Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

Aims The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. Background Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. Methods qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. Results The wild type α+/β+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/β− hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/β+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α−/β+ hTERT and α−/β− hTERT (both p = 0.02). A positive association was observed between BRCA1 and α−/β+ hTERT and α−/β− hTERT expression (p = 0.003 and p = 0.04, respectively). Conclusions These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/β+)

Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model

YesAlopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss.Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945)

Deploying the tolerogenic effects of IDO enzyme and skin fibroblasts in prevention of graft rejection

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme with tolerogenic effects on different immune cells. Our group has previously shown that co-transplantation of IDO-expressing fibroblasts with donor tissues can delay immune rejection by inducing local immunosuppression. We first asked a question whether we can improve this effect by delivering the IDO-fibroblasts through a systemic intraperitoneal approach, instead of local co-transplantation, and secondly whether this effect is only delivered by the immunosuppressive effects of IDO or the fibroblast cells have additional immunosuppressive effects. We employed a systemic approach to improve allograft survival without using any immunosuppressive medication. To achieve this, 10 million lentiviral transduced IDO-expressing donor derived fibroblasts were injected into the peritoneal cavity of allograft recipients. We showed that IDO-fibroblast therapy increases the survival of both islets and skin allografts and decreases the infiltration of immune cells in subcutaneous transplanted skins. Indirect pathway of allo-reactive T cell activation was suppressed more than the direct pathway. Injected IDO-fibroblasts were found in peritoneal cavity and mesenteric lymph nodes of the recipient mice. In conclusion, fibroblasts have tolerogenic effects on DCs and IDO-expressing fibroblast therapy proved to be a novel approach in improving the allogeneic graft survival. There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). In a mouse model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased the expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4+ and CD8+ T cells. Even activation of fibroblast-primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4+ T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and IDO. Fibroblast-primed DCs had a significantly reduced interleukin- 12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin).Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat

Hydatid Cyst of Ovary: A Case Report

Echinococcus granulosus is considered the major cause of humanhydatid cysts. Usually the duration of cyst formation is 10-20 years. This period shortens significantly upon rupture of aprimary cyst. The literature describes low incidence of primaryinvolvement of ovary as a site of hydatid cyst formation. Ourcase is the first report on ovarian hydatid cyst in Iran. A 60-year-old woman was presented with abdominal pain in the leftlower quadrant area. Paraclinical data were suggestive of neoplasiaand preoperative diagnosis was ovarian tumor. Duringlaparotomy, multiple cysts resembling hydatid cysts were observedin the left ovary. Pathological examination confirmed thediagnosis of hydatid cyst. Although there is a small possibilityof secondary ovarian echinococcal disease, it is more probablefor this case to be primary infection, as the patient had developedovarian hydatid cysts 15 years after hepatic involvementand recurrence after 30 months is very uncommon

A COMPARISON BETWEEN DIFFERENT EXISTING METHODS USED TO SEPARATE EPIDERMAL CELLS FROM SKIN BIOPSIES FOR AUTOLOGOUS TRANSPLANTATION

Background: Burn surgeons use autologous skin graft technique for patients, but a challenge remains for large surface wounds. Recently, a method was described which used a small piece of skin to cover a 70 times greater surface by spraying epidermal cells on injured skin. We designed a comparative study to find the best method to make an epidermal cell suspension. Materials and Methods : Eleven discarded skin samples were sent to our laboratory from Ghotboddin Burn Hospital, Shiraz. Each sample was sliced into four small pieces (1 cm 2 ) and each piece was treated with a different chemical including sodium bromide (2N) and (4N), ammonium hydroxide (2N), and trypsin (0.05%) for 20 minutes. The epidermis and dermis were separated using forceps. Trypsin was added to all samples (except the trypsinized sample) to begin the intercellular detachment. Afterward, epidermis was sliced into small pieces followed by filtration and centrifugation. Cells were counted using hemocytometer. Identification of keratinocytes and melanocytes was made through immunocytochemical staining for cytokeratin and melanosome antigens, respectively. Results: There was a significant difference in alive cell counts comparing cells obtained from NaBr (4N) method to other methods. Considering total cell count and alive cell count, NaBr (4N) yielded the most cells. Immunocytochemical staining showed that in all methods, some cells are stained positively for cytokeratin antibody and some for melanosome antibody. Conclusion: Although recent papers had advised trypsin method to make a cell suspension to use for burn patients, we found that NaBr (4N) method yields more alive cells and less toxicity

Parameters that influence the isolation of multipotent mesenchymal stromal cells from human umbilical cord blood

Background and objectives: Umbilical cord blood is an important source of stem cells. However, isolating multipotent mesenchymal stromal cells (MSCs) from umbilical cord blood presents methodological challenges. We compared the effectiveness of six approaches to improve the success rate of MSC isolation and proliferation from umbilical cord blood. Methods: Thirty umbilical cord blood units underwent investigation. In 10 samples, MNCs from each sample were divided into four groups to test the effect of negative immunodepletion (NI) alone (group A); NI plus basic fibroblastic growth factor (bFGF) supplementation together (group B); bFGF supplementation alone (group C); and culture with neither NI nor bFGF (group D).The cells of each group were isolated from 10 mL of umbilical cord blood. For investigating the effect of sample volume (group E) and MesenCult Proliferation Kits (group F), cells were isolated from 45 ± 2 ml. MSCs were identified on the basis of morphological, flow cytometric and differentiation potential characteristics. Results: In groups of A–D, one week after the initial seeding, the cells showed a rounded appearance, and in the fourth week, many of them died. MSCs outgrowth was seen in 40% of the samples from group F, and this yield was further enhanced to 60% in cultures done with the MesenCult Proliferation Kit (group F). The fibroblast-like cells expanded rapidly and showed features of MSCs. Conclusion: Sample volume was the parameter that showed the greatest influence on the isolation yield of MSCs from umbilical cord blood. This could be further enhanced by adding the MesenCult Proliferation Kit

Changes in endothelial progenitor cell subsets in normal pregnancy compared with preeclampsia

Background: The results of studies measuring the number of endothelial progenitor cells (EPCs) in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of cross-sectional designs and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC), colony-forming unit endothelial cell (CFU-ECs), and endothelial colony forming cells (ECFCs). To provide a clear explanation for these underlying controversies, we designed a prospective study to compare the number of all EPC subsets between three trimesters of normal gestation and a case–control study to compare these values as preeclampsia occurs with those from gestational age (GA) matched normal pregnancy. Methods: Samples from peripheral blood of nine women were taken during their three consecutive trimesters of normal pregnancy, and from eight women with preeclampsia. To cover most of the reported phenotypes for CACs and ECFCs in the literature, we enumerated 13 cell populations by quantitative flow cytometry using various combinations of the markers CD34, CD133, CD309, and CD45. We used routine culturing techniques to enumerate CFU-ECs. Results: The numbers of CACs and ECFCs were higher in women with preeclampsia (p = 0.014). By contrast, preeclampsia was associated with a reduced number of CFU-ECs (p = 0.039). The CAC number rose with the increase in GA (p = 0.016) during normal pregnancy, while the number of CFU-ECs and ECFCs did not differ during the trimesters. Conclusion: Although we did demonstrate an increase in absolute counts of CACs and ECFCs in preeclampsia, fewer colony formation capacities indicated a loss in their functional capabilities. By contrast, the number of CACs increased without alterations in colony formation ability in normal pregnancy with the growth of the fetus. Here, by comparing different methodologies to calculate the number of EPC subsets, we could imitate the existing controversy in the literature for such calculations, which may help to elucidate clearer explanations

Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated instability using small molecules

Regulatory T (T-reg) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and T-reg instability after adoptive transfer in inflammatory conditions are major limitations to T-reg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of T-regs. In this research, we evaluated T-reg-like cells obtained from different T-reg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4(+) cells and a naive CD4(+)/T-reg co-culture by using three different protocols - ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor beta (S-TGF) and small molecules N-acetyl puromycin and SR1555 (N-Ac/SR). The results showed that a high yield of a homogeneous population of T-reg-like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-beta, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated T-reg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without T-reg-specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure T-reg-like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for T-reg generation when compared with the other methods, including a higher purity of T-regs, easier procedure, superior suppressive activity during the inflammatory condition and decreased cost