Appropriate reference gene selection for real-time PCR data normalization during rat mesenchymal stem cell differentiation.

Reverse transcription quantitative PCR (RT—qPCR) is one of the best methods for the study of mesenchymal stem cell (MSC) differentiation by gene expression analysis. This technique needs appropriate reference or housekeeping genes (HKGs) to normalize the expression of the genes of interest. In the present study the expression stability of six widely used HKGs including Actb, Btub, Hprt, B2m, Gusb and Tfrc was investigated during rat MSC differentiation into osteocytes, adipocytes and chondrocytes lineages using geNorm and NormFinder software. RT—qPCR data analyzed by geNorm revealed the different sets of suitable reference genes for each cell type. NormFinder also showed similar results. Analysis of the combined data of MSCs with each differentiated cell type revealed the considerable shift in expression of some reference genes during differentiation; for example Gusb and B2m were among the least stable genes in MSCs but the most stable in chondrocytes. Normalization of specific genes for each lineage by different reference genes showed considerable difference in their expression fold change. In conclusion, for the appropriate analysis of gene expression during rat MSC differentiation and also for monitoring differentiation procedures, it is better to consider precisely the reference gene stability and select suitable reference genes for each purpose.

Black cumin seeds, Artemisia leaves (Artemisia sieberi), and Camellia L. plant extract as phytogenic products in broiler diets and their effects on performance, blood constituents, immunity, and cecal microbial population.

Two experiments were conducted to evaluate the effects of using black cumin seeds (BCS), Artemisia leaves (AL), and Camellia L. plant extract (CLE) in the diets of broiler chicks. Experiment 1 was conducted as a completely randomized design in a factorial arrangement (2 × 2) with 8 replicates of 4 chicks in each battery cage. Factors included 2 levels of BCS and AL (0 and 1%). Experiment 2 was conducted as a completely randomized design with 4 treatments (control, 0.3 and 0.5 g/kg of CLE, and 0.5 g/kg of mannanoligosaccharide) of 8 replicates and 4 chicks in each. Body weight and cumulative feed intake were measured at 21, 35, and 42 d of age. Antibody response against SRBC was measured on d 28 and 42. Blood characteristics, relative weight and length of different parts of the carcass, gastrointestinal pH, villi length, and crypt depth were measured at 42 d of age. Artemisia addition did not affect BW and feed conversion ratio (FCR) but decreased feed intake significantly up to 21 d of age (P ≤ 0.01). Black cumin significantly increased BW (P ≤ 0.05) at 21 and 42 d of age and decreased FCR throughout the experimental period (P ≤ 0.01). Artemisia significantly increased monocytes but had no effect on gastrointestinal pH, antibody response, and relative weight and length of different parts of the carcass. Black cumin increased red blood cells, hematocrit, hemoglobin, gizzard relative weight, and pH but decreased antibody response and monocytes percentage (P ≤ 0.01). Artemisia did not affect plasma lipid profile but decreased coliform and Escherichia coli populations of ceca significantly (P ≤ 0.01 and P ≤ 0.05, respectively). Addition of 0.5 g/kg of CLE decreased BW, feed intake, and FCR throughout the experiment (P ≤ 0.01). Camellia increased gizzard and proventriculus pH, villi length, and crypt depth (P ≤ 0.01) but decreased primary antibody response, total white blood cell count, and cholesterol concentration (P ≤ 0.05). The results of this experiment showed that using BCS alone or mixed with AL improved broiler health and performance but CLE negatively affected broiler BW and feed intake and is not a good alternative to commercial mannanoligosaccharide.

Pyrvinium pamoate inhibits proliferation and invasion of human endometriotic stromal cells.

Endometriosis is characterized by the presence of functional endometrial tissue in other pelvic organs. This gynecologic problem occurs in 35-50% of women with pain and infertility. Endometriotic cells share some characteristics such as proliferation, migration, and invasion with tumor cells. Pyrvinium pamoate, an anthelmintic drug approved by the Food and Drug Administration, could inhibit the Wnt/ß-catenin signaling pathway and its anticancer effects were examined by several researchers. In this study, 12 ectopic and eutopic endometrial biopsies from females with ovarian endometrioma and 12 endometrial biopsies from nonendometriotic females were obtained. Ectopic (EESCs), eutopic (EuESCs), and control (CESCs) endometrial stromal cells were isolated. Then, the effect of pyrvinium pamoate on the proliferation and invasiveness of in vitro cultured cells was evaluated. The proliferation of CESCs, EuESCs, and EESCs was significantly decreased after treatment with pyrvinium pamoate. In addition, treatment with pyrvinium pamoate significantly inhibited the invasiveness of CESCs, EuESCs, and EESCs compared to nontreated groups. The results of the present research showed that pyrvinium pamoate inhibits the proliferation and invasion of human endometriotic stromal cells in vitro, further investigations on the therapeutic potential of this compound in endometriosis are required.

Immunomodulatory effects of hydroxychloroquine on Th1/Th2 balance in women with repeated implantation failure.

BACKGROUND: Cellular immune abnormalities such as the imbalance between T-helper (Th) 1 and Th2 cytokines have been implicated as potentially modifiable causes of idiopathic repeated implantation failures (RIF). The purpose of this study was to investigate the effects of hydroxychloroquine on IL-10 and TNF-α secretion, expression of T-bet and GATA-3 transcription factors and cellular localization of TNF-α, IFN-γ, IL-10 and IL-4 in endometrial cells in women with RIF. MATERIALS AND METHODS: A total of 17 women with a history of RIF and elevated TNFα/IL-10 ratio (TNFα/IL-10> = 30.6) were included in the study. The serum levels of TNFα and IL-10, the expression of transcription factors related to Th1 and Th2 cells and the immune-reactivity of TNFα, IFN-γ as Th1 related cytokines and IL-10, IL-4 as Th2 related cytokines in endometrial tissues were evaluated by ELISA, real-time PCR, and fluorescent immunohistochemistry respectively. All, evaluations were done both before and after treatment with hydroxychloroquine (400 mg/orally per day). RESULTS: Hydroxychloroquine treatment significantly decreased (p < 0.0001) serum level of TNF-α and significantly increased serum level of IL-10 (p < 0.0001). T-bet, the Th1 transcription factor, expression was down-regulated and GATA-3, the Th2 transcription factor, expression was up-regulated. IL-10 and IL-4 fluorescent immunoreactivities significantly increased (p < 0.05 and p < 0.001 respectively) and TNFα and IFN-γ fluorescent immunoreactivities significantly decreased (p < 0.05) in endometrial tissue in women with RIF after treatment in comparison with before treatment. CONCLUSION: Hydroxychloroquine administration in women with RIF With a high TNF-α/IL-10 ratio during the implantation window can decrease this ratio and seems to be an effective therapeutic strategy in RIF caused by cellular immune abnormalities through a shift in Th2 responses.

In vitro Growth and Characterization of Stem Cells from Human Dental Pulp of Deciduous Versus Permanent Teeth.

OBJECTIVE: By date investigations have indicated the presence of stem cells within the pulp tissue of both temporary and permanent human teeth. In the present study, these stem cells were compared in terms of their growth kinetics and culture requirements. MATERIALS AND METHODS: Stem cells within the pulp of the human third molar (permanent tooth) and the deciduous incisor (temporary tooth) were isolated, culture-expanded and characterized. Then the proliferation potential of the cells was compared using multiple cell growth indices as PDT (Population doubling time), colonogenic activity and growth curve. Furthermore, the cultures of both cells were optimized for maximal proliferation. RESULTS: Stem cells of either pulp tissue appeared as fibroblastic cells capable of differentiating into osteoblastic, odontoblastic, adipocytic and chondrocytic cell lineages. In contrast to molar stem cells, those from the incisor tooth expressed neurogenic markers of ßIII Tubulin and Tau. Based on in vitro growth data, the cells from third molar tended to have a lower PDT value (20.79, SD=2.8 versus 25.55, SD=2.9 hours), higher colonogenic activity and better growth curve than those from the deciduous incisor (P<0.05). Both cells exhibited high expansion rate when being plated in a medium with 20% phosphate buffer solution at a density of 100 cells/cm(2). CONCLUSION: Given the high proliferation capacity, the stem cells from the human third molar would be an appropriate candidate for use in experimental, preclinical and even clinical setups.

Colorectal carcinoma: analysis of management in two medical eras.

Trends in presentation, diagnosis, management, and outcome were analyzed for 503 patients with colorectal cancer seen at the UCLA Medical Center between 1960 and 1970 (Group A; n = 210) and 1980 and 1985 (Group B; n = 293). Patients in the latter group exhibited a shift in site to the right side of the colon (18% in Group A vs. 31% in Group B; P < .01), an increase in the number of primary resections without colostomy (38% vs. 61%; P < .01), a lower overall complication rate (28% vs. 18%; P = .01), and a decline in 30-day mortality (6.2% vs. 2%; P = .01). Although little difference was seen in detection of asymptomatic tumors, earlier lesions were treated in the latter group, accounting for substantially reduced rate of recurrence (69% in Group A vs. 44% in Group B; P < .01). Future management should include an emphasis on earlier detection in order to continue the trend toward enhanced survival.