PreImplantation Factor (PIF) promotes HLA-G, -E, -F, -C expression in JEG-3 choriocarcinoma cells and endogenous progesterone activity

BACKGROUND: Pregnancy success requires mandatory maternal tolerance of the semi/allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. AIMS: To examine if PIF affects gene expression of human leukocyte antigen (HLA)-G, -E -F and -C in JEG-3 choriocarcinoma cells, and to examine the influence of PIF on local progesterone activity. Methods: PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. RESULTS: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, F confirmed also by Western blotting. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling. CONCLUSION: PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders

Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NF?B activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1? secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1? restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1? secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1? expression, while NOD2 inversely promoted IL-1?. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Mir-15A Reconstitution in Prostate Cancer Cell Line Suppresses Cancer Progression Through Down Regulation of MYB and Androgen Receptor Upregulation

Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. MicroRNAs are noncoding RNAs that have a role of post-transcriptional regulators. In this study we investigated how the tumour suppressor miR-15a modulates main transcription factors like cMYB and AR in androgen sensitive prostate cancer cell line LNCaP. The miR-15a inhibitor, mimic, and their negative controls were transfected into LNCaP cells. Real-time PCR analysis was performed in order to estimate the transcript levels of cMYB and AR. Flow cytometry analysis was performed to measure the protein levels of cMYB and AR. A Cell migration assay was done for cells transfected with miR-15a inhibitor and mimic. We found that cMYB is down-regulated and AR is up-regulated by miR-15a on the transcriptional and protein levels. By reconstituting miR-15a, we found that its down regulation in prostate cancer contributes to cMYB-induced cancer progression and reduced androgen receptivity. The ability of miR-15a to suppress cancer cell viability and migration is a very important phenomenon for understanding cancer heterogeneity in regard to adapted therapeutic approach development

Nalp Signalling is Required in Sertoli Cells for Tight-Junction Protein Interaction

The present study aims to investigate the NALP3 system and its influence on occludin in Sertoli cells, utilising primary murine cells and adult Sertoli cell line as models. Its main goals are the Sertoli cell biology with possible implications on male reproductive functions. Primary and adult Sertoli cells were transfected with NAPL3 siRNA and treated with NOD1 (ie-DAP) and NOD2 (MDP) receptor ligands. There was positive occludin expression levels on transcript (RT-qPCR) and protein (FCS and Immunofluorescence) levels for both cell types. The innate immunity and tight-junction pathways integration serve a protective role for both testis immune barrier and spermatogenesis compartmentalisation maintained by the very same barrier. This integration also points the way for mechanistic research of the disturbances inflicted during an inflammatory response in testis niche

The Role of microRNA-15A in the Development of Prostate Cancer – Effects on Cell Proliferation and Pro-Inflammatory Signalling

Worldwide prostate cancer is the second leading cause of cancer death among men after lung cancer. MicroRNAs are non-coding, endogenous RNAs and they play a role in tumorigenesis, RNA silencing and post-transcriptional regulation of gene expression. In this study we have investigated microRNA-15a impact on transcription factors cMYB and ETS1 in prostate-carcinoma cell line PC3. The PC3 cells were transfected with a synthetic analogue and inhibitor of microRNA-15a. The study was performed using reverse transcription polymerase chain reaction and flow cytometry methods for assessing the transcript and protein levels of cMYB and ETS1, NFκB stable reporter live cell line. Statistical analysis was performed using One–way ANOVA test. We found that cMYB and ETS1 are up-regulated by the synthetic analogue of microRNA-15a at the transcription and protein level. Transfection with microRNA-15a mimic resulted in NFκB transcription factor activation as found by using the live cell reporter system. There was some opportunistic activity exhibited by the synthetic inhibitor, but less pronounced. Our data suggest that microRNA-15a could participate in prostate cancer progression by modulating cell proliferation and pro-inflammatory signaling and paves a way for further in-depth investigation of the gene regulatory networks underneath

Stem Cells in the Reproductive System

This review article summarizes current knowledge on regulation, functions, and capacities of stem cells in the female and male reproductive tract. Major locations in which pluripotent cells reside and from where they can be isolated are the ovaries, the endometrium, the decidua, and the testis. They include oocytes, embryonic stem cells, trophoblast stem cells, and spermatogonial stem cells, but also several side populations, which can be obtained after certain isolation and culture procedures. The potential of pluripotent cells in the reproductive tract to differentiate is manifold, but heterogenous, depending upon their respective origin. As stem cells have a potential for future application in transplantation and regenerative medicine, this article also reviews the literature on major histocompatibility complex expression on stem cells of the reproductive tract, because of its immunogenic effects, but also because of its potential expression of HLA-G, a potent immunomodulator mainly associated with trophoblast cells. © 2012 John Wiley & Sons A/S

HLA-DR Genotyping and Mitochondrial DNA Analysis Reveal the Presence of Family Burials in a Fourth Century Romano-British Christian Cemetery

In Colchester, Britain's oldest recorded town, during the Roman period there were areas which were clearly used solely as cemeteries. One of the most significant is at Butt Road, which includes a late Roman probable Christian cemetery with an associated building, apparently a church, that overlies and developed from a pagan inhumation cemetery. DNA was extracted from the long bones (femurs) of 29 individuals, mostly from a large complex of burials centered on two timber vaults. These were thought to comprise a number of family groupings, deduced from osteological analysis, stratigraphical and other considerations. The use of a modified version of the silica-based purification method recovered nanogram quantities of DNA/gram of bone. Two-stage amplification, incorporating primer-extension preamplification-polymerase chain reaction, permitted simultaneous amplification of both mitochondrial and nuclear DNA. Sequence-specific oligonucleotide probes yielded human leukocyte antigen (HLA)-DR typing of seven samples, with four revealing the infrequent HLA-DR10 genotype. Examination of the control region of mitochondrial DNA (mtDNA) by direct sequencing revealed polymorphisms yet to be reported in the modern population. HLA-DRB typing and mtDNA analysis affirmatively supported kinship among some, if not all, individuals in the “vault complex” and demonstrate a continental European origin of the individuals investigated