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During the normal menstrual cycle, the human endometrium undergoes extensive tissue remodelling under the influence of ovarian-derived hormones. The endometrium has well defined stromal and epithelial compartments with the former containing both a well-developed vasculature as well as a diverse population of immune cells. Mast cells (MCs) are long-lived tissue resident immune cells characterised by the presence of granules containing proteases. Mast cells have been detected in the human uterus but little is known about their regulation or the impact of steroids on their differentiation status. Recently MCs have been implicated as key players in physiological and pathological pain pathways but little is known about their role in endometrial pathologies. Endometriosis is a chronic incurable condition characterized by the presence of endometrial tissue outside the uterine cavity: women with endometriosis can suffer from a debilitating range of symptoms including chronic pain. Whilst the aetiology of endometriosis is uncertain, close proximity between MCs and nerves has implicated them in aberrant activation of pain pathways. The aims of the current project were: 1. To determine the spatial and temporal location of uterine MCs and to explore their phenotype including expression of steroid receptors. 2. To explore the activation status of MCs in women with endometriosis and/or pain, 3. To explore the use of cells and mice as models to investigate the phenotype of mast cells and their regulation by steroids. Mast cell proteases tryptase and chymase were detected by RTPCR and immunohistochemistry in “full thickness” (uterine lumen to endometrial-myometrial junction) biopsies from women undergoing hysterectomy. In agreement with previous findings MCs were most abundant in the myometrium. Uterine MCs were predominantly of the classical MC subtypes: tryptasepos/chymaseneg and tryptasepos/chymasepos but a rare third subtype was also identified as tryptaseneg/chymasepos. Mast cell activation/degranulation was cycle stage dependent and for the first time their steroid receptor phenotype was identified as ERαneg/ERβpos/GRpos, suggesting potential regulation by the uterine steroid microenvironment. Studies on tissue samples from women with endometriosis revealed MCs with an altered activation status in the pelvic peritoneal wall, compared to controls, which showed an intense diffuse immunoexpression of chymase suggestive of MC activation and release of this protease during normal physiology of the peritoneum. Surprisingly, analysis of peritoneal fluids from controls, women with pain but no endometriosis, and pain with endometriosis did not detect differences in numbers of MCs or concentrations of tryptase or chymase. Analysis of peritoneal biopsies also provided the first evidence for a striking increase in immunoexpression of PAR-2, a protease-activated receptor, in women suffering from chronic pelvic pain and/or endometriosis which may provide a mechanism by which mast cell derived factors may alter pain pathways. Studies in a mouse model of endometriosis identified MCs within endometria-llike lesions and offer a platform for future studies. In vitro explorations using MCs derived from peripheral blood precursors and HMC-1, a cell line derived from a patient with MC leukaemia confirmed expression of ERβ but did not support previous studies claiming cells were ERαpos. In summary, this study has provided novel insights into the phenotype of endometrial mast cells in the normal cycling endometrium and contrasted them with those in women with endometriosis and pelvic pain. This is the first study to identify MCs as ERβpos. Further studies are required to determine whether inhibition of PAR- 2 might offer a therapeutic target in women with chronic pelvic pain.

Intracellular signalling molecules create the signalling cascades which enable the transfer of the signal to the cell. In this work we have studied the influence of tick saliva on the cytokine production and the activation of signalling pathways in ionomycin stimulated murine mast cells. We found out that tick saliva inhibits production of several cytokines and affects two important signalling pathways in mast cells possibly involved in the regulation of cytokine induction.

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-11Bitstream added on 2014-06-13T19:41:10Z : No. of bitstreams: 1 costa_ss_dr_botfmvz.pdf: 1745465 bytes, checksum: 6163d7d6432dccda5910a889a705ab7b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O mastocitoma (MCT) cutâneo é uma das neoplasias malignas mais comuns em cães, representa 11% dos tumores de pele nesta espécie e apresenta comportamento biológico variável. Este trabalho dá continuidade a pesquisas com biomarcadores prognósticos em MCTs cutâneos em cães. Foi realizado um estudo em busca de critérios complementares ao exame clínico (ultrassonografia pelo método Doppler), que auxiliem na determinação do potencial de recidiva e metástase do tumor, correlacionando-os com a expressão da proteína KIT, densidade microvascular (DMI), tamanho, número de tumores, tempo de evolução, ulceração, tempo de sobrevida e graduação histopatológica. Além disso, foi realizada uma análise dos parâmetros clínicos, incluindo dados epidemiológicos, achados histológicos e moleculares e a correlação destes com o comportamento biológico do MCT. Foram avaliados 20 cães, que totalizaram 28 tumores. A ultrassonografia (US) pelo método Doppler permitiu a identificação de vasos em 54% dos tumores. Não houve correlação entre a presença de vasos e a DMI, a localização da proteína KIT, os graus histológicos, o tempo de evolução, o tamanho, a ocorrência de recidivas e metástases, e o tempo de sobrevida, mas sim com a presença de ulceração tumoral. Observou-se correlação estatística entre o grau histológico, a DMI, a presença de ulceração e o número de tumores e também da expressão de Ki-67 com os padrões de marcação da proteína KIT. O grau histológico no MCT cutâneo canino não deve ser avaliado isoladamente, mas sim em conjunto com a expressão da proteína KIT, DMI, proliferação celular, presença de ulceração, número de tumores e ocorrência de recidivas e metástases. Indicamos que estudos adicionais... / Cutaneous mast cell tumor (MCT) is one of the most common malignant neoplasms in dogs, representing 11% of skin tumors in this species and shows a variable biological behavior. This article maintains the search on prognostic biomarkers in cutaneous MCTs in dogs. A study was conducted to find additional criteria to the clinical examination (Doppler ultrasound) to add in determining the potential for recurrence and metastasis of tumors, correlating them with KIT protein expression, intratumoral microvessel density (IMD), size, number of tumors, duration time, ulceration, survival rates and histological classification. In addition, we performed an analysis of clinical parameters, including epidemiological data, histological and molecular ones and correlate them with the biological behavior of MCT. We evaluated 20 dogs, in a total of 28 tumors. The Doppler ultrasound (US) allowed the identification of vessels in 54% of tumors. There was no correlation between the presence of vessels and IMD, KIT protein location, histological grades, duration time, tumor size, recurrence and metastasis, and survival rates, but with ulceration. We have observed statistical correlation between histological grade, IDM, presence of ulceration and number of tumors and also the expression of Ki-67 with patterns of KIT protein. The histological grade in canine cutaneous MCT should not be assessed in isolation but in conjunction with expression of KIT protein, IDM, cell proliferation, presence of ulceration, number of tumors and recurrences and metastases rates. We suggest that additional studies should be done to further evaluate the use of Doppler US as a noninvasive method to chacarterize the vascularization and blood flow in... (Complete abstract click electronic access below)

Abstract The mast cells are an integral part of the human immune system. They are important modulators of inflammatory and physiological processes. Mast cells exert their functions through degranulation and release of inflammatory mediators, such as histamine, proteases and cytokines. There are two main pathways leading to the mast cell activation, the immunoglobulin-dependent and the immunoglobulin-independent pathway. The latter pathway can be triggered by several non-immunological stimuli, and two novel receptors responsible for the activation have been identified, the MAS-related G protein-coupled receptor X1 (MRGPRX1) and X2. The MRGPRX1 and MRGPRX2 have two established functions: i) they trigger the degranulation of mast cells and ii) they are involved in pain perception and itch on a specific subset of sensory neurons. These receptors are not expressed in all of the populations of mast cells, only in the tryptase and chymase containing mast cells, contributing to the mast cell heterogeneity. Unlike most G protein-coupled receptors, the MRGPRX1 and MRGPRX2 are quite non-selective, binding an ever growing list of different ligands. Their ligands include endogenous neuropeptides, host defense peptides and protein fragments, as well as synthetic compounds such as different antibiotics. Their endogenous ligands could be a triggering signal in some mast cell-related diseases by degranulating mast cells and thereby inducing inflammation. Due to the non-selectivity of MRGPRX1 and MRGPRX2, they probably still have many hitherto unknown ligands. The aim of this study was to isolate novel endogenous ligands for the MRGPRX1 and MRGPRX2 from human tissues with the “reverse pharmacology approach” and to determine their potential to degranulate mast cells. The starting materials for the isolation, human platelets and plasma, contained MRGPRX1 and MRGPRX2 activating compounds. From the human plasma, three fragments of albumin able to activate the MRGPRX2 were isolated and sequenced. These fragments were dose-dependently activating the MRGPRX2 and degranulating mast cells. Two MRGPRX1 activating hemoglobin β-chain fragments were isolated from human platelets. These fragments were dose-dependently activating the MRGPRX1, but had no effect on mast cell degranulation. / Tiivistelmä Syöttösolut on tärkeä osa ihmisen immuunijärjestelmää. Ne ovat tärkeitä tulehdus- ja fysiologistenprosessien säätelijöitä. Syöttösolujen vaikutus välittyy degranulaation ja siinä vapautuvien tulehdusvälittäjäaineiden kautta. Vapautuviin aineisiin lukeutuu esim. histamiini ja lukuisia sytokiinejä, sekä proteaaseja. Syöttösolujen aktivaatio voi tapahtua immunoglobuliineista riippuvaa tai immunoglobuliineista riippumatonta reittiä pitkin. Monet ei-immunologiset tekijät voivat laukaista jälkimmäisen reitin ja kaksi uutta tähän vaikuttavaa G-proteiinikytkentäistä reseptoria on löydetty, MAS-related G protein-coupled receptor X1 (MRGPRX1) ja X2. MRGPRX1:llä ja MRGPRX2:lla on kaksi tunnettua tehtävää: i) ne laukaisevat syöttösolujen degranulaation ja ii) ne osallistuvat kivun ja kutinan aistimiseen tietyissä tuntohermoissa. Näitä reseptoreita ei ilmennetä kaikissa syöttösoluissa, vaan ainoastaa tryptaasia ja kymaasia sisältävissä syöttösoluissa, ja täten osaltaan selittävät syöttösolujen monimuotoisuutta. Useimmista G-proteiinikytkentäisistä reseptoreista poiketen MRGPRX1 ja MRGPRX2 ovat laajakirjoisia, sitoen monia erilaisia ligandeja. Ligandeihin kuuluu endogeenisia neuropeptidejä, antimikrobiaalisia peptidejä ja proteiinin fragmentteja, sekä synteettisiä yhdisteitä kuten erilaisia antibiootteja. Reseptoreiden endogeeniset ligandit voivat toimia laukaisijana jossain syöttösoluihin liittyvissä sairauksissa, degranuloidessaan syöttösoluja ja aiheuttaen paikallisen tulehdustilan. Reseptoreiden laajakirjoisuudesta johtuen niillä on oletettavasti monia vielä tuntemattomia ligandeja. Tämän tutkimuksen tarkoitus oli eristää uusia endogeenisiä ligandeja MRGPRX1:lle ja MRGPRX2:lle ihmisen kudoksista ”kääteisfarmakologista lähestymistapaa” hyödyntäen ja selvittää ligandien kyky syöttösolujen degranulaatioon. Lähtömateriaalina käytetyt ihmisen verihiutaleet ja plasma sisälsivät MRGPRX1:ta ja MRGPRX2:ta aktivoivia yhdisteitä. Plasmasta eristettiin ja sekvensoitiin kolme albumiinin fragmenttia, jotka aktivoivat MRGPRX2:ta. Nämä fragmentit aktivoivat MRGPRX2:ta ja degranuloivat syöttösoluja annosriippuvaisesti. Kaksi MRGPRX1:tä aktivoivaa hemoglobiinin β-ketjun fragmenttia eristettiin ihmisen verihiutaleista. Nämä fragmentit tunnistettiin hemorfiineiksi ja ne aktivoivat MRGPRX1:tä annosriippuvaisesti, mutta eivät vaikuttaneet syöttösolujen degranulaatioon.

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