A potential role of Sirtuin3 and its target enzyme activities in patients with ovarian endometrioma.

OBJECTIVE: Sirtuin3 (SIRT3) is a NAD+-dependent major mitochondrial deacetylase. In this study, we aimed to investigate SIRT3 levels and their target enzyme activities, including glutamate dehydrogenase (GDH), succinate dehydrogenase (SDH), and manganese superoxide dismutase (MnSOD), also to determine the antioxidant capacity and oxidative stress in tissue, mitochondria and serum samples in ovarian endometrioma patients. METHODS: We collected serum and endometrioma tissue samples from 30 patients. In the control group, we collected serum and eutopic endometrial tissue samples from 26 women without endometriosis. RESULTS: SIRT3 levels were significantly decreased in endometrioma tissue samples compared to the control group. There was no statistically significant difference in SIRT3 levels between patient and control serum samples. Furthermore, there was a decrease in GDH and SDH enzyme activities in both endometrioma tissue homogenate and mitochondria. MnSOD activity was decreased in tissue homogenate but increased in mitochondria and there was no difference in serum. While total SOD activity was decreased, CuZnSOD activity was increased in both tissue and serum samples. Besides these, total antioxidant capacity and advanced oxidation protein products (AOPP) levels were decreased in endometrioma tissue and mitochondria, but there was no difference in serum. CONCLUSIONS: Our results suggested that decreased levels of SIRT3 in endometrioma may be an important factor in the weakening of mitochondrial energy metabolism and antioxidant defense in endometriosis. We think that SIRT3 deficiency may be an important factor underlying the pathogenesis of endometriosis. More detailed studies are needed to reveal the relationship between SIRT3 and metabolism and oxidative stress in ovarian endometrioma.

Involvement of PTEN and FOXO3a Proteins in the Protective Activity of Protocatechuic Acid Against Cisplatin-Induced Ovarian Toxicity in Mice.

The present study evaluated the effects of protocatechuic acid (PCA) after cisplatin-induced ovarian toxicity in mice and if PTEN and FOXO3a proteins are involved in PCA action. The mice were divided into five experimental groups (five animals per group) and treated once a day for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by an intraperitoneal (i.p.) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (150 mg/kg of body weight), or with (4) 20 or (5) 50 mg/kg body weight of PCA, followed by 5 mg/kg body weight (i.p.) of cisplatin. Next, the ovaries were destined to histological (morphology and activation), immunohistochemical (PCNA and cleaved caspase-3 expression), and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. Moreover, the immunoreactivity for p-PTEN and p-FOXO3a was evaluated to investigate a potential mechanism by which PCA could prevent the cisplatin-induced ovarian damage. Pretreatment with N-acetylcysteine or 20 mg/kg PCA before cisplatin preserved the percentage of normal follicles and cell proliferation as observed in the control, reduced apoptosis and ROS levels, and showed higher active mitochondria and GSH levels than the cisplatin treatment (P < 0.05). Moreover, pretreatment with 20 mg/kg PCA decreased cisplatin-induced p-PTEN and increased (P < 0.05) nuclear export of p-FOXO3a. In conclusion, PCA at 20 mg/kg reduced apoptosis, maintained cell proliferation and mitochondrial function, reduced ROS production, and increased GSH expression likely through the involvement of PTEN and FOXO3a proteins.

Cilostazol protects against cyclophosphamide-induced ovarian toxicity in female rats: role of cAMP and HO-1.

Purpose: Cancer rates have been increased among women of reproductive age nowadays. Hence, many young female will be exposed to chemotherapeutic agents as cyclophosphamide (CP), carrying the hazards on female fertility. Cilostazol is a selective phosphodiesterase-3 inhibitor drug which exhibits antioxidant, anti-inflammatory, and anti-apoptotic activities. We aimed in this study to explore the possible protective effects of cilostazol against CP-induced ovarian damage in female rats.Methods: Cilostazol (10 mg/kg/day) was administered orally for 10 days in presence and absence of CP (150 mg/kg IP single dose) treatment. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), and anti-Müllerian hormone (AMH) levels were determined. Ovarian oxidative stress parameters along with inflammatory biomarkers were measured. 3,5-Cyclic adenosine monophosphate (cAMP) ovarian level was detected. Ovarian histopathological examination and caspase-3 immunohistochemical study were evaluated.Results: CP-treated rats showed a significant increase in serum levels of FSH and LH with decreased serum E2 and AMH levels with an increase in the ovarian inflammatory and oxidative stress biomarkers besides a significant decrease in cAMP ovarian level with an evident histopathological picture of ovarian damage and a high caspase-3 immunoexpression. Cilostazol pretreatment significantly restored the distributed hormonal levels, the oxidative stress and inflammatory biomarkers to their normal levels with marked improvement in histopathological picture of ovarian damage with a significant decrease in caspase-3 immunoexpression.Conclusions: These data suggest that cilostazol protects against CP- induced ovarian damage, which may be related to an increase in cAMP with subsequent anti-inflammatory, antioxidant, and anti-apoptotic properties.

Histone deacetylase: A potential therapeutic target for ovarian dysfunction.

Post-partum uterine disorders and reproductive tract infections cause ovarian dysfunction and infertility. Histone deacetylases (HDACs) prevent the relaxation of chromatin, and positively or negatively regulate transcription. Hence, HDACs play a pivotal role in altering the gene expression that impact different signalling pathways underling ovarian dysfunction. Thus, HDAC inhibitors (HDACi) may act as potential therapeutic targets in the treatment of an array of disorders impacting ovarian function.

Serum arylesterase and paraoxonase activities in patients with ovarian tumors.

OBJECTIVE: High levels of toxic reactive oxygen species have been found in many types of cancer cells. Serum arylesterase (ARE) and paraoxonase (PON) are esterase enzymes that have strong antioxidant characteristics. The main purpose of our study was to evaluate the activity of ARE and PON in the sera of patients with ovarian cancer and benign ovarian tumors. MATERIALS AND METHODS: This study included 30 patients with ovarian cancer, 42 patients with benign ovarian tumors, and 19 healthy age- and sex-matched individuals. ARE and PON activities were measured using spectrophotometry. RESULTS: Serum ARE activity was significantly different among the three studied groups (p<0.0001). However, posthoc tests revealed that ARE activity was lower in the benign ovarian tumor group (median, 1.53 U/mL; range, 0.43-2.47 U/mL) than in the other groups. There were no differences in ARE activity between patients with ovarian cancer (1.89 U/mL; range, 1.01-2.56 U/mL) and healthy individuals (2.05 U/mL; range, 0.79-2.44 U/mL). We found no differences in PON activity or the PON:ARE activity ratio between the studied groups. Tumor size in the benign ovarian tumor group was positively correlated with ARE activity (R Spearman=0.46, p=0.003) and negatively correlated with PON activity (R Spearman=-0.50, p=0.001). The ARE and PON activities were not influenced by histological type, ovarian cancer grade, or disease advancement. CONCLUSION: ARE activity is higher in patients with ovarian cancer than in patients with benign ovarian tumors; however, the serum activity of ARE is similar between patients with cancer and healthy individuals.

Effects of chemically induced ovarian failure on voluntary wheel-running exercise and cardiac adaptation in mice.

The role of exercise in decreasing the risk of cardiovascular disease in postmenopausal women has not been studied sufficiently. Accordingly, we investigated the effect of voluntary wheel-running and forced treadmill exercise on cardiac adaptation in mice treated with 4-vinylcyclohexine diepoxide (VCD), which selectively accelerates the loss of primary and primordial follicles and results in a state that closely mimics human menopause. Two-month-old female C57BL/6 mice injected with VCD (160 mg/kg) for 20 consecutive days underwent ovarian failure by 60 to 90 d after injection. Responses to voluntary wheel running and treadmill exercise did not differ between VCD- and vehicle-treated 7-mo-old C57BL/6 or outbred B6C3F1 mice. Moreover, adaptive cardiac hypertrophy, hypertrophic marker expression, and skeletal muscle characteristics after voluntary cage-wheel exercise did not differ between VCD- and vehicle-treated mice. Because 5' AMP-activated protein kinase (AMPK) is a key component for the maintenance of cardiac energy balance during exercise, we determined the effect of exercise and VCD-induced ovarian failure on the AMPK signaling axis in the heart. According to Western blotting, VCD treatment followed by voluntary cage-wheel exercise differently affected the upstream AMPK regulatory components AMPKα1 and AMPKα2. In addition, net downstream AMPK signaling was reduced after VCD treatment and exercise. Our data suggest that VCD did not affect exercise-induced cardiac hypertrophy but did alter cellular cardiac adaptation in a mouse model of menopause.

Interleukin-4 and prostaglandin E2 synergistically up-regulate 3ß-hydroxysteroid dehydrogenase type 2 in endometrioma stromal cells.

CONTEXT: Endometriosis is a chronic inflammatory disease in which immune response and production of estrogen in endometriotic tissues are involved in the development of the disease. Prostaglandin E2 (PGE2) stimulates aromatase (P450arom) expression in endometrioma stromal cells (ESCs) and increases the production of estrogens. On the other hand, an accumulating amount of evidence suggests that IL-4, a typical Th2 cytokine, plays important roles in the disease. OBJECTIVE: The objective of the investigation was to study the effect of IL-4 on the expression of 3ß-hydroxysteroid dehydrogenase (HSD3B2), a pivotal enzyme for estrogen production, in ESCs. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES: ESCs were isolated from ovarian endometrioma tissues and cultured with IL-4 and PGE2. CP-690550, a Janus protein tyrosine kinase 3 inhibitor, and HSD3B2 small interfering RNA were added to the culture. Gene expression of HSD3B2 and P450arom was examined by quantitative RT-PCR. Dehydroepiandrosterone (DHEA) was added to the culture, and then the combined enzyme activity of HSD3B2, which converts DHEA to androstenedione, and P450arom, which converts androstenedione to estrone, was examined by measuring estrone concentration in the supernatants with a specific enzyme immunoassay. RESULTS: IL-4 increased the expression of HSD3B2 mRNA in a dose-dependent manner. CP-650550 inhibited the IL-4-induced increase in HSD3B2 mRNA expression. PGE2 also increased the expression of HSD3B2 mRNA, and the combination of IL-4 and PGE2 synergistically increased the expression of HSD3B2 mRNA. IL-4 had no effect on the expression of P450arom mRNA, whereas PGE2 increased the expression of P450arom mRNA. Although PGE2 alone increased the production of estrone from DHEA, the combination of IL-4 and PGE2 significantly augmented the production of estrone from DHEA. The enhanced production of estrone by the combination of IL-4 and PGE2 was inhibited by CP-690550 and HSD3B2 small interfering RNA. CONCLUSIONS: IL-4 in combination with PGE2 may enhance estrogen production in endometriotic tissues, implying an elaborate mechanism that Th2 immune response augments inflammation-dependent progression of the disease.

Dienogest inhibits aromatase and cyclooxygenase-2 expression and prostaglandin E2 production in human endometriotic stromal cells in spheroid culture.

OBJECTIVE: To determine the effect of dienogest (DNG) on the expression of aromatase and cyclooxygenase-2 (COX-2) and the production of prostaglandin E(2) (PGE(2)) in human endometriotic stromal cells (ESCs). DESIGN: Experimental study in vitro. SETTING: University hospital. PATIENT(S): Seventeen patients with ovarian endometrioma. INTERVENTION(S): ESCs from chocolate cyst linings of ovaries were treated with DNG. MAIN OUTCOME MEASURE(S): Expression of aromatase and COX-2 evaluated in spheroid cultures of human ESCs by real-time quantitative polymerase chain-reaction and immunocytochemistry, production of PGE(2) quantified by enzyme-linked immunosorbent assay (ELISA), and nuclear factor kappa B (NF-κB) DNA-binding examined by ELISA and immunocytochemistry. RESULT(S): The pharmaceutical actions of DNG on the expression of aromatase and COX-2 and the production of PGE(2) were examined using spheroid cultures of human ESCs. More aromatase, COX-2, and PGE(2) were expressed in spheroid cultures than in conventional ESCs monolayers. In the spheroid cultures, DNG (10(-7) M) and progesterone (10(-7) M) inhibited the expression of aromatase, COX-2, and PGE(2). DNG also inhibited NF-κB DNA-binding activity and reduced the immunocytochemical protein expression of aromatase, COX-2, and NF-κB p50 nuclear localization. CONCLUSION(S): Because DNG inhibits aromatase and COX-2 expression as well as PGE(2) production in ESCs, these pharmacologic features might contribute to a therapeutic effect of DNG on endometriosis.

Expression of cyclooxygenase-2 in eutopic endometrium and ovarian endometriotic tissue in women with severe endometriosis.

BACKGROUND/AIMS: The purpose of this study is to evaluate the level and pattern of cyclooxygenase-2 (Cox-2) expression in endometriosis and to investigate the correlation between the expression of Cox-2 and several clinicosurgical parameters. METHODS: Twenty-six patients with endometriosis and 21 patients with other benign gynecological conditions were enrolled. The eutopic endometrium was sampled by pipelle, and fragments of ovarian endometrioma and non-endometriotic ovarian cysts were sampled during surgery. Total RNA isolation and semiquantitative reverse transcriptase polymerase chain reaction was performed. RESULTS: The expression of Cox-2 mRNA (mean +/- SEM) in eutopic endometrium and ovarian endometriotic tissue significantly increased in patients with endometriosis compared with the controls. The expression of Cox-2 increased significantly in the proliferative phase in eutopic endometrium and in the secretory phase in ovarian endometriotic tissue of patients with endometriosis compared with the controls. Cox-2 mRNA expression in the endometrium and ovarian lesions significantly correlated with serum CA-125 and the diameter of the endometrioma. CONCLUSIONS: Cox-2 expression increased in the eutopic endometrium and ovarian endometriotic tissue of the patients with endometriosis. These findings indicate that Cox-2 may be involved in the pathogenesis and progression of endometriosis.

Lipopolysaccharide promoted proliferation and invasion of endometriotic stromal cells via induction of cyclooxygenase-2 expression.

Lipopolysaccharide-enhanced cyclooxygenase 2 (COX-2) expression and prostaglandin E2 (PGE2) production were compared in endometriotic stromal cells (ESCs) and eutopic endometrial stromal cells. Lipopolysaccharide promotes the proliferation and invasion of ESCs via up-regulation of COX-2 and PGE2 expression, suggesting that pelvic inflammation may promote the progression of endometriosis.

Correlation of telomere length and telomerase activity with occult ovarian insufficiency.

BACKGROUND: Occult ovarian insufficiency is associated with infertility, impaired response to ovarian stimulation, and reduced live birth rates in women treated with assisted reproductive technologies. Although a decline in ovarian follicle number is expected with age, the proximate causes of occult ovarian insufficiency in young women remain poorly understood. Abnormalities in telomere length and telomerase activity in human granulosa cells may serve as molecular markers for this condition. METHODS: A cross-sectional study was performed. Subjects (37 yr old or less) undergoing in vitro fertilization were classified as cases of occult ovarian insufficiency or controls with mechanical infertility (male or tubal factor). Granulosa cells were acquired at the time of oocyte retrieval to quantify telomere length and telomerase activity. RESULTS: Fifty-four women were enrolled. Human granulosa cell telomerase activity was demonstrated, and lack of granulosa cell telomerase activity was associated with occult ovarian insufficiency (odds ratio, 11.0; 95% confidence interval, 1.3-495.6; P = 0.02). Telomeres were shorter in women with occult ovarian insufficiency than in controls (relative telomere/single copy gene ratio, 1.88 vs. 3.15; P = 0.039). CONCLUSIONS: Aberrant telomere homeostasis is associated with occult ovarian insufficiency in young women. This finding is consistent with the presence of telomeric attenuation that has been shown in multiple age-related conditions.

Aldose reductase enzyme and its implication to major health problems of the 21(st) century.

Aldose reductase enzyme (ALR2) of the polyol metabolic pathway, apart from its role as detoxifying enzyme towards toxic aldehydes, osmoregulator in the kidney and regulator of sperm maturation, was first found to be implicated in the etiology of the long term diabetic complications. However, to date, emerging reports have suggested that under normal glucose concentration, ALR2 may be up-regulated by factors other than hyperglycemia and therefore be involved also in other pathological processes that have become major threats to human health in the 21(st) century. Such pathologies are a number of cardiac disorders, inflammation, mood disorders, renal insufficiency and ovarian abnormalities. In addition, ALR2 was found to be over-expressed in different human cancers such as liver, breast, ovarian, cervical and rectal cancers. Although several aldose reductase inhibitors (ARIs) have progressed to the clinical level, only one is currently on the market. Thus, attention is currently targeted to discover ARIs of distinct chemical structures, being neither hydantoin nor carboxylic acid derivatives. The present review focuses on the molecular mechanisms by which ALR2 is implicated in a number of pathologies, on various aspects concerning its catalytic mechanism and its active site, and on the main classes of ARIs that have been developed to date, as well as on reported (quantitive) structure-activity relationships. The presented data aim to support the notion that ARIs are of pharmacotherapeutic interest for the pharmaceutical community and highlight essential aspects for the development of efficient and potent ARIs.

Cyclooxygenase-2 overexpression in ovarian endometriomas is associated with higher risk of recurrence.

Cyclooxegenase-2 expression was evaluated by immunohistochemistry in endometrioma tissue samples taken from 109 patients, of whom 53 had recurrence by 30 months after surgery and 56 did not. Cyclooxegenase-2 overexpression, along with previous medical treatment of endometriosis and the presence of adhesion, is predictive of recurrence of ovarian endometrioma within 30 months after surgery, with a resultant sensitivity of 72.5% and a specificity of 72.4%, suggesting that there are intrinsic and identifiable biomarkers that confer recurrence risk.

Prostaglandin E2 via steroidogenic factor-1 coordinately regulates transcription of steroidogenic genes necessary for estrogen synthesis in endometriosis.

CONTEXT: Products of at least five specific steroidogenic genes, including steroidogenic acute regulatory protein (StAR), which facilitates the entry of cytosolic cholesterol into the mitochondrion, side chain cleavage P450 enzyme, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, and aromatase, which catalyzes the final step, are necessary for the conversion of cholesterol to estrogen. Expression and biological activity of StAR and aromatase were previously demonstrated in endometriosis but not in normal endometrium. Prostaglandin E2 (PGE2) induces aromatase expression via the transcriptional factor steroidogenic factor-1 (SF1) in endometriosis, which is opposed by chicken-ovalbumin upstream-transcription factor (COUP-TF) and Wilms' tumor-1 (WT1) in endometrium. OBJECTIVE: The aim of the study was to demonstrate a complete steroidogenic pathway leading to estrogen biosynthesis in endometriotic cells and the transcriptional mechanisms that regulate basal and PGE2-stimulated estrogen production in endometriotic cells and endometrium. RESULTS: Compared with normal endometrial tissues, mRNA levels of StAR, side chain cleavage P450, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, aromatase, and SF1 were significantly higher in endometriotic tissues. PGE2 induced the expression of all steroidogenic genes; production of progesterone, estrone, and estradiol; and StAR promoter activity in endometriotic cells. Overexpression of SF1 induced, whereas COUP-TFII or WT1 suppressed, StAR promoter activity. PGE2 induced coordinate binding of SF1 to StAR and aromatase promoters but decreased COUP-TFII binding in endometriotic cells. COUP-TFII or WT1 binding to both promoters was significantly higher in endometrial compared with endometriotic cells. CONCLUSION: Endometriotic cells contain the full complement of steroidogenic genes for de novo synthesis of estradiol from cholesterol, which is stimulated by PGE2 via enhanced binding of SF1 to promoters of StAR and aromatase genes in a synchronous fashion.

Focal adhesion kinase expression in ovarian endometriosis.

OBJECTIVE: To investigate whether focal adhesion kinase (FAK) is expressed in endometriotic ovarian tissue. METHODS: We collected tissue samples from the ovaries of 41 women with ovarian endometriosis and from the endometrium of 20 healthy women undergoing surgery for benign indications. FAK expression was assessed by immunohistochemistry, Western blot analysis, and reverse-transcription polymerase chain reaction. RESULTS: Immunoreactive staining for FAK was present in the cytoplasm of epithelial and stromal cells in the 2 groups, but FAK expression was significantly higher in ovarian endometriotic tissue than in normal endometrium (P<0.01). FAK expression in ovarian endometriotic tissue was correlated with disease stage, pelvic pain level, and mean diameter of endometriotic cysts (P<0.05). CONCLUSIONS: FAK might contribute to the pathogenesis and progression of endometriosis.

Genome-wide microarray gene expression, array-CGH analysis, and telomerase activity in advanced ovarian endometriosis: a high degree of differentiation rather than malignant potential.

The aim of the present study was to investigate whether endometriosis and cancer share common molecular characteristics. Tissue samples were collected prospectively during diagnostic laparoscopy of patients with primary infertility. Using high-density oligonucleotide microarrays, (Affymetrix Gene Chip HG-U133 Set) the genome-wide gene expression profile of advanced ovarian endometriosis was analyzed compared with matched normal endometrium. Expression of TERT, the gene encoding the telomerase reverse transcriptase subunit, and telomerase activity were analyzed in eutopic and ectopic endometrium. Genome-wide, high-resolution array-CGH was used to screen for genomic aberrations in endometriosis. Expression microarray data were validated quantitatively with RT-PCR. The genes RARRES1 and RARRES2 (retinoic acid receptor responder 1 and 2) were found to be up-regulated in endometriosis, suggesting a high degree of differentiation. Consistently, down-regulated genes included those involved in the cell cycle, cell metabolism and homeostasis. Expression of TERT and telomerase activity were present in eutopic but absent in ectopic endometrium. Array-CGH revealed a normal genomic pattern without gross amplifications and deletions. In conclusion, these data suggest that advanced ovarian endometriosis represents a highly differentiated tissue with minimal or no malignant potential.

An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells.

OBJECTIVE: To examine the molecular basis of aromatase expression in stromal cell culture from endometriotic chocolate cysts. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago Japan. PATIENT(S): Thirty women, selected randomly, who underwent laparoscopy (n = 18) or laparotomy (n = 12). INTERVENTION(S): Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary. MAIN OUTCOME MEASURE(S): Estradiol concentrations in the culture media were measured by means of enzyme immunoassay. Aromatase expression was examined by quantitative real-time polymerase chain reaction. Promoter usage was examined using unique exon I (PII, I.1, I.3, I.4, I.5, and I.6) and exon II primers. To determine the effect of 5-aza-deoxycytidine on endometrial stromal cells, the cells were treated with the agent for 96 hours. RESULT(S): Endometriotic cells secreted a marginal level of estradiol into the culture media, but adding testosterone to the culture produced a pronounced level of estradiol. In endometrial cells, estradiol production was far less efficient than in endometriotic cells even after adding testosterone. Real-time polymerase chain reaction analyses demonstrated the up-regulation of aromatase messenger RNA (mRNA) expression in endometriotic cells. Three proximal promoters, PII, 1.3, and 1.6, drove mRNA expression. In endometrial cells where a marginal level of aromatase mRNA expression was observed, the same promoters as those in the endometriotic cells were used. To determine the role of epigenetic modification of aromatase gene expression in endometriotic cells, endometrial cells were treated with 5-aza-deoxycytidine, which markedly enhanced aromatase mRNA expression, depending on the same proximal promoters as those in endometriotic cells. CONCLUSION(S): An epigenetic disorder may play a role in the pathophysiology of endometriosis.

Prevention of autoimmune gastritis in mice requires extra-thymic T-cell deletion and suppression by regulatory T cells.

BACKGROUND AND AIMS: Autoimmune gastritis is one of the most common autoimmune diseases and is caused by a CD4(+) T-cell response to the gastric H(+)/K(+) ATPase encoded by Atp4a and Atp4b (H(+)/K(+) ATPase). Here, we have elucidated events that result in immunological tolerance to the H(+)/K(+) ATPase and thus the prevention of autoimmune gastritis. METHODS: T cells from H(+)/K(+) ATPase-deficient mice and H(+)/K(+) ATPase-specific T-cell receptor transgenic mice were purified and transferred to wild-type (WT) or H(+)/K(+) ATPase-deficient recipients to assess the impact of exposure to antigen on pathogenicity. RESULTS: The CD4(+) T-cell population from H(+)/K(+) ATPase-deficient mice was highly effective at inducing gastritis when compared with T cells from WT mice and, as a population, was comparatively resistant to the suppressive activity of regulatory T cells. Exposing T cells from H(+)/K(+) ATPase-deficient mice to H(+)/K(+) ATPase in WT mice decreased their ability to induce gastritis and resulted in a population that could be more easily suppressed by T(reg) cells. Transfer of clonotypic antigen-inexperienced H(+)/K(+) ATPase-specific T cells into WT mice resulted in extra-thymic clonal deletion. CONCLUSIONS: Prevention of autoimmune gastritis requires the extra-thymic purging of highly autoaggressive H(+)/K(+) ATPase-specific T cells to produce a T-cell repertoire that is more susceptible to the suppressive activity of regulatory T cells. Taken together with recent published data describing the role of T-cell receptor signalling in the maintenance of regulatory T-cell populations, we propose that exposure of T cells to antigen in the periphery is able to both delete autoaggressive specificities and maintain regulatory T-cell activity, establishing a balance between pathogenicity and regulation.

Expression analysis of the genes involved in estradiol and progesterone action in human ovarian endometriosis.

Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites, and it is well known that endometriosis is an estrogen-dependent disease. The defective formation and metabolism of steroid hormones is responsible for the promotion and development of endometriosis. In the present study we examined the mRNA levels of six enzymes that are involved in the metabolism of estrogen and progesterone--aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2 and 7, sulfatase and sulfotransferase--and of the steroid receptors--estrogen receptors alpha and beta (ERalpha, ERbeta) and progesterone receptors A and B (PRAB)--implicated in human ovarian endometriosis. We analyzed 16 samples of ovarian endometriosis and 9 of normal endometrium. The real-time polymerase chain reaction analyses revealed that six of the nine genes investigated are differentially regulated. Aromatase, 17beta-HSD types 1 and 7, sulfatase and ERbeta were statistically significantly upregulated, while ERalpha was significantly downregulated, in the endometriosis group compared with the control group. There were no significant differences in 17beta-HSD type 2, sulfotransferase and PRAB gene expression. Our results indicate that, in addition to the previously reported upregulation of aromatase, upregulation of 17beta-HSD types 1 and 7 and sulfatase can also increase the local estradiol concentration. This could thus be responsible for the estrogen-dependent growth of endometriotic tissue. Surprisingly ERalpha was downregulated.