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The reconstruction capability of osteochondral (OCD) defects using silk-based scaffolds has been demonstrated in a few studies. However, improvement in the mechanical properties of natural scaffolds is still challengeable. Here, we investigate the in vivo repair capacity of OCD defects using a novel Bombyx mori silk-based composite scaffold with great mechanical properties and porosity during 36 weeks. After evaluation of the in vivo biocompatibility and degradation rate of these scaffolds, we examined the effectiveness of these fabricated scaffolds accompanied with/without autologous chondrocytes in the repair of OCD lesions of rabbit knees after 12 and 36 weeks. Moreover, the efficiency of these scaffolds was compared with fibrin glue (FG) as a natural carrier of chondrocytes using parallel clinical, histopathological and mechanical examinations. The data on subcutaneous implantation in mice showed that the designed scaffolds have a suitable in vivo degradation rate and regenerative capacity. The repair ability of chondrocyte-seeded scaffolds was typically higher than the scaffolds alone. After 36 weeks of implantation, most parts of the defects reconstructed by chondrocytes-seeded silk scaffolds (SFC) were hyaline-like cartilage. However, spontaneous healing and filling with a scaffold alone did not eventuate in typical repair. We could not find significant differences between quantitative histopathological and mechanical data of SFC and FGC. The fabricated constructs consisting of regenerated silk fiber scaffolds and chondrocytes are safe and suitable for in vivo repair of OCD defects and promising for future clinical trial studies.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a plant secondary metabolite, which shows antioxidant activity and is commonly found in many plant-based foods and beverages. Recent evidence suggests that oxidative stress contributes to the development of many human chronic diseases, including cardiovascular and neurodegenerative pathologies, metabolic syndrome, type 2 diabetes and cancer. GA and its derivative, methyl-3-O-methyl gallate (M3OMG), possess physiological and pharmacological activities closely related to their antioxidant properties. This paper describes the antidepressive-like effects of intraperitoneal administration of GA and two synthetic analogues, M3OMG and P3OMG (propyl-3-O-methylgallate), in balb/c mice with post-stroke depression, a secondary form of depression that could be due to oxidative stress occurring during cerebral ischemia and the following reperfusion. Moreover, this study determined the in vivo antioxidant activity of these compounds through the evaluation of superoxide dismutase (SOD) and catalase (Cat) activity, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) levels in mouse brain. GA and its synthetic analogues were found to be active (at doses of 25 and 50 mg/kg) in the modulation of depressive symptoms and the reduction of oxidative stress, restoring normal behavior and, at least in part, antioxidant endogenous defenses, with M3OMG being the most active of these compounds. SOD, TBARS, and GSH all showed strong correlation with behavioral parameters, suggesting that oxidative stress is tightly linked to the pathological processes involved in stroke and PSD. As a whole, the obtained results show that the administration of GA, M3OMG and P3OMG induce a reduction in depressive symptoms and oxidative stress.

Full thickness wound healing with minimal scarring and complete restoration of normal skin properties still remains as a clinical challenge. In this study, a bilayer skin substitute has been fabricated to biomimic the microstructure of natural extracellular matrix of the skin. Human amniotic membrane (HAM) and silk fibroin nano-fibers were combined to produce bilayer construct, which was further treated and characterized. HAM was obtained from healthy mothers and de-epithelized by means of fine enzymatic method to preserve the extracellular structure. Fibroin protein was extracted from fresh Bombyx mori cocoons and transformed to uniform nano-fiberous structure, which was used as a coating layer on the de-epithelized membrane. Surface modification through oxygen plasma treatment was attempted to further induce hydrophilicity. Subsequently, scaffolds were fully characterized in terms of morphology, mechanical properties, hydrophilicity and cell culture response. Histological and immunohistological staining demonstrated localization of fibronectin, cell denudation and structural integrity of HAM after de-epithelization. Scanning electron microscopy images showed bead-free silk fibroin nano-fibers with the average diameter of 250nm. Water contact angle of bilayer scaffolds reduced dramatically to 26.34° after oxygen plasma treatment, which is correlated with more hydrophilic surface. Due to fibroin nano-fiber coating, mechanical properties of HAM improved significantly. Tensile Young's modulus and tensile strength increased from 16.14MPa and 68.46MPa to 25.69MPa and 108.03MPa, respectively. 14days in vitro cultivation of mouse embryonic fibroblasts on the scaffolds revealed that bilayer scaffolds are able to support cell attachment and proliferation. Plasma-etched scaffolds provided the best niche for cell-matrix crosstalk by allowing cells to penetrate beneath the pores and to integrate in fibers direction. The obtained results suggest that the presented nano-fibrous bilayer composite based on HAM is a potential substitute for skin regeneration application., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Scope: Growing evidence suggests that oxidative stress plays a role in the development of chronic diseases such as cardiovascular disease and some psychiatric disorders. Tea consumption exerts beneficial effects against damage induced by cerebral ischemia-reperfusion in ischemic stroke and depressive symptoms in depression. The aim of this study was to evaluate, in vivo, the protective activity of green tea (GT) and GABA green tea (GGT) against post-stroke depression (PSD), a common consequence of stroke., Methods and Results: The antidepressive-like effects of GT and GGT were determined by behavioral tests in a mouse model of post-stroke depression. The antioxidant activity was evaluated by GSH, SOD, and TBARS measurements on mouse brain. The chemical composition of tea extracts was characterized through chromatographic methods. GGT and GT resulted active in the modulation of depressive symptoms and the reduction of oxidative stress, restoring normal behavior, and at least in part, antioxidant endogenous defenses. The higher polyphenol, theanine, glutamine, and caffeine content may justify the higher activity found in GGT., Conclusions: This work represents the first attempt to demonstrate the positive effect of tea, and especially GGT, on post-stroke depression and to correlate this effect with the antioxidant activity and phytochemical composition of tea., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving in vitro and in vivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration. spjba;30/6/793/FIG10885328215601925F1fig1-0885328215601925Figure 1.Illustrative summary of the main methods and findings.RS: regenerated silk; RSF: regenerated fibroin/ silk fiber composite scaffolds; H&E: Hematoxylin and eosin; COX-1: Cyclooxygenase., (© The Author(s) 2015.)

In recent years, the advantages of menstrual blood-derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum-supplemented or serum-free culture media, were also investigated. The sequential differentiation was monitored by real-time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT-4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin-18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte-specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte-like cells. We recommend a complementary serum-free differentiation protocol for enrichment of in vitro production of functional MenSC-derived hepatocyte-like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases., (Copyright © 2013 John Wiley & Sons, Ltd.)

Recently, valuable characteristics of menstrual blood stem cells (MenSCs) have impelled scientists to take its advantages for cell therapy of different diseases including liver disorders. In this study, we examined messenger RNA (mRNA) expression levels of phases I and II drug metabolizing enzymes including glutathione S-transferase (GST) and cytochrome P-450 (CYP) in differentiated hepatocyte-like cells from MenSCs. The isolated MenSCs were characterized and differentiated into hepatocyte-like cells using hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum-free culture media. After primary characterization of hepatocyte markers, mRNA expression of GSTA1, GSTA2, GSTP1, CYP3A4, and CYP7A1 was assessed in differentiated cells in reference to undifferentiated cells using real-time PCR. Based on immunofluorescent staining and real-time PCR data, the differentiated MenSCs could express functional hepatocyte markers at mRNA and/or protein levels suggesting development of hepatocyte-like cells from MenSCs. Moreover, the expression levels of GSTA1, GSTA2, and CYP3A4 mRNA were upregulated in differentiated cells compared to undifferentiated cells. The expression of CYP7A1 gene was also remarkable on the last day of differentiation process. However, the expression level of GSTP1 did not exhibit statistically significant change during differentiation (P = 0.6). Based on accumulative data, MenSCs could be viewed as an accessible population of stem cells with differentiation ability into drug-metabolizing hepatocyte-like cells.

Objectives: To characterize potency of menstrual blood-derived stem cells (MenSCs) for future cell therapies, we examined differentiation potential of MenSCs into adipocytes., Materials and Methods: Differentiation potential of MenSCs in comparison to bone marrow stem cells (BMSCs) was assessed in conventional culture medium. Differentiation potential of MenSCs into adipocytes was improved using different combinations of growth factors and hormones., Results: First, we demonstrated that MenSCs preserve their appearance and karyotypic stability during passages. Although these cells express mesenchymal stem cells markers, they cannot simply be classified as mesenchymal stem cells due to expression of embryonic stem cells marker, OCT-4. Oil red O staining showed that differentiated MenSCs in conventional medium with/without retinoic acid (protocols 1 and 2) did not attain adipocyte characteristics, whereas differentiated BMSCs in conventional medium accumulated oil vacuoles typically. Nevertheless, real-time RT-PCR results showed that LPL gene expression was up-regulated in both protocols 1 and 2, whereas LEPR was up-regulated only in protocol 2 (fortified with retinoic acid). Surprisingly, protocol 3 (including rosiglitazone) had odd influence on mRNA expression of all genes (LEPR, LPL and PPAR-γ). Oil red O staining confirmed fat-producing ability of MenSCs under protocol 3., Conclusions: Presented data suggest an efficient differentiation protocol for in vitro production of MenSC-derived adipocytes. These cells are suggested to be an apt alternative to BMSCs for future stem cell therapy of soft tissue injuries., (© 2014 John Wiley & Sons Ltd.)

Background: Awareness is a postoperative recall of events experienced under general anesthesia. In this study, we compared the incidence of awareness between two routine methods used, inhalation (Isoflurane) and intravenous protocol (Propofol), in elective Cesarean section, and also evaluated the effect of these two different methods on the apgar score of newborns., Materials and Methods: In this prospective, clinical trial study, 90 pregnant women candidates for elective Cesarean section were randomly enrolled, after taking written consent. Induction of anesthesia in both groups was provided by propofol and succinylcholine in the same manner, and maintenance of anesthesia in Group 1 was provided by propofol 100 μg/kg/minute and in Group 2 with isoflurane 1 MAC, to maintain the bispectral index (BIS) between 45 and 60. Blood pressure, heart rate, electrocardiography (ECG), and also Etco2 and o2sat were recorded throughout the surgery and finally analyzed and compared., Results: From 90 patients, four cases of confirmed awareness were found in the propofol group and three cases in the Isoflurane group (8/9% vs. 6/7%), but the apgar scores were comparable between the two groups. Meanwhile there were no significant differences between the two groups in basic information, neonatal apgar scores, hemodynamic changes, and BIS, Electromyography (EMG), and signal quality index (SQI) values., Conclusion: According to the patient's state, diagnosis of the anesthesiologist, and other criteria like price and its availability, we could use these drugs in general anesthesia during Cesarean section, although it is recommended that more studies be done to compare the effect of these two drugs in larger groups.

A metal-organic framework MOF-5 has been synthesized on silk fiber through electrostatic layer-by-layer assembly. The silk surface coating was formed via sequential dipping in an alternating bath of metal and ligand solutions at room temperature by direct mixing. SEM was used to investigate the growth of MOF-5 coating as materials for separation membrane due to their desirable properties in adsorptive removal of congo red (CR) from contaminated water. The adsorption capacity of MOF-5 is remarkable high in the liquid phase. The adsorption of CR at various concentration and contact time in spontaneous process were studied. The silk fibers containing MOF-5 open a wide field of possible applications, such as protection layers or membranes in pollution remediation wastewater and any effluent. Desorption of the dye can be carried out by using NaOH solution with more than about 50% recovery of congo red from MOF-5 coated on silk membrane filtration. In order to investigate the role of sonicating on the morphology of products, one of the reactions was performed with ultrasound irradiation and the crystal growth is completed more than other methods. The samples and adsorption of CR were characterized with SEM, powder X-ray diffraction (XRD) and UV-visible spectroscopy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Menstrual blood is easily accessible, renewable, and inexpensive source of stem cells that have been interested for cell therapy of neurodegenerative diseases. In this study, we showed conversion of menstrual blood stem cells (MenSCs) into clonogenic neurosphere- like cells (NSCs), which can be differentiated into glial-like cells. Moreover, differentiation potential of MenSCs into glial lineage was compared with bone marrow stem cells (BMSCs). Differentiation potential of individual converted NSCs derived from MenSCs or BMSCs into glial-like cells was investigated using immunofluorescence staining and real-time polymerase chain reaction.The fibroblastic morphology of both MenSCs and BMSCs was turned into NSCs shape during first step of differentiation. NSCs derived from both BMSCs and MenSCs expressed higher levels of Olig-2 and Nestin markers compared to undifferentiated cells. The expression levels of myelin basic protein (MBP) mRNA up regulated only in BMSCs-NSCs no in MenSCs-NSCs. However, outgrowth of individual NSCs derived from both MenSCs and BMSCs into glial-like cells led to significant up regulation of glial fibrillary acidic protein,Olig-2 and MBP at mRNA and protein level accompanied with down regulation of Nestin protein.This is the first study demonstrating that MenSCs can be converted to NSCs with differentiation ability into glial-like cells. Accumulative data show different expression pattern of glial markers in differentiated MenSCs compared to BMSCs. The comparable differentiation potential, more accessibility and no invasive technique for sample collection of MenSCs in comparison with BMSCs introduce MenSCs as an apt, consistent and safe alternative to BMSCs for cell therapy of neurodegenerative diseases., (© 2014 International Federation for Cell Biology.)

Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs) possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under protocols developed by different concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB), cytokeratin 18 (CK-18), tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1) at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1) compared to lower concentration of these factors (P2). Moreover, omission of serum during differentiation process (P3) caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

The growth of magnesium hydroxide nanostructures on silk yarn was achieved by sequential dipping steps in alternating bath of magnesium nitrate and potassium hydroxide under ultrasound irradiation. The effects of ultrasound irradiation, concentration, pH and sequential dipping steps on growth of the Mg(OH)(2) nanostructures have been studied. Morphology of the nanostructures, depending on pH and with decreasing pH from 13 to 8, changed from nanoparticle to nanoneedle. Results show a decrease in the particles size as the concentration and sequential dipping steps increased. The physicochemical properties of the nanostructures were determined by powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and wavelength dispersive X-ray (WDX)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Deposition of zinc(II) oxide (ZnO) nanoparticles on the surface of silk fabrics was prepared by sequential dipping steps in alternating bath of potassium hydroxide and zinc nitrate under ultrasound irradiation. This coating involves in situ generation and deposition of ZnO in a one step. The effects of ultrasound irradiation, concentration and sequential dipping steps on growth of the ZnO nanoparticles have been studied. Results show a decrease in the particles size as increasing power of ultrasound irradiation. Also, increasing of the concentration and sequential dipping steps increase particle size. The physicochemical properties of the nanoparticles were determined by powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and wavelength dispersive X-ray (WDX)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The growth of sphere-like trimanganese tetraoxide (Mn(3)O(4)) nanoparticles on silk fiber was achieved by sequential dipping in an alternating bath of potassium hydroxide and manganese(II) nitrate under ultrasound irradiation. Some parameters such as the effect of pH, numerous sequential dipping and ultrasonic irradiation on growth of the nanoparticles have been studied. The samples were characterized with powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and wavelength dispersive X-ray (WDX)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Menstrual blood is easily accessible, renewable, and inexpensive source of stem cells. In this study, we investigated the chondrogenic differentiation potential of menstrual blood-derived stem cells (MenSCs) compared with that of bone marrow-derived stem cells (BMSCs) in two-dimensional culture. Following characterization of isolated cells, the potential for chondrogenic differentiation of MenSCs and BMSCs was evaluated by immunocytochemical and molecular experiments. MenSCs were strongly positive for mesenchymal stem cell markers in a manner similar to that of BMSCs. In contrast to BMSCs, MenSCs exhibited marked expression of OCT4, and higher proliferative capacity. Differentiated MenSCs showed strong immunoreactivity to a monoclonal antibody against Collagen type 2, in a pattern similar to BMSCs. Accumulation of proteoglycans in differentiated MenSCs was also comparable with that in differentiated BMSCs. However, the mRNA expression patterns as judged by RT-PCR of chondrogenic markers such as Collagen 2A1, Collagen 9A1 and SOX9 in MenSCs were different from those in BMSCs. Given these findings, MenSCs appear to be a unique stem cell population with higher proliferation than and comparable chondrogenic differentiation ability to BMSCs in two-dimensional culture. Much quantitative studies at the molecular level may elucidate the reasons for the observed differences in MenSCs and BMSCs.

Background: Prior to the onset of human labour there is an increase in the synthesis of prostaglandins, cytokines and chemokines in the fetal membranes, particular the amnion. This is associated with activation of the transcription factor nuclear factor kappa B (NFκB). In this study we characterised the level of NFκB activity in amnion epithelial cells as a measure of amnion activation in samples collected from women undergoing caesarean section at 39 weeks gestation prior to the onset of labour., Methodology/principal Findings: We found that a proportion of women exhibit low or moderate NFκB activity while other women exhibit high levels of NFκB activity (n = 12). This activation process does not appear to involve classical pathways of NFκB activation but rather is correlated with an increase in nuclear p65-Rel-B dimers. To identify the full range of genes upregulated in association with amnion activation, microarray analysis was performed on carefully characterised non-activated amnion (n = 3) samples and compared to activated samples (n = 3). A total of 919 genes were upregulated in response to amnion activation including numerous inflammatory genes such cyclooxygenase-2 (COX-2, 44-fold), interleukin 8 (IL-8, 6-fold), IL-1 receptor accessory protein (IL-1RAP, 4.5-fold), thrombospondin 1 (TSP-1, 3-fold) and, unexpectedly, oxytocin receptor (OTR, 24-fold). Ingenuity Pathway Analysis of the microarray data reveal the two main gene networks activated concurrently with amnion activation are i) cell death, cancer and morphology and ii) cell cycle, embryonic development and tissue development., Conclusions/significance: Our results indicate that assessment of amnion NFκB activation is critical for accurate sample classification and subsequent interpretation of data. Collectively, our data suggest amnion activation is largely an inflammatory event that occurs in the amnion epithelial layer as a prelude to the onset of labour.

Progesterone is widely used to prolong gestation in women at risk of preterm labour (PTL), and acts at least in part via the inhibition of inflammatory cytokine-induced prostaglandin synthesis. This study investigates the mechanisms responsible for this inhibition in human myometrial cells. We used reporter constructs to demonstrate that interleukin 1beta (IL-1β) inhibits progesterone driven PRE activation via p65 activation and that IL-1β reduced progesterone driven gene expression (FKBP5). Conversely, we found that the activity of a p65-driven NFκB reporter construct was reduced by overexpression of progesterone receptor B (PRB) alone and that this was enhanced by the addition of MPA and that both MPA and progesterone suppressed IL-1β-driven cyclo-oxygenase-2 (COX-2) expression. We found that over-expressed Halo-tagged PRB, but not PRA, bound to p65 and that in IL-1β-treated cells, with no overexpression of either PR or p65, activated p65 bound to PR. However, we found that the ability of MPA to repress IL-1β-driven COX-2 expression was not enhanced by overexpression of either PRB or PRA and that although the combined PR and GR antagonist Ru486 blocked the effects of progesterone and MPA, the specific PR antagonist, Org31710, did not, suggesting that progesterone and MPA act via GR and not PR. Knockdown using siRNA confirmed that both MPA and progesterone acted via GR and not PR or AR to repress IL-1β-driven COX-2 expression. We conclude that progesterone acts via GR to repress IL-1β-driven COX-2 activation and that although the interaction between p65 and PRB may be involved in the repression of progesterone driven gene expression it does not seem to be responsible for progesterone repression of IL-1β-induced COX-2 expression.

Background: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour., Methods: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies., Results: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB.

The uterine expression of the chemokine IL8 increases dramatically with the onset of labour both at term and preterm. The IL8 promoter contains binding sites for the transcription factors nuclear factor-kappa B (NFκB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein (CEBP). In this study we investigated the roles of these transcription factors in IL1B regulation of the IL8 gene in human myometrium. Using chromatin immune precipitation (ChIP) assay, we showed that each of NFκB, CEBP, and AP-1 binds to the IL8 promoter upon IL1B stimulation. To examine the relative importance of each site in IL8 gene expression, site-directed mutagenesis of each of these sites was performed. We found that the NFκB site was essential for basal and IL1B-stimulated gene expression. Mutation of the AP-1 site reduced both basal and IL1B-stimulated expression but to a lesser extent. Mutation of the CEBP site had no effect upon basal expression but eliminated the IL1B response. Small interfering RNA (siRNA) silencing of NFκB abolished the IL8 response to IL1B significantly; siRNA against AP-1 reduced it to a lesser extent whilst knockdown of CEBP enhanced the response. Our data confirms a central and essential role for NFκB in regulation of IL8 in human myometrium.

Uterine activation is associated with increased oxytocin receptor (OXTR) expression and myometrial sensitivity to oxytocin. The OXTR promoter contains binding sites for CCAAT/enhancer-binding protein (CEBP) and nuclear factor-kappa B p65 (RELA). RELA and CEBP beta (CEBPB) play a synergistic role in OXTR promoter activation. We created deletions in a DNA construct consisting of 850 bp upstream of the transcription start site linked to luc reporter to identify the CIS element of the OXTR promoter responsible for the synergistic activation by RELA and CEBPB. Deletion from -712 to -692 bp eliminated synergy, demonstrating that the critical region lies within these 20 bp. Binding studies showed that this sequence binds both RELA and CEBPB. The 20-bp critical region for synergistic activation of OXTR requires full-length RELA but only the basic leucine zipper domain of CEBPB.

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor-κB (NF-κB) activity in myometrium which both up-regulates contraction-associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF-κB p65, or IκB-α between upper- or lower-segment myometrium or before or after labour, there is nuclear localization of serine-256-phospho-p65 and serine-536-phospho-p65 in both upper- and lower-segment myometrium both before and after the onset of labour at term. This shows that NF-κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF-κB we overexpressed p65 in myocytes in culture. This led to NF-κB activation identical to that seen following interleukin (IL)-1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF-κB increased expression of 38 genes principally related to immunity and inflammation. IL-1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL-1β proving a central role for NF-κB. We conclude that NF-κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: Leishmania is an obligate intracellular protozoa, and the sandfly, as a vector, transmits infectious forms of the parasite to the vertebrate host. The etiologic agents of cutaneous leishmaniasis (CL), Leishmania major and Leishmania tropica, are the most prevalent factor in Iran, especially in the Isfahan and Bam regions. Because of the importance of CL in endemic regions and the interaction of species diversity factors in developing control strategies, several isolated Leishmania species from 2 hyperendemic regions of Iran, Isfahan and Bam cities, were examined in this study by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)., Material/methods: In this study, 340 samples were taken from clinically suspected CL patients to prepare slides for direct microscopy and cultures for promastigotes by PCR-RFLP. The internal transcribed spacer 1 (ITS1) region of genomic DNA was extracted and amplified with LITSr and L5.8s primers. Amplification by PCR-RFLP was performed to determine 4 kinds of genotype pattern of the species in the 2 main cities of Isfahan and Bam. Some of the product samples were sequenced and analyzed., Results: Two genotypic groups were detected from L. major isolates, LmA and LmB; also L. tropica showed 2 patterns, LtA and LtB, in comparison with standard species. The most prevalent genotypes related to isolates of Isfahan were LmA and of Bam were LtA. These 2 genotypes were recorded as major etiologic factors of CL in these 2 regions., Conclusions: Leishmania major and L. tropica, the causative agents of zoonotic CL and anthroponotic CL, respectively, in Isfahan and Bam, are genetically highly polymorphic species, and a correlation may exist between genetic heterogeneous and clinical manifestation and geographic regions of the disease in humans.

Investigations of the modulation of prostaglandin F(2alpha) receptor (FP) expression in primary cultures of human uterine myocytes showed that FP mRNA expression was reduced by progesterone, unaltered by cAMP (8-bromo cAMP or forskolin), but increased by the PKA antagonist H89. Interleukin (IL)-1beta, tumour necrosis factor-alpha and oxytocin increased FP mRNA expression and IL-6 and prostaglandin E(2) reduced FP mRNA expression. The changes in FP protein levels were similar to the mRNA responses. We found that the IL-1beta-induced increase in FP expression was mediated at least in part via protein kinase C (PKC), but was independent of mitogen-activated protein kinase, phospholipase C and PI3 kinase. Since IL-1beta activates NFkappaB, AP-1 and C/EBP, we over-expressed these transcription factors alone and in combination and found that only NFkappaB alone increased FP mRNA expression. Finally, we found that the IL-1beta-induced increase in FP expression was unaffected by progesterone and/or cAMP, but was accentuated by H89. These data suggest that the pregnancy-induced down-regulation in myometrial FP expression is mediated by progesterone and cAMP and that the increase with labour is induced by inflammatory cytokine activation of PKC and NFkappaB.

Preterm labour (PTL) is the most important cause of neonatal morbidity and mortality. While some causes have been identified, the mechanisms involved remain elusive. This study investigates whether term labour (TL) is an appropriate model for PTL by examining pro-labour gene expression, using quantitative rtPCR, and protein synthesis, using Western analysis, in preterm and term myometrial samples obtained from the upper and lower uterine segments before and after the onset of labour. In the lower segment, the levels of prostaglandin H synthase type-2 (PGHS-2), interleukin-1beta (IL-1beta), IL-6 and IL-8 mRNA expression were significantly higher in TL compared with PTL samples. Compared with non-labour controls, the expression of IL-1beta and IL-8 mRNA was increased in both PTL and TL samples and the expression of PGHS-2 and IL-6 mRNA was increased in TL samples only. In the upper segment, there were no differences between PTL and TL samples and the mRNA expression of PGHS-2 and IL-1beta was increased in TL compared with term no labour samples. No effect of PTL or TL was seen on either oxytocin receptor or connexin-43 mRNA expression or protein levels. The multiple regression analysis and studies in primary cultures of uterine myocytes suggest that the inflammatory cytokines, IL-1beta and tumour necrosis factor-alpha, are the most important regulators of PGHS-2 and IL-8. Our data show that preterm and term labouring myometrium are significantly different and that the most marked labour-induced changes in gene expression are in the lower segment. These changes may occur in response to the release of inflammatory cytokines by the labour-associated inflammatory infiltration.