Effect of administration of selective progesterone receptor modulators (SPRMs) on uterine and endometrial morphology

Introduction: The human menstrual cycle is regulated by sex-steroid hormones, including oestrogen (E), progesterone (P4) and androgens which act by ligand binding to their cognate receptors. Perturbation of the complex series of events governing the menstrual cycle may lead to heavy menstrual bleeding (HMB). This is a common debilitating condition and often associated with uterine fibroids. There remains an unmet need for effective, long-term medical treatment so women avoid surgery and preserve their fertility. Selective progesterone receptor modulators (SPRMs, e.g. ulipristal acetate, UPA) are synthetic ligands that bind the progesterone receptor (PR). Many SPRMs have been developed but only mifepristone (for the management of unwanted pregnancy) and UPA are in current clinical use. UPA is licensed for the intermittent treatment of symptomatic fibroids. SPRMs have potential utility for treatment of HMB as administration rapidly induces amenorrhoea but the mechanisms by which this is achieved are unknown. SPRM administration results in unique endometrial morphological changes (progesterone receptor modulator-associated endometrial changes; PAEC). Despite endometrial unopposed estradiol exposure these morphological changes do not appear to be associated with malignancy or pre-malignancy risk. Indeed endometrial cell proliferation appears reduced despite relative progesterone-antagonism. Based upon findings with other SPRMs it was hypothesised that: (i) administration of UPA would have an endometrial specific effect upon the reproductive tract, with regard to alteration in morphology, localisation of sex steroid receptors (SSR) and cell proliferation.; (ii) administration of UPA would impact upon progesterone-regulated (Pregulated) genes in the endometrium. Methods: The data presented within this thesis are derived from biopsies obtained at hysterectomy from the endometrium, fallopian tubes and cervices of women with symptomatic fibroids administered UPA for 8-15 weeks. Samples were obtained for histological assessment, immunohistochemistry and RNA extraction for subsequent quantitative RT-qPCR of sex-steroid receptors (SSR) and proliferation markers. In addition key P-regulated genes within the endometrium were investigated by RT-qPCR and selected protein expression. To further interrogate the anti-proliferative effect, RNA was extracted from “paired” endometrial biopsies from the same woman in the proliferative phase of the menstrual cycle and following subsequent treatment with UPA for at least eight weeks and microarray gene analyses undertaken. Results: Morphological alteration of the endometrium with UPA administration was consistent with previously published data, but with a higher prevalence than previously described. There was a striking alteration in expression and localization of SSRs, particularly PR and androgen receptor (AR), and alteration of many P-regulated genes, consistent with UPA acting with low progesteroneagonism within the endometrium. There was no alteration of SSR expression within the cervix and proliferation was unchanged. Fallopian tube morphology and SSR expression was consistent with proliferative phase but cell proliferation was reduced following UPA administration, consistent with secretory phase levels. Microarray analyses identified multiple transcripts altered relative to proliferative phase, with GREM2 the most significantly down-regulated gene and MUC1 one of the most significantly upregulated genes. Consistent with low levels of mitotic figures and cell proliferation, the most down regulated KEGG pathway was the cell cycle. Multiple elements within this were subsequently validated (RT-qPCR) and included key regulators of all elements of the mitotic cell cycle, many of which were novel to those previously described following administration of another SPRM, mifepristone. In summary the novel data presented in this thesis considerably extend the data available to date concerning the actions of the SPRM, UPA, on the female reproductive tract, and increases knowledge regarding a compound with promising utility for the management of the debilitating complaint of HMB

Evidence That a TRPA1-Mediated Murine Model of Temporomandibular Joint Pain Involves NLRP3 Inflammasome Activation

From MDPI via Jisc Publications RouterHistory: accepted 2021-10-14, pub-electronic 2021-10-23Publication status: PublishedFunder: Versus Arthritis; Grant(s): 21541This study investigates the role of transient receptor potential ankyrin 1 (TRPA1) in murine temporomandibular joint (TMJ) inflammatory hyperalgesia and the influence of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. Two distinct murine models of TMJ pain and inflammation (zymosan and CFA) were established. Spontaneous pain-like behaviours were observed as unilateral front paw cheek wipes. Ipsilateral cheek blood flow was used as a measure of ongoing inflammation, which, to our knowledge, is a novel approach to assessing real-time inflammation in the TMJ. Joint tissue and trigeminal ganglia were collected for ex vivo investigation. Both zymosan and CFA induced a time-dependent increase in hyperalgesia and inflammation biomarkers. Zymosan induced a significant effect after 4 h, correlating with a significantly increased IL-1β protein expression. CFA (50 µg) induced a more sustained response. The TRPA1 receptor antagonist A967079 significantly inhibited hyper-nociception. The NLRP3 inhibitor MCC950 similarly inhibited hyper-nociception, also attenuating inflammatory markers. In the trigeminal ganglia, CFA-induced CGRP expression showed trends of inhibition by A967079, whilst lba1 immunofluorescence was significantly inhibited by A967079 and MCC950, where the effect of TRPA1 inhibition lasted up to 14 days. Our results show that stimulation of TRPA1 is key to the TMJ pain. However, the inflammasome inhibitor exhibited similar properties in attenuating these pain-like behaviours, in addition to some inflammatory markers. This indicates that in addition to the therapeutic targeting of TRPA1, NLRP3 inhibition may provide a novel therapeutic strategy for TMJ inflammation and pain

Novel Antibacterial Properties of the Human Dental Pulp Multipotent Mesenchymal Stromal Cell Secretome

It is well recognized that clearance of bacterial infection within the dental pulp precedes pulpal regeneration. However, although the regenerative potential of the human dental pulp has been investigated extensively, its antimicrobial potential remains to be examined in detail. In the current study bactericidal assays were used to demonstrate that the secretome of dental pulp multipotent mesenchymal stromal cells (MSCs) has direct antibacterial activity against the archetypal Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, respectively, as well as the oral pathogens Streptococcus mutans, Lactobacillus acidophilus, and Fusobacterium nucleatum. Furthermore, a cytokine/growth factor array, enzyme-linked immunosorbent assays, and antibody blocking were used to show that cytokines and growth factors present in the dental pulp MSC secretome, including hepatocyte growth factor, angiopoietin-1, IL-6, and IL-8, contribute to this novel antibacterial activity. This study elucidated a novel and diverse antimicrobial secretome from human dental pulp MSCs, suggesting that these cells contribute to the antibacterial properties of the dental pulp. With this improved understanding of the secretome of dental pulp MSCs and its novel antibacterial activity, new evidence for the ability of the dental pulp to fight infection and restore functional competence is emerging, providing further support for the biological basis of pulpal repair and regeneration

Novel Antibacterial Properties of the Human Dental Pulp Multipotent Mesenchymal Stromal Cell Secretome

It is well recognized that clearance of bacterial infection within the dental pulp precedes pulpal regeneration. However, although the regenerative potential of the human dental pulp has been investigated extensively, its antimicrobial potential remains to be examined in detail. In the current study bactericidal assays were used to demonstrate that the secretome of dental pulp multipotent mesenchymal stromal cells (MSCs) has direct antibacterial activity against the archetypal Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, respectively, as well as the oral pathogens Streptococcus mutans, Lactobacillus acidophilus, and Fusobacterium nucleatum. Furthermore, a cytokine/growth factor array, enzyme-linked immunosorbent assays, and antibody blocking were used to show that cytokines and growth factors present in the dental pulp MSC secretome, including hepatocyte growth factor, angiopoietin-1, IL-6, and IL-8, contribute to this novel antibacterial activity. This study elucidated a novel and diverse antimicrobial secretome from human dental pulp MSCs, suggesting that these cells contribute to the antibacterial properties of the dental pulp. With this improved understanding of the secretome of dental pulp MSCs and its novel antibacterial activity, new evidence for the ability of the dental pulp to fight infection and restore functional competence is emerging, providing further support for the biological basis of pulpal repair and regeneration

Evidence That a TRPA1-Mediated Murine Model of Temporomandibular Joint Pain Involves NLRP3 Inflammasome Activation

This study investigates the role of transient receptor potential ankyrin 1 (TRPA1) in murine temporomandibular joint (TMJ) inflammatory hyperalgesia and the influence of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. Two distinct murine models of TMJ pain and inflammation (zymosan and CFA) were established. Spontaneous pain-like behaviours were observed as unilateral front paw cheek wipes. Ipsilateral cheek blood flow was used as a measure of ongoing inflammation, which, to our knowledge, is a novel approach to assessing real-time inflammation in the TMJ. Joint tissue and trigeminal ganglia were collected for ex vivo investigation. Both zymosan and CFA induced a time-dependent increase in hyperalgesia and inflammation biomarkers. Zymosan induced a significant effect after 4 h, correlating with a significantly increased IL-1β protein expression. CFA (50 µg) induced a more sustained response. The TRPA1 receptor antagonist A967079 significantly inhibited hyper-nociception. The NLRP3 inhibitor MCC950 similarly inhibited hyper-nociception, also attenuating inflammatory markers. In the trigeminal ganglia, CFA-induced CGRP expression showed trends of inhibition by A967079, whilst lba1 immunofluorescence was significantly inhibited by A967079 and MCC950, where the effect of TRPA1 inhibition lasted up to 14 days. Our results show that stimulation of TRPA1 is key to the TMJ pain. However, the inflammasome inhibitor exhibited similar properties in attenuating these pain-like behaviours, in addition to some inflammatory markers. This indicates that in addition to the therapeutic targeting of TRPA1, NLRP3 inhibition may provide a novel therapeutic strategy for TMJ inflammation and pain

Government targets for protected area management: will threatened butterflies benefit?

The UK Government has set targets for biodiversity conservation in England based on several indicators, including the status of protected areas [e.g. Sites of Special Scientific Interest (SSSIs)]. Specifically, the Government aims to achieve favourable condition [defined by Common Standards Monitoring (CSM)] on 95% by area of SSSIs by 2010. SSSIs are important for threatened butterflies and management to achieve favourable condition will play a key role in determining future population levels of these high-profile insects. Because only notified features of SSSIs are considered within CSM, we investigated the level of notification for three threatened butterflies. We found that these species were notified at only 15–33% of SSSIs where they occurred; though most site managers did manage for them under broader site conservation objectives. We investigated the relationship between SSSI condition status and population trend for eight butterfly species of conservation concern to assess the benefit to butterflies of sites attaining favourable condition status. The majority (80%) of population trends on SSSIs in favourable condition were positive, suggesting an overall beneficial impact of SSSI management. However, four of the eight species maintained lower populations at favourable condition SSSIs than at sites in one of the unfavourable condition categories. We suggest that current condition assessment based chiefly on notified vegetation communities lacks the sensitivity to identify the complex habitat conditions for these (mosaic) species. As butterflies are good indictors for a wide range of invertebrates, many species requiring fine-scale habitat heterogeneity may be at risk from the Government’s target