TWEAK Appears as a Modulator of Endometrial IL-18 Related Cytotoxic Activity of Uterine Natural Killers

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human

Standardisation of uterine natural killer (uNK) cell measurements in the endometrium of women with recurrent reproductive failure

Considerable work is being carried out on endometrial NK cells to determine whether they play a role in successful pregnancy outcome. In addition there is debate about whether measurements of uNK should be included in the clinical assessment for women with recurrent implantation failure or recurrent miscarriage. A hindrance to taking this forward is the fact that the density of uNK cells reported by different centres is very different. The aim of this study was to determine the reason for these differences and to develop a standardised method. Three centres participated in the study. Each centre exchanged five formalin fixed, wax embedded sections of endometrium from five women. Sections were immunostained for CD56. Images were taken of 10 random fields at ×400 magnification; total stromal and uNK cells were counted using Image J. Results were expressed as % positive uNK cells and the variation in counts obtained in each centre was compared. After initial analysis a standardised protocol was agreed and the process repeated.Significant variation was seen in the counts obtained after initial analysis (Centre A vs.B, mean difference = -0.72 P < 0.001; A vs.C mean difference = -0.47 P < 0.001; B vs.C, mean difference = 0.25 P = 0.085). Analysis suggested that differences may be due to duration of tissue fixation, the embedding and sectioning processes, selection of areas for assessment, definition of immunopositive cells and inclusion or exclusion of blood vessels. Adoption of a standardised protocol reduced the variation (Centre A vs.B mean difference = -0.105 P = 0.744; A vs.C mean difference = 0.219 P = 0.150; B vs.C mean difference = 0.32 P = 0.031). Use of a standardised method is needed to establish a normal range for uNK cells and to develop a meaningful clinical test for uNK cell measurements

Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: Expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells

The chemokine stromal cell-derived factor 1 (SDF-1) stimulates the growth of pre-B cells in vitro, and mice with a disrupted SDF-1 gene have abnormal fetal liver B cell lymphopoiesis. The origin of SDF-1 production has not been determined yet. Using an anti-SDF-1 mAb, we performed immunohistochemical studies in four human embryos and five fetuses to define which cells express the SDF-1 protein at sites of antenatal B cell lymphopoiesis. All mesothelial cells contained SDF-1 at all stages of development, including in the intraembryonic splanchnopleuric mesoderm early into gestation. In fetal lungs and kidneys, SDF-1 was expressed by epithelial cells, and a few B lymphoid precursors, expressing V pre-B chains, were also detected. In the fetal liver, in addition to mesothelial cells, biliary epithelial cells were the only cells to contain SDF-1. Pre-B cells expressing V chains were abundant and exclusively located around the edge of portal spaces, in close contact with biliary ductal plate epithelial cells. They did not colocalize with biliary collecting ducts. Biliary ductal plate epithelial cells and liver B cell lymphopoiesis display a parallel development and disappearance during fetal life. These results indicate that early B cell lymphopoiesis in the splanchnopleura may be triggered by mesothelial cells producing SDF-1. Later into gestation, biliary ductal plate epithelial cells may support B cell lymphopoiesis, thus playing a role similar to that of epithelial cells in the avian bursa of Fabricius, and of thymic epithelial cells for T cell lymphopoiesis

An integrative paradigm to impart quality to correlative science

Correlative studies are a primary mechanism through which insights can be obtained about the bioactivity and potential efficacy of candidate therapeutics evaluated in early-stage clinical trials. Accordingly, well designed and performed early-stage correlative studies have the potential to strongly influence further clinical development of candidate therapeutic agents, and correlative data obtained from early stage trials has the potential to provide important guidance on the design and ultimate successful evaluation of products in later stage trials, particularly in the context of emerging clinical trial paradigms such as adaptive trial design

MKLN1 splicing defect in dogs with lethal acrodermatitis

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of similar to 1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN/:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.Peer reviewe

Therapeutic management of intestinal fibrosis induced by radiation therapy: from molecular profiling to new intervention strategies et vice et versa

Chronic toxicities of locoregional and systemic oncological treatments commonly develop in long-term cancer survivors. Amongst these toxicities, post-radiotherapeutic complications alter patient's quality of life. Reduction of exposure of normal tissues can be achieved by optimization of radiotherapy. Furthermore, understanding of the fibrogenic mechanisms has provided targets to prevent, mitigate, and reverse late radiation-induced damages. This mini-review shows how (i) global molecular studies using gene profiling can provide tools to develop new intervention strategies and (ii) how successful clinical trials, conducted in particular with combined pentoxifylline-vitamin E, can take benefice of biological and molecular evidences to improve our understanding of fibrogenic mechanisms, enhance the robustness of proposed treatments, and lead ultimately to better treatments for patient's benefice