DIRAS3 regulates autophagy in an endometriosis epithelial cell line.

RESEARCH QUESTION: What is the role of DIRAS3 in endometriosis pathogenesis? DESIGN: Prospective patient cohort study combined with experiments in the 12Z human endometriosis epithelial cell line model to determine the role of DIRAS3 in endometriosis. Endometrium and endometriosis lesion samples were collected from premenopausal women from 24 control and 40 endometriosis patients by laparoscopic surgery. The role of DIRAS3 in endometriosis was assessed by siRNA knockdown in 12Z cells followed by proliferation, apoptosis, invasion and autophagy assays. Autophagy was induced by serum starvation and the levels of autophagy determined by assessing changes in the expression levels and localization of autophagy marker proteins, such as LC3. RESULTS: DIRAS3 mRNA showed a large increase in expression in ectopic endometriosis lesions compared with endometrium from control patients, with expression largely localized to the epithelium. DIRAS3 knockdown in 12Z endometriosis epithelial cells caused a significant reduction in the number of proliferating cells (1.6-fold, adjusted P = 0.0007) and increased apoptosis (AnnexinV/7AAD double-positive cells +48%, P = 0.01), indicating an effect on cell proliferation. Induction of autophagy by serum starvation caused significant upregulation in DIRAS3 expression after 24 h (mRNA +2.4-fold [adjusted P = 0.017], protein +8.1-fold (adjusted P = 0.029), reduced LC3I/LC3II ratio (-2.2-fold, adjusted P = 0.044) and an increase in the number of double positive LC3/DIRAS3 puncta (+2.3-fold, P = 0.02). Knockdown of DIRAS3 in serum-starved cells led to a reduction in autophagy, indicated by an overall decrease in LC3 expression and significant increase in LC3I/LC3II ratio. CONCLUSIONS: DIRAS3 is highly upregulated in endometriosis lesions. Studies in an endometriosis epithelial cell line indicate that DIRAS3 facilitates cell survival in this context by inducing autophagy.

Association of the Estrogen Receptor 1 Polymorphisms rs2046210 and rs9383590 with the Risk, Age at Onset and Prognosis of Breast Cancer.

Estrogen receptor α (ERα), encoded by the ESR1 gene, is a key prognostic and predictive biomarker firmly established in routine diagnostics and as a therapeutic target of breast cancer, and it has a central function in breast cancer biology. Genetic variants at 6q25.1, containing the ESR1 gene, were found to be associated with breast cancer susceptibility. The rs2046210 and rs9383590 single nucleotide variants (SNVs) are located in the same putative enhancer region upstream of ESR1 and were separately identified as candidate causal variants responsible for these associations. Here, both SNVs were genotyped in a hospital-based case-control study of 409 female breast cancer patients and 422 female controls of a Central European (Austrian) study population. We analyzed the association of both SNVs with the risk, age at onset, clinically and molecularly relevant characteristics and prognosis of breast cancer. We also assessed the concordances between both SNVs and the associations of each SNV conditional on the other SNV. The minor alleles of both SNVs were found to be non-significantly associated with an increased breast cancer risk. Significant associations were found in specific subpopulations, particularly in patients with an age younger than 55 years. The minor homozygotes of rs2046210 and the minor homozygotes plus heterozygotes of rs9383590 exhibited a several-years-younger age at onset than the common homozygotes, which was more pronounced in ER-positive and luminal patients. Importantly, the observed associations of each SNV were not consistently nullified upon correction for the other SNV nor upon analyses in common homozygotes for the other SNV. We conclude that both SNVs remain independent candidate causal variants.

Co-cultivation of human granulosa cells with ovarian cancer cells leads to a significant increase in progesterone production.

PURPOSE: In humans, granulosa cells (GCs) are part of the follicle and nourish the growing oocyte. GCs produce estrogen and, after ovulation, progesterone. They are embedded in a multicellular tissue structure of the ovary, which consists of a variety of different cell types that are essential for the physiological function of the ovary. However, the extent to which individual ovarian cell types contribute to overall functionality has not yet been fully elucidated. In this study, we aim to investigate the effects of co-culturing human granulosa cells with ovarian cancer cells on their progesterone and estrogen production in an in vitro model. METHODS: After seeding, the cells were stimulated with 200 µM forskolin in DMEM for 72 h and the medium of the different cell culture experiments was collected. Subsequently, progesterone and oestradiol concentrations were determined using an Elisa assay. RESULTS: Morphologically, it was striking that the cells self-organize and form spatially separated areas. Compared to culturing granulosa cells alone, co-culturing human granulosa cells together with the ovarian cancer cell line OvCar-3 resulted in a significant increase in progesterone production (20.3 ng/ml versus 50.2 ng/ml; p < 0.01). CONCLUSIONS: Using a simple in vitro model, we highlight the importance of cellular crosstalk between different ovarian cells in a complex cellular network and that it strongly influences granulosa cell hormone production. This could have potential implications for the procedure of transplanting endocrine tissues after cryopreservation, as it highlights the importance of survival of all cells for the functionality of the transplanted tissue.

LINC01133 Inhibits Invasion and Promotes Proliferation in an Endometriosis Epithelial Cell Line.

Endometriosis is a common gynecological disorder characterized by ectopic growth of endometrium outside the uterus and is associated with chronic pain and infertility. We investigated the role of the long intergenic noncoding RNA 01133 (LINC01133) in endometriosis, an lncRNA that has been implicated in several types of cancer. We found that LINC01133 is upregulated in ectopic endometriotic lesions. As expression appeared higher in the epithelial endometrial layer, we performed a siRNA knockdown of LINC01133 in an endometriosis epithelial cell line. Phenotypic assays indicated that LINC01133 may promote proliferation and suppress cellular migration, and affect the cytoskeleton and morphology of the cells. Gene ontology analysis of differentially expressed genes indicated that cell proliferation and migration pathways were affected in line with the observed phenotype. We validated upregulation of p21 and downregulation of Cyclin A at the protein level, which together with the quantification of the DNA content using fluorescence-activated cell sorting (FACS) analysis indicated that the observed effects on cellular proliferation may be due to changes in cell cycle. Further, we found testis-specific protein kinase 1 (TESK1) kinase upregulation corresponding with phosphorylation and inactivation of actin severing protein Cofilin, which could explain changes in the cytoskeleton and cellular migration. These results indicate that endometriosis is associated with LINC01133 upregulation, which may affect pathogenesis via the cellular proliferation and migration pathways.

The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

CD36 is a scavenger receptor that exhibits pleiotropic functions, including adhesion to thrombospondin, inhibition of angiogenesis, transport of long-chain fatty acids, and clearance of apoptotic cells. In addition, it has been implicated in the host immune response because it acts as a coreceptor for TLR2 and plays a role in Staphylococcus aureus infection. However, its role in other Gram-positive bacterial infections is unclear. In this study, using mice deficient in CD36, we sought to examine the role of CD36 in pneumococcal pneumonia, a major cause of morbidity and mortality worldwide. We show that CD36 is expressed on both alveolar macrophages and respiratory epithelial cells. Early in infection, CD36(-/-) mice have an exaggerated inflammatory response compared with wild-type littermate controls. In vitro studies using CD36(-/-) primary cells confirm the enhanced early inflammation in response to S. pneumoniae and its lipoteichoic acid, demonstrate that S. pneumoniae binds to cells via its phosphocholine residues, and suggest a role for CD36 in reducing inflammation induced by the phosphocholine residues of pneumococcal lipoteichoic acid. Later in infection, although CD36(-/-) mice exhibit impaired bacterial clearance, owing to a decreased capacity of CD36(-/-) macrophages to phagocytose S. pneumoniae, minor effects on mortality occur, in comparison with those in wild-type littermate control mice. These data show that CD36 contributes to the pulmonary host response during S. pneumoniae infection by virtue of its ability to act as a phagocytic receptor and as a modulator of the early innate immune response.

Bbeta(15-42) protects against acid-induced acute lung injury and secondary pseudomonas pneumonia in vivo.

RATIONALE: Acute lung injury (ALI) is a serious condition in critically ill patients that predisposes to secondary bacterial pneumonia. Vascular leak is a hallmark in the pathogenesis of ALI. The fibrin-derived peptide Bbeta(15-42) was shown to preserve endothelial barriers, thereby reducing vascular leak. The potential therapeutic role of Bbeta(15-42) in ALI has not been addressed so far. OBJECTIVES: To investigate the therapeutic potential of Bbeta(15-42) in ALI and secondary pneumonia induced by Pseudomonas aeruginosa. METHODS: The effect of the fibrin-derived peptide Bbeta(15-42) was studied in models of ALI, induced either by pulmonary administration of LPS or hydrochloric acid. Lung inflammation was analyzed by quantifying cell influx, cytokine levels, and oxidized lipids. Vascular leak was determined by Evans Blue extravasations and alveolar protein content. In subsequent two-hit studies, mice were infected with P. aeruginosa 24 hours after induction of aspiration pneumonitis and effects of Bbeta(15-42) on inflammation, bacterial clearance, and survival were evaluated. MEASUREMENTS AND MAIN RESULTS: After LPS or acid inhalation, proinflammatory cytokine levels, neutrophil influx, and vascular leak were found diminished in mice treated with Bbeta(15-42). Acid aspiration impaired macrophage functions and rendered mice more susceptible to subsequent P. aeruginosa infection, whereas mice that received Bbeta(15-42) during acid aspiration and were subsequently challenged with bacteria displayed reduced inflammation, enhanced bacterial clearance, and ultimately improved survival. CONCLUSIONS: The fibrin-derived peptide Bbeta(15-42) exerted protective effects during ALI, resulting in diminished lung injury and preserved antibacterial properties of macrophages, which improved outcome during subsequent P. aeruginosa pneumonia.

TREM-1 activation alters the dynamics of pulmonary IRAK-M expression in vivo and improves host defense during pneumococcal pneumonia.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is an amplifier of TLR-mediated inflammation during bacterial infections. Thus far, TREM-1 is primarily associated with unwanted signs of overwhelming inflammation, rendering it an attractive target for conditions such as sepsis. Respiratory tract infections are the leading cause of sepsis, but the biological role of TREM-1 therein is poorly understood. To determine the function of TREM-1 in pneumococcal pneumonia, we first established TREM-1 up-regulation in infected lungs and human plasma together with augmented alveolar macrophage responsiveness toward Streptococcus pneumoniae. Mice treated with an agonistic TREM-1 Ab and infected with S. pneumoniae exhibited an enhanced early induction of the inflammatory response that was indirectly associated with lower levels of negative regulators of TLR signaling in lung tissue in vivo. Later in infection, TREM-1 engagement altered S. pneumoniae-induced IRAK-M (IL-1R-associated kinase-M) kinetics so as to promote the resolution of pneumonia and remarkably led to an accelerated elimination of bacteria and consequently improved survival. These data show that TREM-1 exerts a protective role in the innate immune response to a common bacterial infection and suggest that caution should be exerted in modulating TREM-1 activity during certain clinically relevant bacterial infections.

Successful treatment of familial Mediterranean fever with Anakinra and outcome after renal transplantation.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent episodes of fever and inflammation. The most severe complication of FMF is the development of AA amyloidosis, which can be life threatening. The only current effective treatment for FMF is colchicine. Regular prophylactic treatment with colchicine at a dose of 1-2 mg daily prevents or substantially reduces the clinical manifestations of FMF in at least 90% of cases. However, approximately 10% of patients are reported to be resistant or non-responsive to colchicine and in these cases there is no consensus as to which second line agents should be used. We describe the first case, to our knowledge, of a patient with FMF and end-stage renal failure due to AA amyloidosis, successfully treated with IL-1 receptor blockade. Our data suggest that the IL-1 receptor antagonist Anakinra (Kineret; r-metHuIL-1 ra) may represent a safe and effective therapy for the treatment of colchicine-resistant FMF, in patients requiring renal replacement therapy, with dialysis or transplantation.

Lipoteichoic acid-induced lung inflammation depends on TLR2 and the concerted action of TLR4 and the platelet-activating factor receptor.

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.

Influence of the washing buffer composition on the sensitivity of an enzyme-linked immunosorbent assay using mycobacterial glycolipids as capture antigens.

Immunogenic glycolipids from the cell wall of Mycobacterium tuberculosis are potential capture antigens in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnostis of tuberculosis. Typically, washing steps in ELISAs are performed with buffers containing a detergent. However, Tween-20, the most commonly added detergent, was reported to be able to remove the coating of certain glycolipid antigens from microtitre wells. In order to determine the influence of the washing buffer composition on the results, we measured serum immunoglobulin G (IgG) against three mycobacterial glycolipids by ELISA, conducting three separate experiments with three different buffers: Tris-buffered saline (TBS), TBS plus 0.02% Tween-20 (TBS-Tween), or TBS plus 0.3% bovine serum albumin (TBS-BSA). The capture antigens applied were lipoarabinomannan with the basic arabinose-containing motif (AraLAM), the mannose-capped version of lipoarabinomannan (ManLAM), and trehalose-6,6'-dimycolate (cord factor). All ELISAs achieved acceptable specificities around 95%. The sensitivities, however, varied widely, depending upon the sort of washing buffer used. In 38 patients with sputum smear-positive pulmonary tuberculosis and control groups of 79 patients with non-tuberculosis lung disease and 92 healthy volunteers, the anti-cord factor ELISA achieved 100%, 31.6%, and 60.5% with TBS, TBS-Tween, and TBS-BSA, respectively. Corresponding sensitivity values for AraLAM were 39.5%, 26.3%, and 23.7%, and for ManLAM 94.7%, 65.8%, and 55.3%. We conclude that Tween-20 or BSA should be omitted from the washing buffer in ELISAs, when the capture antigen is of lipid nature.