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Inflammation is a multifaceted process involving a host of resident and recruited immune cells that eliminate the insult or injury and initiate tissue repair. In the female reproductive tract (FMRT), inflammation-mediated alterations in epithelial, vascular, and immune functions are important components of complex physiological processes and many local and systemic pathologies. It is well established that intracoital and postcoital function of seminal fluid (SF) goes beyond nutritive support for the spermatozoa cells. SF, in particular, the inflammatory bioactive lipids, and prostaglandins present in vast quantities in SF, have a role in localized immune modulation and regulation of pathways that can exacerbate inflammation in the FMRT. In sexually active women SF-mediated inflammation has been implicated in physiologic processes such as ovulation, implantation, and parturition while also enhancing tumorigenesis and susceptibility to infection. This review highlights the molecular mechanism by which SF regulates inflammatory pathways in the FMRT and how alterations in these pathways contribute to physiology and pathology of the female reproductive function. In addition, based on findings from TaqMan® 96-Well Plate Arrays, on neoplastic cervical cells treated with SF, we discuss new findings on the role of SF as a potent driver of inflammatory and tumorigenic pathways in the cervix.

Background An increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F 2α , where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF 2α via the FP receptor. Methods Human endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF 2α -treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA. Results ADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2α-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation. Conclusions These data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF 2α -FP receptor mediated induction of ADAMTS1.

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A (VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Enzyme-linked immunosorbent spot analysis is frequently used to investigate immune responsiveness during clinical trials. However, ELISpot classically utilizes peripheral blood mononuclear cell isolates from whole blood, requiring relatively high blood draw volumes and removing both granulocytes and bound drug. Here, we describe a novel protocol whereby CD45 cells are magnetically isolated from human whole blood and co-incubated with serum isolated from the same subject. Infliximab is a well characterized anti-tumor necrosis factor α (TNF-α) antibody in clinical use since the late 1990s. We demonstrated that TNF-α inhibition by infliximab in spiked whole blood is lost on peripheral blood mononuclear cell isolation but remains in serum, and that combining serum from infliximab spiked whole blood with magnetically isolated CD45 immune cells inhibited PMA/ionomycin-stimulated TNF-α secretion. This novel protocol has important implications for enzyme-linked immunosorbent spot analysis in clinical trials in which blood volume is limited, and keeping drug responses intact provides critical information.

Background In July 2010, the Western Australian AIDS Council established the 'M Clinic', a peer-led STI testing service for MSM. This study describes trends in HIV notifications among MSM in WA from 2004 to 2013, particularly the impact of the M Clinic on newly acquired HIV diagnoses. The number and proportion of MSM HIV cases with newly acquired infection were compared for the 2004-2006, 2007-2009 and 2011-2013 time periods. Data from 2010 were excluded as the M Clinic opened in July 2010. Between the 2004-2006 and 2007-2009 periods, the number of MSM with newly acquired HIV increased by 50% (23 to 33 cases) and the number of newly acquired cases as a proportion of all new HIV diagnoses among MSM increased from 27% to 35% (30% increase) (P=0.25). In the 2011-2013 period, the number of newly acquired HIV cases among MSM more than doubled to 70 cases and comprised 53% of all new HIV diagnoses among MSM (P<0.05). Of the 70 newly acquired HIV cases in the 2011-2013 period, 30% (n=21) were diagnosed at the M Clinic. The proportion of MSM HIV notifications that were newly acquired increased between 2004 and 2013 in WA, with the greatest increase seen after the M Clinic commenced operation. A peer-led approach to HIV testing should be considered in order to achieve early diagnosis and treatment of HIV among MSM.

Cyclooxygenase (COX) enzymes catalyze the rate-limiting biosynthesis of prostaglandins (PG). Following biosynthesis PG exert an autocrine/paracrine function locally by coupling to specific G-protein coupled receptors. Over the past decade, many epidemiological, pharmacological and laboratory studies using in vitro and in vivo model systems of targeted gene disruption in mice have provided conclusive evidence for a role for COX enzymes, PG and PG receptors in pathophysiology. We have established elevated expression of COX enzymes (COX-1 and COX-2), PG (PGE2 and PGF2α) and PG receptors (EP2/EP4 and FP receptor) in pathologies of the female reproductive tract. In cervical and endometrial carcinomas, the expression of COX enzymes, PG and PG receptors are up-regulated in neoplastic epithelial cells as well as endothelial cells of the microvasculature compared with normal tissue. We elucidated the role of COX-1 and COX-2 in cervical and endometrial carcinomas using an in vitro HeLa Tet-Off system to inducibly express COX-1 or Ishikawa cell line stably expressing COX-2. We found that elevated COX enzyme expression in vitro, up-regulated the biosynthesis of PG and induced the expression of PG receptors. Coincident with the up-regulation of PG and PG receptors, we also observed up-regulation of potent angiogenic factors and down-regulation of anti-angiogenic factors. These findings lead to the suggestion that COX enzyme inhibitors may be of potential benefit as therapeutic regimens for cervical and endometrial carcinomas as has been recommended for other types of carcinomas highly expressing COX enzymes. Subsequently we have elucidated the signal transduction pathways mediating the role of PG via their specific receptors in cervical and endometrial carcinomas. For example, activation of the FP receptor in endometrial cancer cells can promote rapid cytoskeletal reorganization, formation of focal adhesion complexes and integrin-extracellular matrix engagement to promote cancer cell adhesion and migration to facilitate metastasis. Moreover, we have discovered that coincident with these dynamic changes in cell morphology, adhesion and migration, FP receptor signaling also promotes the production of potent angiogenic factors such as VEGF and FGF2. Following release, these angiogenic factors act in a paracrine manner to promote vascular branching and sprouting and endothelial cell proliferation. Taken together, our research has elucidated the role of COX enzymes and PG receptor signaling in cervical and endometrial carcinomas as potent regulators of cell movement and vascular function. These findings have highlighted the use of specific PG receptor antagonists or inhibitors of key signal transduction pathways to inhibit the adverse effects of elevated PG signaling in uterine carcinomas.

Doc number: 8 Abstract Background: Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation. Methods and results: Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE 2 and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling. Conclusion: SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.

An increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F.sub.2[alpha] , where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF.sub.2[alpha] via the FP receptor. Human endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF.sub.2[alpha] -treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA. ADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2[alpha]-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation. These data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF.sub.2[alpha] -FP receptor mediated induction of ADAMTS1.

The pleiotropic cytokine interleukin-11 (IL-11) up-regulates the proliferative and invasive capacity of many human cancer cells in vitro. Co-expression of gp130 and IL-11 receptor alpha (IL-11rα) is necessary for high affinity binding of IL-11 to IL-11rα. Recently we have established a role for prostaglandin F2α (PGF) receptor (FP) signalling in the regulation of tumorigenic genes in endometrial adenocarcinomas. This study investigated the expression of IL-11 and role of PGF-FP receptor signaling in the modulation of IL-11 expression in endometrial adenocarcinoma cells. Human adenocarcinoma biopsies were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Localisation of IL-11, IL-11rα and gp130 expression was performed by immunohistochemistry. IL-11 mRNA expression was determined by real-time RT-PCR analysis using Ishikawa endometrial adenocarcinoma cells expressing the FP receptor to the levels observed in endometrial adenocarcinomas (FPS cells). IL-11 mRNA expression was significantly elevated in endometrial adenocarcinoma samples compared to normal endometrium (0.4 ± 0.1 vs 0.03 ± 0.01 arbitrary units; p<0.05) and localized with IL-11rα and gp130 in the glandular epithelium of endometrial adenocarcinomas. To investigate PGF-FP receptor signaling to IL-11 in endometrial adenocarcinoma cells, FPS cells were treated with vehicle, 100nM PGF or 100nM PGF in the absence/presence of the FP receptor antagonist AL8810 or chemical inhibitors of protein kinase A (PKA; 4C3MQ), protein kinase C (PKC; RO318220), calcineurin (cyclosporine A), Calcium (EGTA) or NFAT (INCa6). Treatment of FPS cells with PGF for 24 hours significantly elevated the expression of IL-11 mRNA (95.1 ± 6.4 fold above vehicle treated cells, P<0.05). Co-incubation of FPS cells with PGF and the FP receptor antagonist AL8810 (8.4 ± 6.4 fold above vehicle treated cells; P<0.01) or chemical inhibitors of PKC (9.6 ± 4.1 fold; P<0.01), calcineurin (17.7 ± 3.7 fold; P<0.01), calcium (15.8 ± 9.5 fold; P<0.05) or NFAT (18.3 ± 10.2 fold; P<0.01) but not PKA (96.5 ± 32.7 fold; P<0.01) significantly reduced the PGF-mediated increase in IL-11 mRNA expression. These data demonstrate that PGF regulation of IL-11 mRNA is mediated via the activation of PKC and the calcium-calcineurin pathway and suggest a possible mechanism for control of endometrial tumorigenesis by PGF-FP receptor via activation of signaling to cytokines such as IL-11.