Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

The altered mononuclear cell-derived cytokine response to glucose ingestion is not regulated by excess adiposity in polycystic ovary syndrome.

CONTEXT: Excess adipose tissue is a source of inflammation. Polycystic ovary syndrome (PCOS) is a proinflammatory state and is often associated with excess abdominal adiposity (AA) alone and/or frank obesity. OBJECTIVE: To determine the effect of glucose ingestion on cytokine release from mononuclear cells (MNC) in women with PCOS with and without excess AA and/or obesity. DESIGN: A cross-sectional study. SETTING: Academic medical center. PATIENTS: Twenty-three women with PCOS (seven normal weight with normal AA, eight normal weight with excess AA, eight obese) and 24 ovulatory controls (eight normal weight with normal AA, eight normal weight with excess AA, eight obese). INTERVENTION: Three-hour 75-g oral glucose tolerance test (OGTT). MAIN OUTCOME MEASURES: Body composition was measured by dual energy x-ray absorptiometry. Insulin sensitivity was derived from the OGTT (ISOGTT). TNFα, IL-6, and IL-1ß release was measured in supernatants of cultured MNC isolated from blood samples drawn while fasting and 2 hours after glucose ingestion. RESULTS: Insulin sensitivity was lower in obese subjects regardless of PCOS status and in normal-weight women with PCOS compared with normal-weight controls regardless of body composition status. In response to glucose ingestion, MNC-derived TNFα, IL-6, and IL-1ß release decreased in both normal-weight control groups but failed to suppress in either normal-weight PCOS group and in obese women regardless of PCOS status. For the combined groups, the cytokine responses were negatively correlated with insulin sensitivity and positively correlated with abdominal fat and androgens. CONCLUSIONS: Women with PCOS fail to suppress MNC-derived cytokine release in response to glucose ingestion, and this response is independent of excess adiposity. Nevertheless, a similar response is also a feature of obesity per se. Circulating MNC and excess adipose tissue are separate and distinct sources of inflammation in this population.

Evidence of mononuclear cell preactivation in the fasting state in polycystic ovary syndrome.

OBJECTIVE: We evaluated mononuclear cell (MNC) preactivation in women with polycystic ovary syndrome (PCOS) by examining the effect of in vitro lipopolysaccharide (LPS) exposure on cytokine release in the fasting state. STUDY DESIGN: Twenty women with PCOS (10 lean, 10 obese) and 20 weight-matched controls (10 lean, 10 obese) volunteered for study participation. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release was measured from mononuclear cells isolated from fasting blood samples and cultured in the presence and absence of LPS. Plasma IL-6 was measured from the same fasting blood samples. Insulin sensitivity was derived from an oral glucose tolerance test using the Matsuda index, and truncal fat was measured by dual-energy x-ray absorptiometry. RESULTS: The percent change from baseline in TNF-α and IL-6 release from MNC following LPS exposure was increased (P < .04) in lean and obese women with PCOS and obese controls compared with lean controls. Plasma IL-6 was increased (P < .02) in obese women with PCOS compared with lean women with PCOS, which in turn was increased (P < .02) compared with lean controls. The MNC-derived TNF-α and IL-6 responses from MNCs were negatively correlated with insulin sensitivity (P < .03) and positively correlated with testosterone (P < .03) and androstenedione (P < .006) for the combined groups. Plasma IL-6 was positively correlated with percentage truncal fat (P < .008). CONCLUSION: In PCOS, increased cytokine release from MNCs following LPS exposure in the fasting state reveals the presence of MNC preactivation. Importantly, this phenomenon is independent of obesity and may contribute to the development of insulin resistance and hyperandrogenism in PCOS. In contrast, the source of plasma IL-6 elevations in PCOS may be excess adiposity.

Glucose and lipopolysaccharide regulate proatherogenic cytokine release from mononuclear cells in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation, which can increase the risk of atherogenesis. We examined the effect of glucose ingestion and lipopolysaccharide (LPS) on markers of proatherogenic inflammation in the mononuclear cells (MNC) and plasma of women with PCOS. Sixteen women with PCOS (8 lean, 8 obese) and 15 weight-matched controls (8 lean, 7 obese) underwent a 3-h oral glucose tolerance test (OGTT). Interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) release from MNC cultured in the presence of LPS and plasma IL-6, C-reactive protein (CRP), and soluble vascular adhesion molecule-1 (sVCAM-1) were measured from blood samples drawn while fasting and 2h after glucose ingestion. Truncal fat was measured by dual-energy absorptiometry (DEXA). Lean women with PCOS and obese controls failed to suppress LPS-stimulated IL-6 and IL-1ß release from MNC after glucose ingestion. In contrast, obese women with PCOS suppressed these MNC-derived cytokines under the same conditions. In response to glucose ingestion, plasma IL-6 and sVCAM-1 increased and CRP suppression was attenuated in both PCOS groups and obese controls compared with lean controls. Fasting plasma IL-6 and CRP correlated positively with percentage of truncal fat. The absolute change in plasma IL-6 correlated positively with testosterone. We conclude that glucose ingestion promotes proatherogenic inflammation in PCOS with a systemic response that is independent of obesity. Based on the suppressed MNC-derived cytokine responses suggestive of LPS tolerance, chronic low-grade inflammation may be more profound in obese women with PCOS. Excess abdominal adiposity and hyperandrogenism may contribute to atherogenesis in PCOS.

Elevated circulating levels of tissue factor in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have coagulation disturbances and inflammation, which increases the risk of atherothrombosis. We evaluated the status of circulating tissue factor (TF), the receptor for coagulation factor VII involved in atherothrombosis, in women with PCOS and weight-matched controls. Two-way analysis of variance models were fit to evaluate the effect of PCOS status and weight class on TF and other parameters. The TF levels were significantly higher in lean women with PCOS compared to lean controls. Plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher in obese participants compared to lean participants after controlling for PCOS status. The TF levels directly correlated with percentage of truncal fat and plasma levels of PAI-1, testosterone, androstendione, and dehydroepiandrosterone-sulfate; and inversely correlated with insulin sensitivity index-OGTT(IS(OGTT)). Circulating TF is elevated in PCOS independent of obesity, but both PCOS and obesity contribute to a prothrombotic state. In PCOS, abdominal adiposity and hyperandrogenism may exacerbate the risk of atherothrombosis.

Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation that can increase the risk of atherothrombosis. We performed a cross-sectional study to examine the effect of glucose ingestion on markers of atherothrombotic inflammation in mononuclear cells (MNC) of 16 women with PCOS (8 lean, 8 obese) and 16 weight-matched controls. Activator protein-1 (AP-1) activation and the protein content of early growth response-1 (EGR-1), matrix matalloproteinases-2 (MMP2), and tissue factor (TF) were quantified from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. Plasma MMP9 and C-reactive protein (CRP) were measured from fasting blood samples. Truncal fat was determined by DEXA. Lean women with PCOS exhibited greater AP-1 activation and MMP2 protein content after glucose ingestion and higher plasma MMP9 and CRP levels than lean controls. Obese women with PCOS exhibited greater EGR-1 and TF protein content after glucose ingestion, and plasma CRP levels were even higher compared with lean subjects regardless of PCOS status. Truncal fat correlated with MMP9 and CRP levels and glucose-stimulated increases in AP-1 activation and EGR-1 and TF protein content. Testosterone correlated with glucose-stimulated AP-1 activation, and androstenedione correlated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. Thus, both PCOS and obesity contribute to an atherothrombotic state in which excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombosis.

Hyperglycemia-induced oxidative stress is independent of excess abdominal adiposity in normal-weight women with polycystic ovary syndrome.

STUDY QUESTION: What is the effect of glucose ingestion on leukocytic reactive oxygen species (ROS) generation in normal-weight women with polycystic ovary syndrome (PCOS) with and without excess abdominal adiposity (AA)? SUMMARY ANSWER: Normal-weight women with PCOS exhibit an increase in leukocytic ROS generation in response to glucose ingestion, and this increase is independent of excess AA. WHAT IS KNOWN ALREADY: Excess adipose tissue is a source of oxidative stress. Normal-weight women with PCOS exhibit oxidative stress and can have excess AA. STUDY DESIGN AND SIZE: This is a cross-sectional study involving 30 reproductive-age women. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fourteen normal-weight women with PCOS (6 normal AA, 8 excess AA) and 16 body composition-matched controls (8 normal AA, 8 excess AA) underwent body composition assessment by dual-energy absorptiometry and an oral glucose tolerance test (OGTT) at a university medical center. Insulin sensitivity was derived from the OGTT (IS(OGTT)). Blood was drawn while fasting and 2 h after glucose ingestion to measure leukocytic ROS generation and p47(phox) protein content and plasma thiobarbituric acid-reactive substances (TBARS) and C-reactive protein (CRP). MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, both PCOS groups exhibited lower IS(OGTT) (43-54%) and greater percentage change (% change) in ROS generation (96-140%), p47(phox) protein (18-28%) and TBARS (17-48%). Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels (29%) and lower CRP levels (70%). For the combined groups, IS(OGTT) was negatively correlated with the % change in ROS generation and p47(phox) protein. CRP was positively correlated with abdominal fat. The % change in p47(phox) protein was positively correlated with CRP and androgens. LIMITATIONS, REASONS FOR CAUTION: Although this study is adequately powered to assess differences in ROS generation between the women with PCOS and control participants, the modest sample size merits caution when interpreting the corroborative results of the additional measures of oxidative stress and inflammation. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the unique pro-oxidant contribution of circulating leukocytes in the development of insulin resistance and hyperandrogenism in PCOS. STUDY FUNDING/COMPETING INTEREST(S): Supported by NIH grant HD-048535 to F.G. The authors have nothing to disclose.

Inflammation in response to glucose ingestion is independent of excess abdominal adiposity in normal-weight women with polycystic ovary syndrome.

CONTEXT: Inflammation and excess abdominal adiposity (AA) are often present in normal-weight women with polycystic ovary syndrome (PCOS). OBJECTIVE: We determined the effects of hyperglycemia on nuclear factor-κB (NFκB) activation in mononuclear cells (MNC) of normal-weight women with PCOS with and without excess AA. DESIGN: This was a prospective controlled study. SETTING: The study was conducted at an academic medical center. PATIENTS: Fifteen normal-weight, reproductive-age women with PCOS (seven normal AA, eight excess AA) and 16 body composition-matched controls (eight normal AA, eight excess AA) participated in the study. MAIN OUTCOME MEASURES: Body composition was measured by dual-energy absorptiometry. Insulin sensitivity was derived from an oral glucose tolerance test (IS(OGTT)). Activated NFκB and the protein content of p65 and inhibitory-κB were quantified from MNC, and TNFα and C-reactive protein (CRP) were measured in plasma obtained from blood drawn while fasting and 2 h after glucose ingestion. RESULTS: Compared with controls, both PCOS groups exhibited lower IS(OGTT), increases in activated NFκB and p65 protein, and decreases in inhibitory-κB protein. Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels and lower TNFα and CRP levels. For the combined groups, the percent change in NFκB activation was negatively correlated with IS(OGTT) and positively correlated with androgens. TNFα and CRP were positively correlated with abdominal fat. CONCLUSION: In normal-weight women with PCOS, the inflammatory response to glucose ingestion is independent of excess AA. Circulating MNC and excess AA are separate and unique sources of inflammation in this population.

Progesterone receptor-A and -B have opposite effects on proinflammatory gene expression in human myometrial cells: implications for progesterone actions in human pregnancy and parturition.

CONTEXT: Progesterone promotes uterine relaxation during pregnancy and its withdrawal induces labor. Progesterone withdrawal in human parturition is mediated in part by changes in the relative levels of the nuclear progesterone receptor isoforms, PR-A and PR-B, in myometrial cells. Parturition also involves myometrial inflammation; however, the functional link between nuclear PR-mediated progesterone actions and inflammation in human myometrial cells is unclear. OBJECTIVE: Our objective was to determine how PR-A and PR-B regulate progesterone action in human myometrial cells and specifically the expression of genes encoding contraction-associated proteins and proinflammatory mediators. DESIGN: Effects of PR-A and PR-B on the capacity for progesterone to modulate gene expression was determined using an immortalized human myometrial cell line stably transfected with inducible PR-A and PR-B expression transgenes and conditioned to express various PR-A and PR-B levels. Gene expression was assessed by genome wide transcriptome analysis, quantitative RT-PCR and immunoblotting. RESULTS: PR-A and PR-B were each transcriptionally active in response to progesterone and affected the expression of distinct gene cohorts. The capacity for progesterone to affect gene expression was dependent on the PR-A to PR-B ratio. This was especially apparent for the expression of proinflammatory genes. Progesterone decreased proinflammatory gene expression when the PR-A to PR-B ratio favored PR-B and increased proinflammatory gene expression when the ratio favored PR-A. Progesterone via PR-B increased expression of inhibitor-κBα, a repressor of the nuclear factor-κB transcription factor, and inhibited basal and lipopolysaccharide-induced proinflammatory gene expression. Both of those PR-B-mediated effects were inhibited by PR-A. CONCLUSIONS: Our data suggest that during most of human pregnancy, when myometrial cells are PR-B dominant, progesterone promotes myometrial quiescence through PR-B-mediated antiinflammatory actions. At parturition, the rise in PR-A expression promotes labor by inhibiting the antiinflammatory actions of PR-B and stimulating proinflammatory gene expression in response to progesterone.

Increased resistance to apoptosis during differentiation and syncytialization of BeWo choriocarcinoma cells.

Transition from mononuclear villous cytotrophoblast into multinuclear syncytiotrophoblast in the human placenta is accompanied by changes in apoptosis-related proteins and an apparent increased resistance to induced apoptosis. We investigated the specific nature and timing of changes in Bcl-2, Bax, p53, and caspases 3 and 8 in forskolin-treated BeWo choriocarcinoma cells, a model for villous cytotrophoblast differentiation. BeWo cells were treated with forskolin or vehicle alone for up to 72 h and evaluated at 24 h intervals for syncytialization and quantitative expression specific apoptosis-related proteins and mRNAs. Syncytialization was quantified using fluorescent staining of intercellular membranes and enumeration of the percentage of nuclei in multinucleate cells, and differential localization of apoptosis-related proteins to multinuclear or mononuclear cells was determined by quantitative immunofluorescence. Forskolin treatment for up to 72 h resulted in 80% syncytialization, increased expression of Bcl-2 protein (P < 0.01) and mRNA (P < 0.05), and significantly decreased expression of protein and mRNA for Bax, p53, and caspases 3 and 8. Syncytialized cells expressed higher levels of Bcl-2 protein concurrent with increased resistance to cisplatin-induced apoptosis. Thus, syncytialization of BeWo cells was accompanied by altered transcription of apoptotic-related proteins characteristic of increased apoptosis resistance secondary to increased expression of the anti-apoptotic protein Bcl-2 and diminish expression of pro-apoptotic proteins.

Elevated circulating levels of macrophage migration inhibitory factor in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have chronic low level inflammation which can increase the risk of atherogenesis. We evaluated the status of circulating macrophage migration inhibitory factor (MIF), a proinflammatory cytokine involved in atherogenesis, in women with PCOS and weight-matched controls. Two-way analysis of variance models adjusted for age were fit to evaluate the effect of PCOS status (PCOS vs. controls) and weight-class (obese vs. lean) on MIF and other parameters. MIF levels were significantly (p<0.001) higher in women with PCOS (lean: 37.7+/-10.6 ng/ml; obese: 54.6+/-15.2 ng/ml) compared to controls (lean: 4.8+/-0.6 ng/ml; obese: 17.5+/-8.0 ng/ml) regardless of weight-class. CRP levels were significantly (p<0.001) higher in obese subjects (PCOS: 6.2+/-1.9 mg/l; controls: 6.7+/-1.4 mg/l) compared to lean subjects (PCOS: 0.9+/-0.4 mg/l; controls: 0.2+/-01 mg/l) after controlling for PCOS status. MIF levels directly correlated with % truncal fat (r=0.41, p<0.05), and plasma levels of CRP (r=0.42, p=0.05), LH (r=0.45, p=0.04), testosterone (r=0.53, p<0.008), androstendione (r=0.58, p<0.005). IS(OGTT) inversely correlated with plasma levels of MIF (r=-0.51, p<0.02) and CRP (r=-0.73, p<0.001). Circulating MIF is elevated in PCOS independent of obesity, but both PCOS and obesity contribute to a proatherogenic state. In PCOS, abdominal adiposity and hyperandrogenism may exacerbate the risk of atherosclerosis.

Evidence of proatherogenic inflammation in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have chronic low-level inflammation that can increase the risk of atherogenesis. We measured circulating proatherogenic inflammatory mediators in women with PCOS (8 lean: body mass index, 18-25 kg/m(2); 8 obese: body mass index, 30-40 kg/m(2)) and weight-matched controls (8 lean, 8 obese). Blood samples were obtained fasting and 2 hours after glucose ingestion to measure interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemotactic protein-1 (MCP-1), C-reactive protein (CRP), matrix metalloproteinase-2, plasminogen activator inhibitor-1 (PAI-1), and activated nuclear factor kappaB in mononuclear cells. Truncal fat was determined by dual-energy x-ray absorptiometry. Fasting MCP-1 levels were elevated in lean women with PCOS compared with lean controls (159.9 +/- 14.1 vs 121.2 +/- 5.4 pg/mL, P < .02). Hyperglycemia failed to suppress matrix metalloproteinase-2 in lean women with PCOS compared with lean controls (1.7 +/- 1.2 vs -4.8 +/- 1.6 pg/mL, P < .002). Among women with PCOS, obese individuals exhibited higher fasting sICAM-1 (16.1 +/- 0.8 vs 10.5 +/- 1.0 ng/mL, P < .03) and PAI-1 (6.1 +/- 0.7 vs 3.4 +/- 0.8 ng/mL, P < .03) levels. Trend analysis revealed higher (P < .005) IL-6, sICAM-1, CRP, PAI-1, systolic and diastolic blood pressures, triglycerides, fasting insulin, and homeostasis model assessment of insulin resistance index in women with PCOS compared with weight-matched controls, and the highest levels in the obese regardless of PCOS status. Fasting MCP-1 levels correlated with activated nuclear factor kappaB during hyperglycemia (P < .05) and androstenedione (P < .004). Truncal fat correlated with fasting IL-6 (P < .004), sICAM-1 (P < .006), CRP (P < .0009), and PAI-1 (P < .02). We conclude that both PCOS and obesity contribute to a proatherogenic state; but in women with PCOS, abdominal adiposity and hyperandrogenism may exacerbate the risk of atherosclerosis.

Obese reproductive-age women exhibit a proatherogenic inflammatory response during hyperglycemia.

OBJECTIVE: The objective was to determine if physiological hyperglycemia induces a proatherogenic inflammatory response in mononuclear cells (MNCs) in obese reproductive-age women. RESEARCH METHODS AND PROCEDURES: Seven obese and 6 age-matched lean women (20 to 39 years of age) underwent a 2-hour 75-g oral glucose tolerance test. The release of interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) from MNCs cultured in the presence of lipopolysaccharide (LPS) was measured after isolation from blood samples drawn fasting and 2 hours after glucose ingestion. Reactive oxygen species (ROS) generation and intra-nuclear nuclear factor kappaB (NFkappaB) from MNCs were quantified from the same blood samples. Insulin resistance was estimated by homeostasis model assessment of insulin resistance (HOMA-IR). Total body fat and truncal fat were determined by DXA. RESULTS: Obese women had a higher (p < 0.03) total body fat (42.2 +/- 1.1 vs. 27.7 +/- 2.0%), truncal fat (42.1 +/- 1.2 vs. 22.3 +/- 2.4%), and HOMA-IR (3.3 +/- 0.5 vs. 1.8 +/- 0.2). LPS-stimulated IL-6 release from MNCs was suppressed during hyperglycemia in lean subjects (1884 +/- 495 vs. 638 +/- 435 pg/mL, p < 0.05) but not in obese women (1184 +/- 387 vs. 1403 +/- 498 pg/mL). There was a difference (p < 0.05) between groups in the hyperglycemia-induced MNC-mediated release of IL-6 (-1196 +/- 475 vs. 219 +/- 175 pg/mL) and IL-1beta (-79 +/- 43 vs. 17 +/- 12 pg/mL). In addition, the obese group exhibited increased (p < 0.05) MNC-derived ROS generation (39.3 +/- 9.9 vs. -1.0 +/- 12.8%) and intra-nuclear NFkappaB (9.4 +/- 7.3 vs. -23.5 +/- 13.5%). Truncal fat was positively correlated with the MNC-derived IL-6 response (rho = 0.58, p < 0.05) and intra-nuclear NFkappaB (rho = 0.64, p < 0.05). DISCUSSION: These data suggest that obese reproductive-age women are unable to suppress proatherogenic inflammation during physiological hyperglycemia. Increased adiposity may be a significant contributor to this pro-inflammatory susceptibility.

Decreased expression of P2X7 in endometrial epithelial pre-cancerous and cancer cells.

OBJECTIVES: To understand the potential role of P2X(7) as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X(7). METHODS: Feasibility clinical experimental study. Normal (28), simple or complex hyperplasia (7), complex hyperplasia with atypia (6) and cancer endometrial discarded tissues (40) were obtained from a total of 81 women, ages 25-75. Endpoint for P2X(7) protein was average pixel signal density of tissue immunoreactivity with anti-P2X(7) antibody. Endpoint for P2X(7) mRNA was one-step quantitative Real-Time PCR. Experiments in-vitro included normal (hEVEC) and cancerous cervical epithelial cells (HeLa) transfected with reporter plasmid containing luciferase-3' untranslated region (3'UTR)-P2X(7) cDNA, using as endpoint steady-state luciferase mRNA levels. RESULTS: Levels of P2X(7) protein and mRNA were significantly lower in vivo, in tissues of complex hyperplasia with atypia or endometrial adenocarcinoma, than in tissues of normal endometrium, simple hyperplasia or complex hyperplasia tissues (sensitivity and specificity of 89-100%, p<0.0001-0.01). Steady-state levels of luciferase mRNA increased over a 6 h incubation period in hEVEC cells transfected with the 3'UTR-P2X(7)-luciferase vector, but decreased in HeLa cells transfected with the reporter plasmid. CONCLUSIONS: Tissue levels of P2X(7) protein and mRNA can differentiate effectively and accurately between normal and benign hyperplastic endometrial tissues from pre-cancerous and cancer tissues. Cancerous epithelial cells degrade P2X(7) mRNA by activation of instability domains located at the 3'UTR of the P2X(7).

Increased activation of nuclear factor kappaB triggers inflammation and insulin resistance in polycystic ovary syndrome.

CONTEXT: Insulin resistance and chronic low level inflammation are often present in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to determine the effects of hyperglycemia on nuclear factor kappaB (NFkappaB) activation and inhibitory kappaB (IkappaB) from mononuclear cells (MNC) in PCOS. DESIGN AND SETTING: This was a prospective controlled study conducted at an academic medical center. PATIENTS: The study population consisted of 16 reproductive-age women with PCOS (eight lean, eight obese) and 16 age- and body composition-matched controls (eight lean, eight obese). MAIN OUTCOME MEASURES: Insulin sensitivity (IS) was derived from a 2-h 75-g oral glucose tolerance test (IS(OGTT)). Intranuclear NFkappaB and IkappaB protein expression were quantitated from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. RESULTS: IS(OGTT) was lower in PCOS compared with controls (3.3 +/- 0.3 vs. 6.4 +/- 0.9, P < 0.004). The percent change in intranuclear NFkappaB was higher in lean and obese PCOS compared with lean controls (42.5 +/- 19.1 and 54.5 +/- 12.5 vs. -14.1 +/- 10.9, P < 0.006). The percent change in intranuclear NFkappaB correlated positively with 2-h post-glucose ingestion levels (r = 0.37; P < 0.04) and plasma testosterone (r = 0.49; P < 0.006) and correlated negatively with IS(OGTT) (r = 0.39; P < 0.04). The percent change in IkappaB was lower in lean and obese PCOS compared with lean controls (-22.3 +/- 3.2 and -17.0 +/- 5.0 vs. 8.4 +/- 11.8, P < 0.02). CONCLUSION: In response to hyperglycemia, intranuclear NFkappaB increases and IkappaB decreases in MNC of women with PCOS independent of obesity. This may represent a cardinal inflammatory signal that contributes to the induction of insulin resistance and hyperandrogenism in PCOS.

Altered tumor necrosis factor alpha release from mononuclear cells of obese reproductive-age women during hyperglycemia.

The aim of the study was to determine whether lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) release from mononuclear cells (MNCs) is altered in obese reproductive-age women in response to hyperglycemia. Six obese and 8 age-matched normal-weight women (18-40 years) underwent a 2-hour 75-g oral glucose tolerance test. Tumor necrosis factor alpha release was measured from MNCs cultured in the presence of LPS after isolation from blood samples drawn fasting and 2 hours after glucose ingestion. Insulin resistance was derived by homeostasis model assessment of insulin resistance. Total body fat (%) and truncal fat (%) were determined by dual-energy absorptiometry. Obese women had a higher (P < .03) body mass index (34.1 +/- 1.1 vs 21.9 +/- 0.8 kg/m2), percentage of total body fat (42.4% +/- 1.3% vs 28.7% +/- 1.8%), and percentage of truncal fat (42.1% +/- 1.2% vs 24.7% +/- 2.2%). Homeostasis model assessment of insulin resistance was greater in the obese group (58.0 +/- 10.6 vs 27.8 +/- 4.3, P < .02). Fasting plasma C-reactive protein (7787 +/- 884 vs 236 +/- 79 ng/mL, P < .0001) and TNF-alpha (2.37 +/- 0.09 vs 0.54 +/- 0.04 pg/mL, P < .05) were both elevated in obese women. Hyperglycemia resulted in a suppression of LPS-stimulated TNF-alpha release from MNCs of normal-weight subjects (154 +/- 21 vs 57 +/- 28 pg/mL, P < .003), but no change in obese women (148 +/- 36 vs 173 +/- 49 pg/mL). The TNF-alpha response was different between groups (-97 +/- 21 vs +24 +/- 22 pg/mL, P < .003). There was also a positive association between the incremental change in MNC-derived TNF-alpha and percentage of truncal fat (r = 0.75, P < .002). In conclusion, these data suggest that there is an absence of the "normal" suppression of TNF-alpha in MNCs after hyperglycemia in obese women, and this response may contribute to impaired glucose disposal and insulin resistance.

Reactive oxygen species-induced oxidative stress in the development of insulin resistance and hyperandrogenism in polycystic ovary syndrome.

CONTEXT: Insulin resistance and chronic low level inflammation are often present in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to determine the effects of hyperglycemia on reactive oxygen species (ROS) generation from mononuclear cells (MNCs) in PCOS. DESIGN: This was a prospective controlled study. SETTING: The study was conducted at an academic medical center. PATIENTS: The study population consisted of 16 women with PCOS (eight lean, eight obese) and 15 age- and body composition-matched controls (eight lean, seven obese). MAIN OUTCOME MEASURES: Insulin sensitivity was derived from a 2-h, 75-g oral glucose tolerance test (IS(OGTT)). ROS generation and p47(phox) protein expression were quantitated from MNCs obtained from blood drawn fasting and 2 h after glucose ingestion. RESULTS: IS(OGTT) was lower in PCOS, compared with controls (3.1 +/- 0.3 vs. 6.3 +/- 0.9, P < 0.003). The percent change in ROS generation from MNCs was higher in lean and obese PCOS, compared with lean controls (138.8 +/- 21.3 and 154.2 +/- 49.1 vs. 0.6 +/- 12.7, P < 0.003). The percent change in ROS generation from MNCs correlated positively with glucose area under the curve (r = 0.38, P < 0.05), and plasma levels of testosterone (r = 0.59, P < 0.002) and androstenedione (r = 0.50, P < 0.009). The percent change in p47(phox) from MNCs was also higher in lean and obese PCOS, compared with lean controls (36.2 +/- 18.2 and 39.1 +/- 8.0 vs. -13.7 +/- 8.7, P < 0.02), and correlated negatively with IS(OGTT) (r = -0.39, P < 0.05). CONCLUSION: ROS generation from MNCs in response to hyperglycemia is increased in PCOS independent of obesity. The resultant oxidative stress may contribute to a proinflammatory state that induces insulin resistance and hyperandrogenism in women with this disorder.

Hyperglycemia alters tumor necrosis factor-alpha release from mononuclear cells in women with polycystic ovary syndrome.

CONTEXT: Women with polycystic ovary syndrome (PCOS) are often insulin resistant and have chronic low-level inflammation. OBJECTIVE: The purpose of this study was to determine the effects of hyperglycemia on lipopolysaccharide (LPS)-stimulated TNFalpha release from mononuclear cells (MNC) in PCOS. DESIGN: The study was designed as a prospective controlled study. SETTING: The study was carried out at an academic medical center. PATIENTS: Sixteen reproductive age women with PCOS (eight lean, eight obese) and 14 age-matched controls (eight lean, six obese) participated in the study. MAIN OUTCOME MEASURES: Insulin sensitivity (IS) was derived from a 2-h 75-g oral glucose tolerance test (IS(OGTT)). Percentage of truncal fat was determined by dual-energy absorptiometry. TNFalpha release was measured from MNC cultured in the presence of LPS from blood samples drawn fasting and 2 h after glucose ingestion. RESULTS: IS(OGTT) was lower in women with PCOS compared with controls (3.9 +/- 0.4 vs. 6.3 +/- 1.0; P < 0.03) and was negatively correlated with percentage of truncal fat (r = 0.56; P < 0.002). Truncal fat was greater in lean women with PCOS compared with lean controls (29.8 +/- 2.6 vs. 23.8 +/- 2.5%; P < 0.04). The TNFalpha response was different between obese and lean controls (-96.9 +/- 21.2 vs. 24.4 +/- 21.6 pg/ml; P < 0.03) and obese and lean women with PCOS (-94.1 +/- 34.5 vs. 30.4 +/- 17.6 pg/ml; P < 0.002). Fasting plasma C-reactive protein was elevated (P < 0.003) in obese PCOS and obese controls compared with lean controls. CONCLUSION: An increase in abdominal adiposity and increased TNFalpha release from MNC after hyperglycemia may contribute to insulin resistance in lean PCOS patients. In contrast, obese PCOS patients have more profound chronic inflammation, and thus may have LPS tolerance that protects them from relatively mild excursions in blood glucose.