleostemin in the bladder cancer cell line 5637,21-mer oligoes against NS were transfected into cells and suppression of NS was confirmed by RT-PCR, immunocytochemistry [ICC] and western blotting. Proliferation assays were done by counting the cell numbers. Changes in cell cycle and apoptosis induction were assessed by staining cells with Propidium Iodide and Annexin V-FITC respectively. Our results revealed that nucleostemin is highly expressed in 5637 carcinoma cells. Expression of NS, by semi-quantitative RT-PCR, after treatment with specific oligoes against it, decreased around 85% in comparison to control groups. In addition, ICC and western blotting further confirmed the suppression of nucleostemin at the protein levels. Analysis of cell proliferation assays showed a considerable decline in the number of cells, specially, 72 h after treatment of cells with oligoes against NS. Cell cycle analysis after NS suppression showed increase of sub-diploid events, characteristic of apoptotic cells that were confirmed by staining cells with Annexin V-FITC dye. It seems that nucleostemin expression plays an important role in the induction of cell proliferation as well as inhibition of apoptosis occurrence in 5637 cells. Further studies focusing on suppression of this gene in vivo, may help to find potential therapeutic strategies to cure bladder cancer