Thrombin Receptors on Human Platelets INITIAL LOCALIZATION AND SUBSEQUENT REDISTRIBUTION DURING PLATELET ACTIVATION

Platelet responses to thrombin are at least partly mediated by a G-protein-coupled receptor whose NH2 terminus is a substrate for thrombin. In the present studies we have examined the location of thrombin receptors in resting platelets and followed their redistribution during platelet activation. The results reveal several new aspects of thrombin receptor biology. 1) On resting platelets, approximately two-thirds of the receptors were located in the plasma membrane. The remainder were present in the membranes of the surface connecting system. 2) When platelets were activated by ADP or a thromboxane analog, thrombin receptors that were initially in the surface connecting system were exposed on the platelet surface, increasing the number of detectable receptors by 40% and presumably making them available for subsequent activation by thrombin. 3) Platelet activation by thrombin rapidly abolished the binding of the antibodies whose epitopes are sensitive to receptor cleavage and left the platelets in a state refractory to both thrombin and the agonist peptide, SFLLRN. This was accompanied by a 60% decrease in the binding of receptor antibodies directed COOH-terminal to the cleavage site irrespective of whether the receptors were activated proteolytically by thrombin or nonproteolytically by SFLLRN. 4) The loss of antibody binding sites caused by thrombin was due in part to receptor internalization and in part to the shedding of thrombin receptors into membrane microparticles, especially under conditions in which aggregation was allowed to occur. However, at least 40% of the cleaved receptors remained on the platelet surface. 5) Lacking the ability to synthesize new receptors and lacking an intracellular reserve of preformed receptors comparable to that found in endothelial cells, platelets were unable to repopulate their surface with intact receptors following exposure to thrombin. This difference underlies the ability of endothelial cells to recover responsiveness to thrombin rapidly while platelets do not, despite the presence on both of the same receptor for thrombin

Peroxisome proliferator-activated receptor delta limits the expansion of pathogenic Th cells during central nervous system autoimmunity.

Peroxisome proliferator-activated receptors (PPARs; PPAR-alpha, PPAR-delta, and PPAR-gamma) comprise a family of nuclear receptors that sense fatty acid levels and translate this information into altered gene transcription. Previously, it was reported that treatment of mice with a synthetic ligand activator of PPAR-delta, GW0742, ameliorates experimental autoimmune encephalomyelitis (EAE), indicating a possible role for this nuclear receptor in the control of central nervous system (CNS) autoimmune inflammation. We show that mice deficient in PPAR-delta (PPAR-delta(-/-)) develop a severe inflammatory response during EAE characterized by a striking accumulation of IFN-gamma(+)IL-17A(-) and IFN-gamma(+)IL-17A(+) CD4(+) cells in the spinal cord. The preferential expansion of these T helper subsets in the CNS of PPAR-delta(-/-) mice occurred as a result of a constellation of immune system aberrations that included higher CD4(+) cell proliferation, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. We also show that the effect of PPAR-delta in inhibiting the production of IFN-gamma and IL-12 family cytokines is ligand dependent and is observed in both mouse and human immune cells. Collectively, these findings suggest that PPAR-delta serves as an important molecular brake for the control of autoimmune inflammation

Endothelial Cell Thrombin Receptors and PAR-2 TWO PROTEASE-ACTIVATED RECEPTORS LOCATED IN A SINGLE CELLULAR ENVIRONMENT

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled receptors. Because previous studies have shown that the biology of the human thrombin receptor varies according to the cell in which it is expressed, we have taken advantage of the presence of both receptors in endothelial cells to examine the enabling and disabling interactions with candidate proteases likely to be encountered in and around the vascular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of thrombin receptor and PAR-2 clearance and replacement in a common cellular environment. Of the proteases that were tested, only trypsin activated both receptors. Cathepsin G, which disables thrombin receptors, had no effect on PAR-2, while urokinase, kallikrein, and coagulation factors IXa, Xa, XIa, and XIIa neither substantially activated nor noticeably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin-sensitive phospholipase C activation as well as activation of phospholipase A2, leading to the release of PGI2. Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent response to the PAR-2 agonist peptide, SLIGRL, while only partially inhibiting the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not require protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that the expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can respond than it does to extend the range of potential responses. The results also show that proteases that can disable these receptors can distinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of thrombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin

Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1.

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage

Polyelectrolyte/surfactant films: from 2D to 3D structural control

Reversible control of the 3D structure of polyelectrolyte/surfactant films at the air/water interface is showcased. A recently discovered mechanism is exploited to form highly efficient, stable and biocompatible films by spreading aggregates composed of poly-L-lysine and sodium dodecyl sulfate on the surface of water. Reversible control of: (1) the surface monolayer coverage, (2) the switching on or off discrete extended structures, and (3) the extended structure coverage is demonstrated for the first time. The intricacy by which the film structures can be controlled is unprecedented and opens exciting potential to optimize film properties by chemical design for novel biomedical transfer applications.We thank the Institut Laue-Langevin for beam time on FIGARO (DOIs: https://doi.org/10.5291/ILL-DATA.9-12-614 and https://doi.org/10.5291/ILL-DATA.9-12-631), Simon Wood for technical assistance and the Partnership for Soft Condensed Matter (PSCM) for lab support. IV acknowledges the financial support from the Hungarian National Research, Development and Innovation Office (NKFIH K116629). AM acknowledges the financial support from MICINN under grant PID2021-129054NA-I00 and the IKUR Strategy of the Basque Government.Peer reviewe

Fbxw7 Deletion Accelerates KrasG12D-Driven Pancreatic Tumorigenesis via Yap Accumulation

AbstractPancreatic cancers driven by KRAS mutations require additional mutations for tumor progression. The tumor suppressor FBXW7 is altered in pancreatic cancers, but its contribution to pancreatic tumorigenesis is unknown. To determine potential cooperation between Kras mutation and Fbxw7 inactivation in pancreatic tumorigenesis, we generated P48-Cre;LSL-KrasG12D;Fbxw7fl/fl (KFCfl/fl) compound mice. We found that KFCfl/fl mice displayed accelerated tumorigenesis: all mice succumbed to pancreatic ductal adenocarcinoma (PDA) by 40 days of age, with PDA onset occurring by 2 weeks of age. PDA in KFCfl/fl mice was preceded by earlier onset of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions, and associated with chromosomal instability and the accumulation of Fbxw7 substrates Yes-associated protein (Yap), c-Myc, and Notch. Using KFCfl/fl and FBXW7-deficient human pancreatic cancer cells, we found that Yap silencing attenuated growth promotion by Fbxw7 deletion. Our data demonstrate that Fbxw7 is a potent suppressor of KrasG12D-induced pancreatic tumorigenesis due, at least in part, to negative regulation of Yap

Attitudes towards microbicide use for bacterial vaginosis in pregnancy

Background Bacterial vaginosis (BV) is the most common reproductive tract infection (RTI) and is a significant risk factor for preterm birth. Microbicides could be an option for the prevention and treatment of BV in pregnancy, and understanding use of the product will be crucial. The present study explored attitudes of women in the third trimester of pregnancy regarding topical microbicide use for the prevention and treatment of BV. METHODS: Twenty-six women in their third trimester were interviewed regarding their knowledge and beliefs about RTIs during pregnancy and attitudes concerning the use of topical microbicides for prevention and treatment of BV. RESULTS: Participants had a mean age of 24.9 years, were largely under-represented minorities and the majority had had past pregnancies. Participants had knowledge and experience with RTIs but not BV. They were open to the use of microbicides for prevention or treatment of BV, but believed that women requiring treatment would be more motivated. Rationales for acceptability were most commonly related to the baby's health. Practical issues that may interfere with use were often, but not always, related to pregnancy. There was a range of attitudes about partner involvement in decision-making and the practicalities of product use. CONCLUSION: Pregnant women are knowledgeable about RTIs but not necessarily BV. The women in this study found microbicide use acceptable, particularly for treatment. To improve acceptability and use, education would be needed about BV and possible complications, how to overcome practical problems and the value of involving partners in the decision

How do economic crises affect migrants’ risk of infectious disease? A systematic-narrative review

Background: It is not well understood how economic crises affect infectious disease incidence and prevalence, particularly among vulnerable groups. Using a susceptible-infected-recovered framework, we systematically reviewed literature on the impact of the economic crises on infectious disease risks in migrants in Europe, focusing principally on HIV, TB, hepatitis and other STIs. Methods: We conducted two searches in PubMed/Medline, Web of Science, Cochrane Library, Google Scholar, websites of key organizations and grey literature to identify how economic changes affect migrant populations and infectious disease. We perform a narrative synthesis in order to map critical pathways and identify hypotheses for subsequent research. Results: The systematic review on links between economic crises and migrant health identified 653 studies through database searching; only seven met the inclusion criteria. Fourteen items were identified through further searches. The systematic review on links between economic crises and infectious disease identified 480 studies through database searching; 19 met the inclusion criteria. Eight items were identified through further searches. The reviews show that migrant populations in Europe appear disproportionately at risk of specific infectious diseases, and that economic crises and subsequent responses have tended to exacerbate such risks. Recessions lead to unemployment, impoverishment and other risk factors that can be linked to the transmissibility of disease among migrants. Austerity measures that lead to cuts in prevention and treatment programmes further exacerbate infectious disease risks among migrants. Non-governmental health service providers occasionally stepped in to cater to specific populations that include migrants. Conclusions: There is evidence that migrants are especially vulnerable to infectious disease during economic crises. Ring-fenced funding of prevention programs, including screening and treatment, is important for addressing this vulnerability

Sex-Specific Expression of Alternative Transcripts in \u3ci\u3eDrosophila\u3c/i\u3e

Background: Many genes produce multiple transcripts due to alternative splicing or utilization of alternative transcription initiation/termination sites. This \u27transcriptome expansion\u27 is thought to increase phenotypic complexity by allowing a single locus to produce several functionally distinct proteins. However, sex, genetic and developmental variation in the representation of alternative transcripts has never been examined systematically. Here, we describe a genome-wide analysis of sex-specific expression of alternative transcripts in Drosophila melanogaster. Results: We compared transcript profiles in males and females from eight Drosophila lines (OregonR and 2b, and 6 RIL) using a newly designed 60-mer oligonucleotide microarray that allows us to distinguish a large proportion of alternative transcripts. The new microarray incorporates 7,207 oligonucleotides, satisfying stringent binding and specificity criteria that target both the common and the unique regions of 2,768 multi-transcript genes, as well as 12,912 oligonucleotides that target genes with a single known transcript. We estimate that up to 22% of genes that produce multiple transcripts show a sex-specific bias in the representation of alternative transcripts. Sexual dimorphism in overall transcript abundance was evident for 53% of genes. The X chromosome contains a significantly higher proportion of genes with female-biased transcription than the autosomes. However, genes on the X chromosome are no more likely to have a sexual bias in alternative transcript representation than autosomal genes. Conclusion: Widespread sex-specific expression of alternative transcripts in Drosophila suggests that a new level of sexual dimorphism at the molecular level exists