Role of mast cells in women's health and disorders of the endometrium

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

Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERβ and glucocorticoid receptor

Background: Human mast cells (MCs) are long-lived tissue-resident immune cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium) surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular) surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1) To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2) To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ), progesterone (PR) and glucocorticoids (GR). Methods: Tissue samples from women (n=46) were used for RNA extraction (n=26) or fixed (n=20) for immunohistochemistry. Results: Messenger RNAs encoded by TPSAB1 (tryptase) and CMA1 (chymase) were detected in endometrial tissue homogenates. Immunohistochemistry revealed the relative abundance of tryptase MCs was myometrium>basal endometrium>functional endometrium. We show for the first time that uterine MCs are predominantly of the classical MC subtypes: (positive, +; negative, -) tryptase+/chymase- and tryptase+/chymase+, but a third subtype was also identified (tryptase-/chymase+). Tryptase+ MCs were of an ERβ+/ERα-/PR-/GR+ phenotype mirroring other uterine immune cell populations, including natural killer cells. Conclusions: Endometrial tissue resident immune MCs have three protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors that of other immune cells in the endometrium and suggests that MC function may be altered by the local steroid microenvironment

Pelvic pain correlates with peritoneal macrophage abundance not endometriosis

Endometriosis is a chronic neuroinflammatory pain condition affecting ~180 million women worldwide. Surgical removal or hormonal suppression of endometriosis lesions only relieves pain symptoms in some women and symptomatic relapse following treatment is common. Identifying factors that contribute to pain is key to developing new therapies. We collected peritoneal fluid samples and clinical data from a cohort of women receiving diagnostic laparoscopy for suspected endometriosis (n = 52). Peritoneal fluid immune cells were analysed by flow cytometry and data compared with pain scores determined using the pain domain of the Endometriosis Health Profile Questionnaire (EHP-30) in order to investigate the association between peritoneal immune cells and pain symptoms. Pain scores were not different between women with or without endometriosis, nor did they differ according to disease stage; consistent with a poor association between disease presentation and pain symptoms. However, linear regression and correlation analysis demonstrated that peritoneal macrophage abundance correlated with the severity of pelvic pain. CD14(high) peritoneal macrophages negatively correlated with pain scores whereas CD14(low) peritoneal macrophages were positively correlated, independent of diagnostic outcome at laparoscopy. Stratification by pain subtype, rather than endometriosis diagnosis, resulted in the most robust correlation between pain and macrophage adundance. Pain score strongly correlated with CD14(high) (P = 0.007) and CD14(low) (P = 0.008) macrophages in patients with non-menstrual pain and also in patients who reported dysmennorhea (CD14(high) P = 0.021, CD14(low) P = 0.019) or dysparunia (CD14(high) P = 0.027, CD14(low) P = 0.031). These results provide new insight into the association between peritoneal macrophages and pelvic pain which may aid the identification of future therapeutic targets. LAY SUMMARY: Endometriosis is a common condition where cells similar to those that line the womb are found elsewhere in the body. It is associated with inflammation and pain in the pelvis and affects ~180 million women worldwide. Current treatments are not effective for all patients and we, therefore, need to understand what causes pain in order to develop new treatments. We investigated the types of immune cells present within the pelvis of women undergoing investigation for suspected endometriosis. Disease diagnosis and stage (I–IV) was recorded along with pain score determined by questionnaire. We characterised the immune cells present and compared them to disease stage and pain score. We found that pelvic pain was linked to the abundance of immune cells but, surprisingly, not to disease stage. These findings suggest that immune cells are closely associated with pain severity in endometriosis and may be good targets for future endometriosis treatments

Oral geranylgeranylacetone treatment increases heat shock protein expression in human atrial tissue

BACKGROUND Heat shock proteins (HSPs) are important chaperones that regulate the maintenance of healthy protein quality control in the cell. Impairment of HSPs is associated with aging-related neurodegenerative and cardiac diseases. Geranylgeranylacetone (GGA) is a compound well known to increase HSPs through activation of heat shock factor-1 (HSF1). GGA increases HSPs in various tissues, but whether GGA can increase HSP expression in human heart tissue is unknown. OBJECTIVE The purpose of this study was to test whether oral GGA treatment increases HSP expression in the atrial appendages of patients undergoing cardiac surgery. METHODS HSPB1, HSPA1, HSPD1, HSPA5, HSF1, and phosphorylated HSF1 levels were measured by western blot analysis in right and left atrial appendages (RAAs and LAAs, respectively) collected from patients undergoing coronary artery bypass grafting (CABG) who were treated with placebo (n = 13) or GGA 400 mg/da(n = 13) 3 days before surgery. Myofilament fractions were isolated from LAAs to determine the levels of HSPB1 and HSPA1 present in these fractions. RESULTS GGA treatment significantly increased HSPB1 and HSPA1 expression levels in RAA and LAA compared to the placebo group, whereas HSF1, phosphorylated HSF1, HSPD1, and HSPA5 were unchanged. In addition, GGA treatment significantly enhanced HSPB1 levels at the myofilaments compared to placebo. CONCLUSION Three days of GGA treatment is associated with higher HSPB1 and HSPA1 expression levels in RAA and LAA of patients undergoing CABG surgery and higher HSPB1 levels at the myofilaments. These findings pave the way to study the role of GGA as a protective compound against other cardiac diseases, including postoperative atrial fibrillation

Postradiation Matrix Metalloproteinase-20 Expression and Its Impact on Dental Micromorphology and Radiation-Related Caries

Recent evidence suggests that head-and-neck radiotherapy (HNRT) increases active forms of matrix metalloproteinase-20 (MMP-20) in human tooth crowns, degrading the dentin-enamel junction (DEJ) and leading to enamel delamination, which is a pivotal step in the formation of radiation-related caries (RRC). Additional participation of enzymatic degradation of organic matrix components in caries progression was attributed to MMP-20 in dentin. Therefore, the current study tested the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes to the enamel and dentin. Thirty-six teeth were studied, including 19 post-HNRT specimens and 17 nonirradiated controls. Optical light microscopy was used to investigate the micromorphological components of the DEJ, dentin-pulp complex components, and carious dentin. The samples were divided into 2 subgroups: nondemineralized ground sections (n = 20) and demineralized histological sections (n = 16). In addition, immunohistochemical analysis using the immunoperoxidase technique was conducted to semiquantitatively assess MMP-20 expression in the DEJ, dentin-pulp complex components, and carious dentin. No apparent damage to the DEJ microstructure or other dentin-pulp complex components was observed and no statistically significant differences were detected in MMP-20 expression (p > 0.05) between the irradiated and control groups. This study rejected the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes. Hence, direct effects of radiation may not be regarded as an independent factor to explain aggressive clinical patterns of RRC. (C) 2017 S. Karger AG, BaselPeer reviewe

Differential item functioning of the Functional Independence Measure in higher performing neurological patients

OBJECTIVE: When comparing outcomes of the Functional Independence Measure (FIM ) between patient groups, item characteristics of the FIM should be consistent across groups. The purpose of this study was to compare item difficulty of the FIM in 3 patient groups with neurological disorders. SUBJECTS: Patients with stroke (n=295), multiple sclerosis (n=150), and traumatic brain injury (n=88). METHODS: FIM scores were administered in each group. The FIM consists of a motor domain (13 items) and a cognitive domain (5 items). Rasch rating scale analysis was performed to investigate differences in item difficulty (differential item functioning) between groups. RESULTS: Answering categories of the FIM items were reduced to 3 (from the original 7) because of disordered thresholds and low answering frequencies. Two items of the motor domain ("bladder" and "bowel") did not fit the Rasch model. For 7 out of the 11 fitting motor items, item difficulties were different between groups (i.e. showed differential item functioning). All cognitive items fitted the Rasch model, and 4 out of 5 cognitive items showed differential item functioning. CONCLUSION: Differential item functioning is present in several items of both the motor and cognitive domain of the FIM. Adjustments for differential item functioning may be required when FIMdata will be compared between groups or will be used in a pooled data analysi

Effects of Boswellia Serrata Roxb. and Curcuma longa L. in an In Vitro Intestinal Inflammation Model Using Immune Cells and Caco-2

Inflammatory bowel diseases, which consist of chronic inflammatory conditions of the colon and the small intestine, are considered a global disease of our modern society. Recently, the interest toward the use of herbal therapies for the management of inflammatory bowel diseases has increased because of their effectiveness and favourable safety profile, compared to conventional drugs. Boswellia serrata Roxb. and Curcuma longa L. are amongst the most promising herbal drugs, however, their clinical use in inflammatory bowel diseases is limited and little is known on their mechanism of action. The aim of this work was to investigate the effects of two phytochemically characterized extracts of B. serrata and C. longa in an in vitro model of intestinal inflammation. Their impact on cytokine release and reactive oxygen species production, as well as the maintenance of the intestinal barrier function and on intestinal mucosa immune cells infiltration, has been evaluated. The extracts showed a good protective effect on the intestinal epithelium at 1 µg/mL, with TEER values increasing by approximately 1.5 fold, compared to LPS-stimulated cells. C. longa showed an anti-inflammatory mechanism of action, reducing IL-8, TNF-α and IL-6 production by approximately 30%, 25% and 40%, respectively, compared to the inflammatory stimuli. B. serrata action was linked to its antioxidant effect, with ROS production being reduced by 25%, compared to H₂O₂-stimulated Caco-2 cells. C. longa and B. serrata resulted to be promising agents for the management of inflammatory bowel diseases by modulating in vitro parameters which have been identified in the clinical conditions

Identification of Altered Evoked and Non-Evoked Responses in a Heterologous Mouse Model of Endometriosis-Associated Pain

The aim of this study was to develop and refine a heterologous mouse model of endometriosis-associated pain in which non-evoked responses, more relevant to the patient experience, were evaluated. Immunodeficient female mice (N = 24) were each implanted with four endometriotic human lesions (N = 12) or control tissue fat (N = 12) on the abdominal wall using tissue glue. Evoked pain responses were measured biweekly using von Frey filaments. Non-evoked responses were recorded weekly for 8 weeks using a home cage analysis (HCA). Endpoints were distance traveled, social proximity, time spent in the center vs. outer areas of the cage, drinking, and climbing. Significant differences between groups for von Frey response, climbing, and drinking were detected on days 14, 21, and 35 post implanting surgery, respectively, and sustained for the duration of the experiment. In conclusion, a heterologous mouse model of endometriosis-associated evoked a non-evoked pain was developed to improve the relevance of preclinical models to patient experience as a platform for drug testing

Human and animal fertility studies in cystinosis reveal signs of obstructive azoospermia, an altered blood-testis barrier and a subtherapeutic effect of cysteamine in testis

Cystinosis is an inherited metabolic disorder caused by autosomal recessive mutations in the CTNS gene leading to lysosomal cystine accumulation. The disease primarily affects the kidneys followed by extra-renal organ involvement later in life. Azoospermia is one of the unclarified complications which are not improved by cysteamine, which is the only available disease-modifying treatment. We aimed at unraveling the origin of azoospermia in cysteamine-treated cystinosis by confirming or excluding an obstructive factor, and investigating the effect of cysteamine on fertility in the Ctns−/− mouse model compared with wild type. Azoospermia was present in the vast majority of infantile type cystinosis patients. While spermatogenesis was intact, an enlarged caput epididymis and reduced levels of seminal markers for obstruction neutral α-glucosidase (NAG) and extracellular matrix protein 1 (ECM1) pointed towards an epididymal obstruction. Histopathological examination in human and mouse testis revealed a disturbed blood-testis barrier characterized by an altered zonula occludens-1 (ZO-1) protein expression. Animal studies ruled out a negative effect of cysteamine on fertility, but showed that cystine accumulation in the testis is irresponsive to regular cysteamine treatment. We conclude that the azoospermia in infantile cystinosis is due to an obstruction related to epididymal dysfunction, irrespective of the severity of an evolving primary hypogonadism. Regular cysteamine treatment does not affect fertility but has subtherapeutic effects on cystine accumulation in testis