The present study was designed to assess the importance of ovine neosporosis in abortion of Iraninan sheep. Seventy aborted foetuses and dams from ovine dairy farms in northwest of Iran were analyzed to investigate the role of Neospora caninum (N. caninum) in ovine abortion. Diagnosis of the infection was determined by serology and polymerase chain reaction (PCR). A total of 70 aborted dairy ovine were blood sampled and used to evaluate serological status for N. caninum infection by enzymelinked immunosorbent assay (ELISA) and extracted DNA from the same aborted foetuses were subjected to PCR. Data were compared using Kruscal-Wallis test. From A total of the 70 sheeps, four (5.7 %) of the dams were seropositive. DNA from aborted foetuses was extracted primarily from placenta and CNS tissues. Extracted DNA from foetuses were analyzed using PCR with primers Np21 ? and Np6 ? . Out of the 70 ovine fetuses 8.5 % were considered to be infected by PCR. This study confirms the importance of N. caninum as an important cause of ovine abortion in northwest of Iran.
Neospora caninum is one of the most significant parasitic organisms causing bovine abortion worldwide. Despite the economic impact of this infection, relatively little is known about the genetic diversity of this parasite. In this study, using Nc5 and ITS1 nested PCR, N. caninum has been detected in 12 brain samples of aborted fetuses from 298 seropositive dairy cattle collected from four different regions in Tehran, Iran. These specimen (Nc-Iran) were genotyped in multilocus using 9 different microsatellites markers previously described (MS4, MS5, MS6A, MS6B, MS7, MS8, MS10, MS12 and MS21). Microsatellite amplification was completely feasible in 2 samples, semi-completely in 8 samples, and failed in 2 samples. Within the two completely performed allelic profiles of Nc-Iran strains, unique multilocus profiles were obtained for both and novel allelic patterns were found in the MS8 and MS10 microsatellite markers. The Jaccard's similarity index showed significant difference between these two strains and from other standard isolates derived from GenBank such as Nc-Liv, Nc-SweB1, Nc-GER1, A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 KBA1, and KBA2. All samples originating from the same area showed identical allelic numbers and a correlation between the number of repeats and geographic districts was observed.
CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016.
CanCer -TargeTed gene and Cell Therapy I fibroblasts has respectively profound effects on the invasive ability of breast cancer cells. By applying both HIF-1α wild-type (WT) and knockout (KO) fibroblasts and breast cancer cells and using an in vitro invasion assay through commercially precoated invasion inserts, we found that fibroblasts, regardless their HIF-1α status, stimulated cell invasion of breast cancer cells that have intact HIF-1α. In contrast, only HIF-1α-expressing fibroblasts, not HIF-1α-nonexpressing ones, stimulated HIF-1α KO breast cancer cell invasion. These results suggest the complicated and respective HIF-1α contribution from breast cancer per se and surrounding fibroblasts to the tumor invasion and malignancy. Moreover, by using a Tet-on inducible system, we found that ectopic p16 gene transfer is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer cells. These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia/HIF-1α-mediated cancer cell migration and invasion. Furthermore, our current ongoing study implies an interaction between p16 and HIF-1α that may elucidate a novel p16-mediated tumor suppression pathway via HIF-1α-regulated signaling. These studies together may provide new information of the convergence of several important cell-growth regulation pathways on tumor invasion that would yield potential novel targets for effective cell-or gene-therapy strategy for treatment of breast cancer.
Gene-directed enzyme prodrug therapy (GDEPT) is an approach that delivers a suicidal gene to cancer cells for the expression of prodrug-activating enzyme which converts prodrug into cytotoxic drug. One of the major impediments of GDEPT is to have the therapeutic gene specifically target on cancer cells prior to the administration of prodrug. Among various gene delivery methods, mesenchymal stem cells (MSCs) have emerged as potential carriers for gene delivery. They exhibit remarkable tumor-tropic and low immunogenic capacities. In this study, MSCs was engineered to express thymidine phosphorylase (TP) which has the capability of converting the prodrug doxifluridine into toxic 5-fluorouracil. TP expression in the MSCs post-transfection of TP cDNA was confirmed by immunoblotting analysis and its activity was determined by spectrophotometric assay. Our results showed the anticancer effectiveness of human HT29 colorectal adenocarcinoma cells co-cultured with TP-expressing MSCs was enhanced with the addition of various dosages of doxifluridine. Citation Format: Nasrin Salehi, Ching-An Peng. Mesenchymal stem cell as delivery carrier for prodrug gene therapy against colorectal cancer cell. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5352. doi:10.1158/1538-7445.AM2015-5352
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