The Effect of Estradiol and Progesterone on Toll Like Receptor Gene Expression in A Human Fallopian Tube Epithelial Cell Line

OBJECTIVE: Toll like receptors (TLRs) are one of the main components of the innate im- mune system. It has been reported that expression of these receptors are altered in the female reproductive tract (FRT) during menstrual cycle. Here we used a fallopian tube epithelial cell line (OE-E6/E7) to evaluate the effect of two sex hormones in modulating TLR expression. MATERIALS AND METHODS: In this experimental study, initially TLR gene expression in OE- E6/E7 cells was evaluated and compared with that of fallopian tube tissue using quanti- tative real time-polymerase chain reaction (qRT-PCR) and immunostaining. Thereafter, OE-E6/E7 cells were cultured with different concentrations of estradiol and progesterone, and combination of both. qRT-PCR was performed to reveal any changes in expression of TLR genes as a result of hormonal treatment. RESULTS: TLR1-10 genes were expressed in human fallopian tube tissue. TLR1-6 genes and their respective proteins were expressed in the OE-E6/E7 cell line. Although estradiol and progesterone separately had no significant effect on TLR expression, their combined treatment altered the expression of TLRs in this cell line. Also, the pattern of TLR expres- sion in preovulation (P), mensturation (M) and window of implantation (W) were the same for all TLRs with no significant differences between P, M and W groups. CONCLUSION: These data show the significant involvement of the combination of es- tradiol and progesterone in modulation of TLR gene expression in this human fal- lopian tube cell line. Further experiments may reveal the regulatory mechanism and signalling pathway behind the effect of sex hormones in modulating TLRs in the hu- man FRT

Variable localization of Toll-like receptors in human fallopian tube epithelial cells

Objective: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results: Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos

Upregulation of circulating cancer stem cell marker, DCLK1 but not Lgr5, in chemoradiotherapy-treated colorectal cancer patients

Cancer stem cell (CSC) markers have attracted considerable attention in tumor diagnostic, prognostic, and therapeutic implications. Detection of cancer stem cells in circulating blood using cancer stem cell markers has received remarkable attention recently. In this study, we aimed to investigate the messenger RNA (mRNA) expression level of Lgr5 and DCLK1 as most proposed colorectal CSC markers in blood circulation also determine the subsequent association to patients� clinical and pathological findings. Peripheral blood mononuclear cells (PBMCs) of 58 patients with colorectal cancer at stage I�IV with 33 out of 58 patients undergoing preoperative chemoradiotherapy (CRT), as well as 58 healthy controls have been isolated and the extracted RNAs were analyzed using real-time PCR. The mRNA expression pattern of CSC markers of patients and controls was compared using ��Ct method. The expression level of Lgr5 was significantly higher in colorectal cancer (CRC) patients comparing to healthy group (4.8-fold change, p < 0.001). Also there was a significant increase in expression level of Lgr5 in patients at stages III and IV comparing to stages I and II (p = 0.031) and higher grades (p = 0.039) of CRC. The expression of DCLK1 was also elevated in patients significantly (2.7-fold change, p < 0.001) and the related expression was increased by increasing disease stage (p = 0.025). Combination of DCLK1 and Lgr5 markers was analyzed by logistic regression and proved to be a slightly better marker compared to each marker alone. Interestingly the DCLK1 expression level was significantly higher in patients undergoing preoperative CRT (p = 0.041); however, no association to neoadjuvant CRT was observed for Lgr5. Considering the over-expression of DCLK1 and Lgr5 in circulating blood of CRC patients comparing to controls, our results might emphasize on the presence of CSCs in blood of these patients which might be attributed to their clinical and pathological characteristics and may lead to apply in future clinical implications. Moreover, the higher expression level of DCLK1 in patients undergoing CRT can propose it as a more relevant candidate among CSC markers comparing to Lgr5 for CRC patients. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Drug-related mutational patterns in hepatitis B virus (HBV) reverse transcriptase proteins from Iranian treatment-Naïve chronic HBV patients

Background: Immunomodulators and Nucleotide analogues have been used globally for the dealing of chronic hepatitis B virus (HBV) infection. However, the development of drug resistance is a major limitation to their long-term effectiveness. Objectives: The aim of this study was to characterize the hepatitis B virus reverse transcriptase (RT) protein variations among Iranian chronic HBV carriers who did not receive any antiviral treatments. Materials and Methods: Hepatitis B virus partial RT genes from 325 chronic in active carrier patients were amplified and directly sequenced. Nucleotide/amino acid substitutions were identified compared to the sequences obtained from the database. Results: All strains belonging to genotype D.365 amino-acid substitutions were found. Mutations related to lamivudine, adefovir, telbivudine, and entecavir occurred in (YMDD) 4% (n = 13), (SVQ) 17.23% (n = 56), (M204I/V + L180M) 2.45% (n = 8) and (M204I) 2.76% (n = 9) of patients, respectively. Conclusions: RT mutants do occur naturally and could be found in HBV carriers who have never received antiviral therapy. However, mutations related to drug resistance in Iranian treatment-naïve chronic HBV patients were found to be higher than other studies published formerly. Chronic HBV patients should be monitored closely prior the commencement of therapy to achieve the best regimen option. © 2013, KOWSAR Corp

Efficacy of biological agents and fillers seed coating in improving drought stress in anise

Many plants, including anise, have tiny, non-uniform seeds with low and light nutrient reserves. The seeds also show a weak establishment, especially under stressful conditions where their accurate planting in the soil and optimal yield are tough. This study sought to improve anise seeds' physical and physiological characteristics under drought stress. To this end, two factorial experiments under laboratory and greenhouse conditions were performed in a completely randomized design with 4 and 3 replications, respectively. Five levels of seed inoculation (inoculation with T36 and T43 of Trichoderma harzianum, and CHA0 and B52 of Pseudomonas fluorescent, and non-inoculation which means that control seeds were not treated with microbial inoculant), three levels of coating (K10P20, K10P10V5, and non-coating), and three levels of drought stress (0, -3, and -6 bars) were considered as the factorial experiment [vermiculite (V), kaolin (K), and perlite (P) numbers refer to the amount of material used in grams]. The laboratory experiment revealed that the combined treatments of bio-agents with coating increased the physical and germination characteristics of anise seeds compared to the control treatment. The greenhouse experiment showed that drought stress reduced the initial growth indices. Still, the combination treatments of biological agents and coating (fillers) could alleviate the destructive effects of drought stress to some extent and improve these indices. The best treatment was provided by T36 and K10P20 in both experiments, which significantly increased morphological indices.Peer reviewe

Sex hormones alter the response of Toll-like receptor 3 to its specific ligand in fallopian tube epithelial cells.

Objective: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. Methods: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. Results: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). Conclusion: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line

Rapid differential diagnosis of vaginal infections using gold nanoparticles coated with specific antibodies

Vaginal infections caused by bacteria, Candida and Trichomonas vaginalis, affect millions of women annually worldwide. Symptoms and signs have limited value in differential diagnosis of three causes of vaginitis. Current laboratory methods for differential diagnosis are either expensive or time consuming. Therefore, in this work, development of a method based on gold nanoparticles has been investigated for rapid diagnosis of vaginal infections. Specific antibodies against three main causes of vaginal infections were raised in rabbits. The antibodies were then purified and conjugated to gold nanoparticles and used in an agglutination test for detection of vaginal infections. Finally, sensitivity and specificity of this test for diagnosis of vaginal infections were estimated using culture method as gold standard. Purification of antibodies from sera was confirmed by electrophoresis. Construction of nanoparticles was proved by TEM and FT-IR methods. Conjugation of antibodies to gold nanoparticles was confirmed using XPS method. Sensitivity and specificity of gold nanoparticles for diagnosis of Candida species were 100%, for Gardnerella were 100% and 93%, and for T. vaginalis was 53.3% and 100%, respectively. Gold nanoparticle-based method is a simple, rapid, accurate, and cost-effective test for differential laboratory diagnosis of vaginal infection