Clinical Characteristics and Predictors of Outcomes of Hospitalized Patients With Coronavirus Disease 2019 in a Multiethnic London National Health Service Trust: A Retrospective Cohort Study.

BACKGROUND: Emerging evidence suggests ethnic minorities are disproportionately affected by coronavirus disease 2019 (COVID-19). Detailed clinical analyses of multicultural hospitalized patient cohorts remain largely undescribed. METHODS: We performed regression, survival, and cumulative competing risk analyses to evaluate factors associated with mortality in patients admitted for COVID-19 in 3 large London hospitals between 25 February and 5 April, censored as of 1 May 2020. RESULTS: Of 614 patients (median age, 69 [interquartile range, 25] years) and 62% male), 381 (62%) were discharged alive, 178 (29%) died, and 55 (9%) remained hospitalized at censoring. Severe hypoxemia (adjusted odds ratio [aOR], 4.25 [95% confidence interval {CI}, 2.36-7.64]), leukocytosis (aOR, 2.35 [95% CI, 1.35-4.11]), thrombocytopenia (aOR [1.01, 95% CI, 1.00-1.01], increase per 109 decrease), severe renal impairment (aOR, 5.14 [95% CI, 2.65-9.97]), and low albumin (aOR, 1.06 [95% CI, 1.02-1.09], increase per gram decrease) were associated with death. Forty percent (n = 244) were from black, Asian, and other minority ethnic (BAME) groups, 38% (n = 235) were white, and ethnicity was unknown for 22% (n = 135). BAME patients were younger and had fewer comorbidities. Although the unadjusted odds of death did not differ by ethnicity, when adjusting for age, sex, and comorbidities, black patients were at higher odds of death compared to whites (aOR, 1.69 [95% CI, 1.00-2.86]). This association was stronger when further adjusting for admission severity (aOR, 1.85 [95% CI, 1.06-3.24]). CONCLUSIONS: BAME patients were overrepresented in our cohort; when accounting for demographic and clinical profile of admission, black patients were at increased odds of death. Further research is needed into biologic drivers of differences in COVID-19 outcomes by ethnicity.

Binge-eating disorder is associated with an unfavorable body mass composition in patients with non-alcoholic fatty liver disease.

The interaction between eating disorders and non-alcoholic fatty liver disease (NAFLD) remains unexplored, especially with regards to binge-eating disorder (BED). Our team conducted a service evaluation project in order to assess risk factors for the presence of BED among patients with NAFLD and the impact of BED on body mass composition. The overall prevalence of patients screening positive to BED Screener-7 (BEDS-7) was 28.4%, while a previous diagnosis of depression and marital status (as single or separated) were independently associated with positive BED. Furthermore, patients with positive BEDS-7 had higher BMI, with greater visceral component and overall lower muscle mass. There was no difference in terms of liver disease severity as assessed by noninvasive markers of fibrosis. However, as body mass composition and sarcopenia have been shown to be associated to disease progression in patients with NAFLD, further studies are required to ascertain the long-term impact of BED in these patients. Moreover, further work is warranted to identify to implement multidisciplinary approach within clinical psychology for the management of patients with BED, who may be particularly challenging in terms of achieving lifestyle modifications. As a hepatology community, we should address NAFLD with a more holistic approach.

Implementation of hepatitis C screening within a pharmacist-managed medication-assisted therapy opioid use disorder protocol program.

OBJECTIVES: (1) To describe an innovative medication-assisted therapy (MAT) opioid use disorder (OUD) protocol in an independent community pharmacy, (2) to assess patient retention of the protocol, and (3) to describe the implementation of pharmacist-initiated hepatitis C virus (HCV) screenings of patients enrolled in the protocol. SETTING: Independent pharmacy affiliated with a detox and rehabilitation center in Louisville, KY. PRACTICE DESCRIPTION: A postgraduate year 1 (PGY-1) pharmacy resident-led OUD and HCV screening protocol. PRACTICE INNOVATION: Under the Kentucky MAT OUD protocol, pharmacists at St. Matthews Community Pharmacy recognized the lack of HCV screening as a protocol requirement. To provide comprehensive care, the pharmacists added an HCV screening assessment for all patients enrolled in the pharmacy MAT OUD program. EVALUATION: The pharmacy was the first in Kentucky to implement the MAT OUD protocol after state board approval in January 2018. Patient retention rates of the MAT OUD protocol were evaluated during the 2018-2019 PGY-1 pharmacy residency program. HCV screening was implemented and assessed during this time. RESULTS: The service was implemented by the pharmacy practice resident with 77 patients enrolled in the MAT OUD program and 36 consenting to the HCV screening assessments. More than half (52%) of the study participants remained in the MAT OUD program for the recommended duration of 6 months or more. All study participants (n = 36) had recent HCV screenings. CONCLUSION: This practice innovation, led through the PGY-1 pharmacy residency program, allowed patients to enroll in a MAT OUD program in the privacy of their community pharmacy. The patient retention rate was similar to those found in physician-provided OUD programs. HCV positive screenings were found in individuals with no previous history of intravenous drug use. This provides reasoning to consider adding HCV screenings as a requirement to OUD protocols.

Modified Histopathological Grading Optimizes Prediction of Survival Outcomes in Small Intestinal Neuroendocrine Tumours.

CONTEXT: One of the major prognostic indices in neuroendocrine tumours (NETs) is Ki67 proliferation index. OBJECTIVE: To identify optimal grading Ki-67 cut-offs to delineate differences in prognosis of patients with small intestinal NETs (SI-NETs). DESIGN, SETTING, PARTICIPANTS: Multicentre retrospective cohort analysis of 551 SI-NET patients diagnosed from 1993 through 2021 at five European referral centres with a mean(±SD) follow-up time of 51.5(±52.9) months. MAIN OUTCOME MEASURES: Overall- and event-free survival (OS and EFS) rates. RESULTS: Median age at baseline was 62.3(range:17-90) years; 252(45.7%) patients were female. All SI-NETs were well-differentiated with 326 being grade 1(G1; 59.2%), 169G2(30.7%), and only 8G3(1.5), while 48 tumours were of unspecified grade (8.7%). The median Ki67 was 2%(range:1-70%). Two-hundred forty-seven patients (44.8%) had distant metastases at baseline (stage IV), 217 locoregional disease (41.1%; stage III), whereas 29(7.1%) and 25(4.5%) presented at stages II and I, respectively. The median OS was 214.7(95%CI:152.7-276.6) months and the median EFS was 79.8(95%CI:68.2-91.5) months, respectively. In multivariable Cox-regression OS analysis, the proposed modified histopathological Ki67 grading system (K67:5-10% group: HR=2.2, 95%CI:1.15-4.31; p=0.018 and K67≥10% group: HR=5.11, 95%CI:2.87-9.09; p<0.001), age (HR=1.07, 95%CI:1.04-1.09; p<0.001), Charlson Comorbidity Index (HR=1.08, 95%CI:1-1.16; p=0.028) and TNM stage (HR=1.79, 95%CI:1.05-3.06; p=0.034) were independent predictors for death. Pertinent EFS analysis, confirmed the proposed modified histopathological Ki67 grading system (K67≥10% group: HR=4.01, 95%CI:2.6-6.37; p<0.001) and age (HR=1.04, 95%CI:1.02-1.05; p<0.001) as independent predictors for recurrence, progression and/or death. CONCLUSIONS: Ki-67 proliferation index was a strong and independent predictor of OS and EFS. A modified histopathological grading system applying Ki-67 cut-offs of 5 and 10% could be superior to predict differences in SI-NET patient survival outcomes.

Monitoring prolongation of QT interval in patients with multidrug-resistant tuberculosis and non-tuberculous mycobacterium using mobile health device AliveCor.

Multidrug resistant tuberculosis and non-tuberculous mycobacterium infections present challenges due to complex treatment regimens. Extended treatment regimes expose patients to higher risks of toxic side-effects. A high drug toxicity profile necessitates closer monitoring. One of the more challenging issues is QTc prolongation with non-injectable regimens. This study investigates the portable AliveCor device to record and measure the QTc on a 6-lead ECG. An automated QTc readout from 12-Lead ECG for each patient (n = 13) and mean QTc value calculated from each patients' respective AliveCor tracing were compared. The general trend suggests AliveCor underestimates QTc - 92% cases calculated the AliveCor QTc as lower than their corresponding 12-Lead QTc readout. The use of AliveCor could potentially be translated into current clinical practice with caution of percentage variation either side. This could facilitate the use of AliveCor as a promising and convenient screening tool before further evaluation by a 12-Lead ECG is required.

Perceptions of Digital Teaching During COVID-19; A National Survey of 359 Internal Medicine Trainees.

Introduction: The Covid-19 pandemic brought significant disruption to post-graduate medical education. Lecture-based training days were rapidly converted to webinars. This study aims to assess the perceptions of digital training in internal medical trainees. Methods: IMTs (internal medicine trainees) nationally were surveyed on their perceptions of digital training, ease of access, engagement, and interactivity via a 10-item questionnaire. A mixed-method approach using qualitative and quantitative questions was used. Likert scales were analysed using a mean result of above 3 to indicate agreement. Results: 359 trainees responded. Trainees agreed that they preferred digital training to face-to-face teaching (mean 3.68); digital training was more engaging (mean 4.25), easier to access (mean 4.49), and as effective for learning as face-to-face teaching (mean 4.69). The most reported advantages were no travel (89%) and the ability to watch later on (88%). 63% of trainees reported loss of social interaction as a disadvantage. Discussion: This survey suggests that digital teaching has a potential role in IMT training beyond the pandemic.

Endoscopic submucosal dissection: an update on tools and accessories.

Endoscopic submucosal dissection (ESD) is a minimally invasive therapeutic procedure to remove larger polyps or early non-metastatic lesions. It has long been used in Asia, but is now fast growing in popularity in the West. There are multiple challenges faced by ESD practitioners. While the practice of ESD in gastric lesions is relatively well established, the oesophagus with its narrow lumen and challenging workspace, and the colon with its tortuous course and folds are more challenging frontiers. The nature of performing a procedure endoscopically means that conventional methods offer no mechanism for providing counter-traction while performing dissection, impeding visibility and increasing the rate of complications. There are a multitude of tools available to those performing ESD for the different stages of the procedure. This article reviews the accessories currently used in regular ESD practice including the knives used to cut and dissect lesions, the cap and hood devices used to improve visibility and safety, injection fluids to lift the submucosal plane, haemostatic devices, generators, and finally, emerging traction apparatus. There is some evidence behind the use of these tools, however, ESD remains the domain of a small number of practitioners and the practice relies heavily on expert experience. Evolution of the ESD toolbox will make the procedure more accessible to more endoscopists, which in turn will drive the development of a more substantial evidence base to evaluate efficacy and safety of the multitude of tools.

Rifampicin induced shock during re-exposure for treatment of latent tuberculosis.

We present a case of a young Asian female with rheumatoid arthritis who received latent tuberculosis infection (LTBI) treatment prior to treatment with a biologic agent, and developed shock with resistant hypotension on re-exposure to rifampicin. We discuss the epidemiology, pathophysiology and management of rifampicin induced shock, concluding that clinicians should be aware of this rare, but potential adverse effect, and be aware that adverse reactions to rifampicin are more frequent during re-exposure or longer dosing interval regimes. The evidence for desensitisation following such a reaction is lacking and this approach is not currently recommended. We would suggest close collaboration between specialties prescribing immunosuppression and the tuberculosis team when LTBI treatment is required after a reaction, with patient involvement to discuss the risks and benefits of treatment options.

Oestrogen formation and connective tissue growth factor expression in rat granulosa cells.

Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450(aromatase) (P450(arom))) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450(arom) mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450(arom) mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450(arom). Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450(arom). Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSH-stimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10(-7)-10(-5) M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450(arom) diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.

Quality of Life and Medication Adherence of Independently Living Older Adults Enrolled in a Pharmacist-Based Medication Management Program.

This study sought to understand the medication adherence and quality of life (QOL) of recipients of a pharmacist-based medication management program among independently living older adults. Using a cross-sectional, quasi-experimental study design, we compared older adults enrolled in the program to older adults not enrolled in the program. Data were collected via face-to-face interviews in independent-living facilities and in participants' homes. Independently living older adults who were enrolled in the medication management program (n = 38) were compared to older adults not enrolled in the program (control group (n = 41)). All participants were asked to complete questionnaires on health-related quality of life (QOL, using the SF-36) and medication adherence (using the four-item Morisky scale). The medication management program recipients reported significantly more prescribed medications (p < 0.0001) and were more likely to report living alone (p = 0.01) than the control group. The medication management program recipients had a significantly lower SF-36 physical functioning score (p = 0.03) compared to the control group, although other SF-36 domains and self-reported medication adherence were similar between the groups. Despite taking more medications and more commonly living alone, independent living older adults enrolled in a pharmacist-based medication management program had similar QOL and self-reported medication adherence when compared to older adults not enrolled in the program. This study provides initial evidence for the characteristics of older adults receiving a pharmacist-based medication management program, which may contribute to prolonged independent living and positive health outcomes.

Targeting lysyl oxidase reduces peritoneal fibrosis.

BACKGROUND: Abdominal surgery and disease cause persistent abdominal adhesions, pelvic pain, infertility and occasionally, bowel obstruction. Current treatments are ineffective and the aetiology is unclear, although excessive collagen deposition is a consistent feature. Lysyl oxidase (Lox) is a key enzyme required for crosslinking and deposition of insoluble collagen, so we investigated whether targeting Lox might be an approach to reduce abdominal adhesions. METHODS: Female C57Bl/6 mice were treated intraperitoneally with multiwalled carbon nanotubes (NT) to induce fibrosis, together with chemical (ß-aminoproprionitrile-BAPN) or miRNA Lox inhibitors, progesterone or dexamethasone. Fibrotic lesions on the diaphragm, and expression of fibrosis-related genes in abdominal wall peritoneal mesothelial cells (PMC) were measured. Effects of BAPN and dexamethasone on collagen fibre alignment were observed by TEM. Isolated PMC were cultured with interleukin-1 alpha (IL-1α) and progesterone to determine effects on Lox mRNA in vitro. RESULTS: NT-induced fibrosis and collagen deposition on the diaphragm was ameliorated by BAPN, Lox miRNA, or steroids. BAPN and dexamethasone disrupted collagen fibres. NT increased PMC Lox, Col1a1, Col3a1 and Bmp1 mRNA, which was inhibited by steroids. Progesterone significantly inhibited IL-1α induced Lox expression by PMC in vitro. CONCLUSION: Our results provide proof-of-concept that targeting peritoneal Lox could be an effective approach in ameliorating fibrosis and adhesion development.

Local estrogen metabolism in epithelial ovarian cancer suggests novel targets for therapy.

Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumors, and estrogen is often implicated in disease progression. We therefore compared the potential for gating of estrogen action via pre-receptor metabolism in normal human ovarian surface epithelium (OSE), EOC and selected EOC cell lines (SKOV3 and PEO1). Steroid sulphatase (STS), estrogen sulfotransferase (EST), 17ß-hydroxysteroid dehydrogenases 2 (17BHSD2) and 5 (17BHSD5) mRNAs, proteins and enzymatic activities were all detectable in primary cell cultures of OSE and EOC, whereas aromatase and 17BHSD1 expression was negligible. qRT-PCR assay on total mRNA revealed significantly higher EST mRNA expression in OSE compared to EOC (P<0.05). Radioenzymatic measurements confirmed reduced sulfoconjugation (neutralization) of free estrogen in EOC relative to OSE. OSE cells were more effective at converting free [(3)H]-E1 to [(3)H]-E1S or [(3)H]-E2S, while EOC cell lines mainly converted [(3)H]-E1 to [(3)H]-E2 with minimal formation of [(3)H]-E1S or [(3)H]-E2S. IL1α treatment suppressed EST (P<0.01) and 17BHSD2 (P<0.001) mRNA levels in OSE and stimulated STS mRNA levels (P<0.001) in cancer (SKOV3) cells. These results show that estrogen is differentially metabolized in OSE and EOC cells, with E2 'activation' from conjugated estrogen predominating in EOC. Inflammatory cytokines may further augment the local production of E2 by stimulating STS and suppressing EST. We conclude that local estrogen metabolism may be a target for EOC treatment.

FSH and TGF-beta superfamily members regulate granulosa cell connective tissue growth factor gene expression in vitro and in vivo.

Connective tissue growth factor (CTGF) is a heparin-binding growth factor implicated in diverse epithelial cell types as a paracrine regulator of mitosis, angiogenesis, cellular taxis, and remodeling of the extracellular matrix. To understand the possible roles of CTGF in the ovarian paracrine system, we studied CTGF gene expression by granulosa cells in relation to their stage of cellular differentiation using both in vitro and in vivo methodologies. Untreated monolayer granulosa cell cultures from immature rats abundantly expressed the approximately 2.5-kb CTGF mRNA transcript (determined by Northern analysis), but had low levels of aromatase activity (an index of granulosa cell differentiation). Treatment for 48 h with FSH (0.1-10 ng/ml) dose-dependently inhibited (>or=50%) CTGF mRNA expression, but enhanced aromatase enzyme activity. This in vitro observation of CTGF mRNA down-regulation coinciding with FSH-induced granulosa cell maturation is substantiated by studies of in vivo ovarian CTGF expression in FSHbeta knockout mice. Northern blot and in situ hybridization analyses demonstrate high levels of CTGF expression in the granulosa cells of preantral follicles blocked from further development by the absence of FSH. The action of FSH (10 ng/ml) was mimicked in vitro by 8-bromo-cAMP (1.0 mM) and was augmented by the additional presence of androgen (1 micro M 5alpha-dihydrotestosterone), consistent with mediation by intracellular cAMP. Conversely, treatment of granulosa cell cultures with TGFbeta1 (0.1-10 ng/ml) dose-dependently increased CTGF mRNA levels up to 12-fold at a dose of 10 ng/ml, without affecting aromatase activity. Cotreatment with FSH (0.1-10 ng/ml) dose-dependently suppressed the stimulatory action of TGFbeta1 (10 ng/ml) on CTGF mRNA, but substantially enhanced aromatase activity beyond levels induced by FSH alone. Importantly, other TGFbeta superfamily members known to be produced in the ovary (growth/differentiation factor-9 and activin A; 10 ng/ml) stimulated granulosa cell CTGF mRNA in a similar fashion as TGFbeta1 (10 ng/ml), and this was also inhibited by FSH (10 ng/ml). These data show that granulosa cell CTGF gene expression is inversely related to the stage of granulosa cell differentiation, being directly inhibited by FSH via cAMP-mediated signaling. CTGF mRNA abundance in nondifferentiated granulosa cells is up-regulated in vitro by TGFbeta1, growth/differentiation factor-9, and activin, suggesting paracrine roles for these growth/differentiation factors in the regulation of CTGF synthesis in mammalian ovaries.

Lysyl oxidase gene expression and enzyme activity in the rat ovary: regulation by follicle-stimulating hormone, androgen, and transforming growth factor-beta superfamily members in vitro.

Lysyl oxidase (LOX) catalyzes the final enzymatic reaction required for cross-linking of collagen and elastin fibers and therefore has a crucial role in regulating the formation and maintenance of extracellular matrix in the ovary. LOX mRNA is abundantly expressed in rat granulosa cells. To examine how regulation of LOX in the ovary might influence follicular development, we studied LOX mRNA expression and enzyme activity in rat granulosa cells from late preantral/early antral follicles in vitro. FSH dose dependently inhibited LOX mRNA and enzyme activity (50% reduction at 10 ng/ml) in vitro, and FSH action was mimicked by 8-bromo-cAMP, suggesting FSH action via elevation of cAMP. Dihydrotestosterone alone enhanced LOX mRNA and enzyme activity, but potentiated the effect of FSH, causing a further reduction. TGFbeta1 alone dose dependently enhanced LOX mRNA (5-fold increase at 10 ng/ml) and activity (1.5-fold increase). FSH dose dependently inhibited the increase in LOX mRNA and activity caused by TGFbeta1 (by up to 84% and 80%, respectively). Growth differentiation factor-9 (GDF-9) and activin A, at the same concentration as TGFbeta1 (10 ng/ml), stimulated LOX mRNA and activity within 6 h, although overall expression was higher at 48 h. All three factors when combined with FSH further reduced both mRNA and enzyme activity (by up to 60%) compared with FSH alone. These findings indicate control of LOX at endocrine, paracrine, and autocrine levels within the ovary and suggest coordinated regulation of ovarian extracellular matrix during follicular development, with FSH determining whether local factors act as stimulators or inhibitors of LOX.

Antiinflammatory steroid action in human ovarian surface epithelial cells.

The human ovarian surface epithelium (OSE) is subject to serial injury and repair during ovulation, which is a natural inflammatory event. We asked whether there is a compensatory antiinflammatory component to this process, involving steroid hormones produced locally at the time of ovulation. Quantitative RT-PCR analysis of total RNA from cultured human OSE cell monolayers showed that exposure to proinflammatory IL1alpha (500 pg/ml) increased mRNA levels of cyclooxygenase-2 (COX-2) (P < 0.01) at 48 h. The COX-2 mRNA response to IL1alpha was associated with an approximate 18-fold (P < 0.01) increase in mRNA levels of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), encoding the steroid dehydrogenase that reversibly reduces cortisone to antiinflammatory cortisol. Addition of cortisol to OSE cell culture medium dose-dependently suppressed the COX-2 mRNA response to IL1alpha (P < 0.01) but reciprocally enhanced the 11betaHSD1 mRNA response (P < 0.05), with both effects strongest at 1 microm cortisol. Presence of glucocorticoid receptor-alpha mRNA and protein was established in OSE cell monolayers and treatment with IL1alpha shown to significantly up-regulate the glucocorticoid receptor-alpha mRNA level (P < 0.05). Glucocorticoid receptor antagonist (RU486, 10 microm) fully reversed the inhibitory effect of 1 microm cortisol on IL1alpha-stimulated COX-2 mRNA expression. Progesterone also suppressed IL1alpha-induced COX-2 mRNA expression but had no significant effect on IL1alpha-stimulated 11betaHSD1 expression. These data provide direct evidence for antiinflammatory actions of cortisol and progesterone in human OSE cells.

Thrombospondin-1 inhibits angiogenesis and promotes follicular atresia in a novel in vitro angiogenesis assay.

Thrombospondin-1 (TSP-1) is a putative antiangiogenic factor, but its role in regulating physiological angiogenesis is unclear. We have developed a novel in vitro angiogenesis assay to study the effect of TSP-1 on follicular angiogenesis and development. Intact preantral/early antral follicles dissected from 21-d-old rat ovaries were cultured for 6 d in the presence or absence of TSP-1. At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. Follicles were fixed and sectioned, and follicular apoptosis was assessed by immunohistochemistry for activated caspase-3 in granulosa cells. The results showed that TSP-1 inhibited follicular angiogenesis (P < 0.01) and promoted follicular apoptosis (P < 0.001) in a dose-dependent manner. To determine whether the proapoptotic activity of TSP-1 is mediated by direct effects on granulosa cells, isolated granulosa cells were cultured with TSP-1 (0, 10, 100, and 1000 ng/ml) for 48 h. Apoptosis was quantified using a luminescent caspase-3/7 assay. TSP-1 promoted apoptosis of granulosa cells in a dose-dependent manner (P < 0.05), suggesting that TSP-1 can act independently of the angiogenesis pathway to promote follicular apoptosis. These results show that TSP-1 can both inhibit follicular angiogenesis and directly induce apoptosis of granulosa cells. As such, it may have potential as a therapeutic for abnormal ovarian angiogenesis and could facilitate the destruction of abnormal follicles observed in polycystic ovary syndrome.

Glucocorticoid receptor-mediated regulation of MMP9 gene expression in human ovarian surface epithelial cells.

OBJECTIVE: To obtain proof-of-concept that locally produced anti-inflammatory steroids suppress ovulation-associated extracellular matrix proteases in human ovarian surface epithelial (OSE) cells. DESIGN: Primary OSE cell cultures treated with interleukin-1alpha (IL-1alpha) (500 pg/mL) as proxy for inflammation, with/without anti-inflammatory steroid (cortisol or progesterone [P], 0.01-1.0 microM). SETTING: Academic medical center. PATIENT(S): Sixteen premenopausal women (29-46 years) undergoing surgery for nonmalignant gynecological conditions. MAIN OUTCOME MEASURE(S): Semiquantitative extracellular matrix protease gene expression profiling with verification by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and gelatinase zymography. RESULT(S): Treatment with IL-1alpha stimulated messenger RNA (mRNA) expression of several ovulation-associated matrix metalloproteinase genes by OSE cell cultures, including gelatinase B (MMP9) but not gelatinase A (MMP2). The IL-1alpha-stimulated MMP9 mRNA production was suppressed by cortisol but not P. Cortisol but not P also dose-dependently suppressed IL-1alpha-stimulated MMP9 gelatinase activity and this effect was blocked by the glucocorticoid receptor antagonist RU486. CONCLUSION(S): In human OSE cells, stimulation of MMP9 gene expression and proteolytic activity by IL-1alpha is suppressed by anti-inflammatory cortisol through a glucocorticoid receptor-mediated mechanism. Because IL-1alpha also generates cortisol formation in OSE by stimulating cortisone reductase activity, these results support a role for intracrine cortisol in minimizing proteolytic damage to the OSE at ovulation.

Pre-ovulatory granulosa cells of infertile women with endometriosis are less sensitive to luteinizing hormone.

PROBLEM: Reduced fertilization rates in women with minor endometriosis may be the result of direct effects on the ovary or to primary dysfunction within the hypothalamic-pituitary-ovarian axis. This controlled study was designed to examine the steroidogenic potential of luteinized granulosa cells in women with minor endometriosis. METHOD OF STUDY: Granulosa cells were harvested at oocyte recovery and incubated for 3 hr in increasing concentrations of luteinizing hormone (LH). The dissociation constant for added concentrations of LH was computed (as Km LH) and the results were compared between women with endometriosis and controls. RESULTS: Women with minor endometriosis had a higher dissociation constant than women with tubal damage [Km 0.98 (0.58-9.24) versus 0.33 (0.28-0.72) ng/mL, P = 0.019], indicating reduced sensitivity to LH. CONCLUSIONS: In women with endometriosis, granulosa cells were less sensitive to LH stimulation. This provides further evidence for primary ovarian dysfunction as a significant contributory cause of the associated subfertility.

Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression in human ovarian surface epithelial cells by interleukin-1.

BACKGROUND: Local modulation of 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity, to promote increased availability of anti-inflammatory glucocorticoids, is proposed as a compensatory response to inflammatory stimuli. Human 11betaHSD type 1 (11betaHSD1) is principally an 11-oxoreductase that reversibly reduces cortisone to cortisol. METHODS: Since ovulation is an acute inflammatory process, we examined the influence of pro-inflammatory cytokines on expression of 11betaHSD1 mRNA and metabolism of cortisone to cortisol by human ovarian surface epithelium (HOSE) in vitro. RESULTS: Northern analysis showed an approximately 1.5 kb-sized 11betaHSD1 mRNA transcript in total RNA that was up-regulated approximately 3-fold by interleukin (IL)-1alpha (0.5 ng/ml) at 24 h. By real-time RT-PCR, induction of 11betaHSD1 mRNA by IL-1alpha was measurable at 6 h and maximal at 12 h. Primary HOSE cell cultures also showed low-level 11-oxoreductase activity that was stimulated time- and dose-dependently by IL-1alpha and IL-1beta. The 11betaHSD1 mRNA and 11-oxoreductase responses to 0.5 ng/ILalpha were both suppressed by IL-1 receptor antagonist (25 ng/ml). CONCLUSIONS: Cultured HOSE cells express IL-1-responsive 11betaHSD1 and 11-oxoreductase activity mRNA in vitro. An 11betaHSD1-catalysed increase in anti-inflammatory glucocorticoid activity caused by pro-inflammatory cytokines could contribute to the local resolution of inflammation during ovulation.