Reza Aflatoonian

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells(1-8); therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation(6,9,10). Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers & also their differentiation capacity. Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,(11-13) i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 & CD44), hematopoietic/endothelial Markers (CD34, CD45 & CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) & Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE & dentin sialophosphoprotein; DSPP).(14).

Role of the innate immunity in female reproductive tract

The mucosal immune system in the female reproductive tract (FRT) is well equipped to meet the sexually transmitted pathogens, allogeneic sperm, and the immunologically distinct fetus. Analysis of the FRT indicates that epithelial cells provide a physical barrier against pathogens and microbial infections as well as secretions containing anti-microbial peptides, cytokines, and chemokines which recruit and activate immune cells. Epithelial and immune cells confer protection in part through Toll-like receptors. The aim of this literature is to review the diverse components of the innate immune system, contributing to an exclusive protection system throughout the FRT.

Th17 cells and related cytokines in unexplained recurrent spontaneous miscarriage at the implantation window

Unexplained recurrent spontaneous abortion (RSA) might be caused by the mothers immunological rejection of the fetus. In this cross-sectional study, the percentage of T helper 17 (Th17), T regulatory (Treg) cells and their cytokines as the main players of immunomodulation in peripheral blood lymphocytes during the luteal phase of 20 women with unexplained RSA were compared with 20 normal non-pregnant women. The percentage of Treg cells in the former was significantly lower compared with controls. The percentage of Th17 cells in the former was higher than controls. Expression of IL-23, IL-17, IL-6 cytokines in the former was significantly higher than controls, but the higher expression of IL-21 was not significant. The gene expression of TGF-β and FoxP3 in the former was lower than controls. Significant positive correlations were found between the percentage of Th17 cells with IL-23, IL-6 and IL-17 and between expression of IL-23 and IL-6 and IL-17. IL-6 gene expression showed a significant positive correlation with IL-17. Therefore, imbalance of Th17-Treg cells and the consequent changes in cytokine expression might be implicated in the pathogenesis of unexplained RSA and may provide new insight into the immunoregulatory events at the maternal-fetal interface.

Sperm protection in the male reproductive tract by Toll-like receptors

Sperm function can be affected by infection. Our understanding of innate immune system molecular mechanisms has been expanded, by the discovery of ‘Toll‐like receptors’ (TLRs). It seems that these receptors could play a critical role in the protection of spermatozoa. This study seeks to examine the presence and distribution of TLRs in different parts of the human male reproductive tract and spermatozoa. So, TLR gene expression was examined by RT‐PCR. Quantitative real‐time PCR (Q‐PCR) analysis used to compare the expression of TLRs in all sections of the male reproductive tract and TLRs 2, 3 and 4 in testicular sperm extraction (TESE) samples, which contained spermatozoa (TESE+) and those that did not (TESE−). Results showed that all TLR genes were expressed in different parts of the human male reproductive tract and spermatozoa. Moreover, Q‐PCR indicated that the relative expression of TLRs did not significantly change in different parts of the male reproductive tract but this technique has shown only relative TLR2 expression in TESE− is lower than TESE+ samples. It could be concluded that TLRs may provide a broad spectrum of protection from infection in the male reproductive tract. Furthermore, TLRs may influence on the developmental process during spermatogenesis.

Effects of melatonin on oocyte maturation in PCOS mouse model

The purpose of oocyte in vitro maturation is generation of mature oocytes that could support future development. Efforts have been made to enhance oocyte developmental competence by developing optimal culture conditions. The present study is conducted to determine melatonin effects on quality of polycystic ovarian syndrome (PCOS) oocytes when it has been added during in vitro maturation, and immature oocytes were cultured in defined conditioned medium with and without different melatonin concentrations. Melatonin could significantly improve nuclear maturation of PCOS oocytes (81.1% vs. 56.3%, P < 0.05 were achieved with 10-6 mol/L concentration). Cleavage rate was significantly higher in 10-5 mol/L concentration compared to untreated oocytes in PCOS (54% vs. 35%, respectively) and it was significantly higher with 10-6 mol/L concentration in the control group, 55% versus 38%, compared to untreated oocytes. This study showed that melatonin has the potential to induce oocyte nuclear maturation and guarantee fertilization potential.

Evaluation of immunological interaction between spermatozoa and fallopian tube epithelial cells

Toll‐like receptors (TLR) are one of the major compartments of innate immune system. It was revealed that the TLR have relevance in ovulation, sperm capacitation and fertilisation. So, in this study, the expression of TLR, their adaptor molecules and cytokines in human fallopian tube cell line under the effect of human normal spermatozoa was evaluated. TLR mRNA and protein were evaluated in OE‐E6/E7 cell line. Semen samples from 10 donors were collected and co‐incubated with OE‐E6/E7 cell line and used as sperm group, and cell line without spermatozoa was used as control group. Afterwards, the level of TLR, their adaptor molecule and cytokine mRNA expression was compared using qPCR in sperm and control groups, and supernatant was used for ELISA. To determine whether elevated cytokine reaction to spermatozoa in OE‐E6/E7 cell line is mediated via TLR, TLR3 function‐blocking antibody was used. OE‐E6/E7 cell line expressed TLR1–6 genes and proteins. TLR expressions, especially TLR3 and TLR5, in OE‐E6/E7 cell line under the effect of spermatozoa were significantly higher. Also, levels of adaptor molecules and cytokine production were increased in sperm group than in control group (P < 0.05). So, it may be hypothesised that TLR are essential for spermatozoa and fallopian tube immunological interaction and for preparing safe environment for important events in fallopian tube.

Estrogen and progesterone receptor subtype expression in granulosa cells from women with polycystic ovary syndrome

Abstract We evaluated gene expression of estrogen and progesterone nuclear receptors in granulosa cells (GCs) of polycystic ovary syndrome (PCOS) women compared to women with normal cycling ovaries (control group) to achieve a better understanding of ovarian steroid status in patients with PCOS. In this prospective study, 40 patients with PCOS and 40 women with normal ovulatory function who underwent in vitro fertilization (IVF) for treatment of tubal and/or male infertility were recruited. Follicular fluid was collected from patients and GCs were isolated from follicular fluid and then were purified with Micro Beads conjugated to monoclonal anti-human CD45 antibodies. RNA was extracted and reverse transcription was performed. Gene expression of estrogen and progesterone receptors was determined by quantitative real time PCR (qRT-PCR). Estrogen receptor β (ERβ) expression was significantly higher than ERα expression in both groups (p < 0.002). ERα and ERβ mRNA expression in PCOS was significantly lower than control group (p < 0.002). The expression levels of PRA and PRB in PCOS was significantly lower than control group (p < 0.002). In conclusion, a significant reduction of these genes in GCs from follicles of women with PCOS could be considered as a sign for maturation defect or follicular arrest in GCs.

Down-regulation of inflammatory signaling pathways despite up-regulation of Toll-like receptors; the effects of corticosteroid therapy in brain-dead kidney donors, a double-blind, randomized, controlled trial

Background: The brain death of a potential organ donor induces a systemic inflammatory response, resulting in inferior organ quality and function. Our study aimed to evaluate the effects of methylprednisolone (MPN) therapy on pattern recognition receptor (PRR) signaling in potential brain‐dead (BD) kidney donors. Material and methods: To evaluate the effects of MPN therapy on PRR signaling in BD kidney donors we performed a prospective randomized treatment‐versus‐control study. Fifty‐one potential kidney donors were randomly divided into three groups: brain‐dead donors (BDDs) who received 15mg/kg/d of methylprednisolone (group T1, n=17), BDDs who received 15mg/kg/d of MPN at the time of filling consent for kidney donation and 100mg/2h until kidney harvest (group T2, n=17), and normal donors as controls n=17. Gene expression for Toll‐like receptors (TLRs) 1–9 and their signaling pathway molecules including MYD88, TRIF, NF‐KB1, IRAK, IRF3, and IRF7, as well as the inflammatory cytokines RANTES, IL‐1&bgr;, TNF‐&agr;, IL‐6, CXCL8, IL‐18, IFN‐&agr;, and IFN‐&bgr; was determined by PCR array. Due to the crucial role of TLRs 2 and 4 in pattern recognition, surface expression of these molecules was analyzed by flow cytometry. Plasma levels of inflammatory cytokines were measured by immunoassay. Finally, serum creatinine and cystatin C were measured in 100 kidney recipients one week and one, three, and six months after transplant. Result: Polymerase chain reaction (PCR) array gene expression revealed greater expression of TLRs and signaling molecules in group T1 than in the controls. Surface expression of TLRs 2 and 4 were significantly greater in group T2 than in group T1 (P<.05). Plasma concentrations of inflammatory cytokines were significantly greater in group T1 than in controls (P<.05). The recipients that received kidneys from group T1 had significantly higher levels of creatinine and cystatin C than the recipients of kidneys from both group T1 and controls (P<0.05). Conclusion: Administration of MPN to BDDs at specified periods until kidney harvest resulted in less systemic inflammation in the BDDs and improved renal function in kidney graft recipients compared with common MPN therapy. HIGHLIGHTSExpression of TLR2 and TLR 4 is increased following MPN therapy in BDDs.Probably, low‐dose MPN cannot control signaling pathways of PRRs in BDDs.Brain death‐induced inflammation may increase the antigenicity of donated organs.Innate alloimmunity pre and post transplantation may affect graft outcome.High‐dose MPN in BDDs results lower creatinine and cystatin C in kidney recipients.

Effect of FTY720 (fingolimod) on graft survival in renal transplant recipients: a systematic review protocol

Introduction Studies have shown that FTY720 has inconsistent effects in kidney transplant recipients. Several review articles on FTY720 have been published, but most have focused on the mechanism of action of FTY720. Therefore, this review aims to evaluate and determine the beneficial and harmful effects of FTY720 therapy in kidney transplant recipients. Methods and analysis We electronically searched the following databases: PubMed, Scopus, the Web of Sciences, EMBASE, Cochrane databases and the Cochrane Central Registry of Controlled Trials. Any clinical, randomised controlled trials relating to FTY720 for treating kidney transplant recipients were included without publication status or language restriction. Study selection, data extraction and assessment of study quality were performed independently by two researchers. Data were synthesised by either the fixed effects or the random effects model according to a heterogeneity test. If the extracted data were suitable for meta-analysis, STATA software was used to combine the relative risks for dichotomous outcomes, and the mean differences for continuous outcomes with 95% CIs were measured. Death, loss of function and incidence of acute kidney rejection were assessed as the primary outcomes. Renal graft function, malignancy, delayed graft function and infection were evaluated as secondary outcomes. Ethics/dissemination This review does not require formal ethics approval because the data are not individualised. The resulting review article will be submitted for publication in a peer-reviewed journal. Trial registration number CRD42015024648.

Innate inflammatory gene expression profiling in potential brain-dead donors: detailed investigation of the effect of common corticosteroid therapy

Our study aimed to assess the influence of common methylprednisolone therapy on innate inflammatory factors in potential brain-dead organ donors (BDDs). The study groups consisted of 50 potential BDDs who received 15 mg/kg/d methylprednisolone and 25 live organ donors (LDs) as control group. Innate immunity gene expression profiling was performed by RT-PCR array. Soluble serum cytokines and chemokines, complement components, heat shock protein 70 (HSP70) and high mobility group box-1 (HMGB1) were measured by ELISA. Surface expression of TLR2 and TLR4 were determined using flow cytometry. Gene expression profiling revealed up-regulation of TLRs 1, 2, 4, 5, 6, 7 and 8, MYD88, NF-κB, NF-κB1A, IRAK1, STAT3, JAK2, TNF-α, IL-1β, CD86 and CD14 in the BDD group. Remarkably, the serum levels of C-reactive protein and HSP70 were considerably higher in the BDD group. In addition, serum amounts of IL-1β, IL-6, TNF-α, HMGB1, HSP70, C3a and C5a, but not IL-8, sCD86 or monocyte chemoattractant protein-1, were significantly increased in the BDD group. Significant differences were observed in flow cytometry analysis of TLR2 and TLR4 between the two groups. In summary, common methylprednisolone therapy in BDDs did not adequately reduce systemic inflammation, which could be due to inadequate doses or inefficient impact on other inflammatory-inducing pathways, for example oxidative stress or production of damage-associated molecules.